Shorea robustaFrom Wikipedia, the free encyclopedia

This article is about the plant commonly known as "sal". For other uses, see Sal (disambiguation).

Sal

Conservation status

Least Concern (IUCN 2.3)

Scientific classification

Kingdom: Plantae

(unranked): Angiosperms

(unranked): Eudicots

(unranked): Rosids

Order: Malvales

Family: Dipterocarpaceae

Genus: Shorea

Species: S. robusta

Binomial name

Shorea robustaRoth

Shorea robusta, also known as sal or shala tree, is a species of tree belonging to

the Dipterocarpaceae family.

Contents

 [hide]

1 Distribution and description

2 Religious significance

3 Uses

4 Gallery

5 See also

6 References

7 External links

[edit]Distribution and description

new leaves with flower buds at Jayantiin Buxa Tiger Reserve in Jalpaiguri district ofWest Bengal, India.

This tree is native to southern Asia, ranging south of the Himalaya, from Myanmar in the east

toNepal, India and Bangladesh. In Nepal it is found mostly in the terai region from east to west, especially, in

the Churia range (The Shivalik Hill Churia Range) in the sub-tropical climate zone. There are many protected

ares such as Chitwan National Park, Bardiya National Park Bardia National Park, Shukla Phat National Parks

etc. where there are dense forest of huge sal trees. It is also found in the lower belt of hilly region and inner

terai. In India it extends from Assam, Bengal,Orissa and Jharkhand west to the Shivalik Hills in Haryana, east

of the Yamuna. The range also extends through the Eastern Ghats and to the

eastern Vindhya and Satpura ranges of central India. It is often the dominant tree in the forests where it occurs.

Sal is moderate to slow growing, and can attain heights of 30 to 35 m and a trunk diameter of up to 2-2.5 m.

The leaves are 10–25 cm long and 5–15 cm broad. In wetter areas, it is evergreen; in drier areas, it is dry-

season deciduous, shedding most of the leaves in between February to April, leafing out again in April and

May.

[edit]Religious significance

Queen Māyā giving birth to the Buddha.

In Hindu tradition the sal tree is said to be favoured by Vishnu.[1] Its name "shala", "shaal" or "sal", comes

from Sanskrit; other names in the Sanskrit language are Ashvakarna, Chiraparna and Sarja, among many

others.[2]

The sal tree is often confused with the ashoka tree (Saraca indica) in the ancient literature of theIndian

Subcontinent.

In Buddhist tradition, it is said that Queen Māyā of Sakya gave birth to Gautama Buddha under a sal tree or an

asoka tree in a garden in Lumbini, in south Nepal while grasping its branch. When this event took place Queen

Māyā was en route to birth him in his grandfather's kingdom.

There is a standard decorative element of Hindu Indian sculpture which originated in a yakshigrasping the

branch of a flowering tree while setting her foot against its roots.[3] This decorative sculptural element was

integrated into Indian temple architecture as salabhanjika or "sal tree maiden", although it is not clear either

whether it is a sal tree or an asoka tree.[4]

In Kathmandu Valley of Nepal, we can find typical Nepali Pagoda Temple Architectures with very rich wooden

carvings. And most of all the temples such as Nyatapol Temple Nyatapolaare made of Bricks and Wood of Sal

Tree.

[edit]Uses

Sal is one of the most important sources of hardwood timber in India, with hard, coarse-grained wood that is

light in colour when freshly cut, and becoming dark brown with exposure. The wood is resinous and durable,

and is sought after for construction, although not well suited to planing and polishing. The wood is specially

suitable for constructing frames for doors and windows. The dry leaves of Sal are a major source for the

productions of leaf plates and leaf bowls in Northern and Eastern India.The leaves are also used fresh to serve

ready made paan(betelnut preparations) and small snacks such as boiled black grams, gol gappa...etc.The

used leaves/plates are readily eaten by goats and cows that roam the streets freely.The tree has therefore

protected Northern India from a flood of styrofoam and plastic plates that would have caused tremendous

pollution.In South India fresh plantain and Banana leaves are used instead.

Sal resin of the sal tree, is known as ṛla in Sanskrit and is used as an astringent in Ayurvedic medicine.[5] It is

also burned as incense in Hindu ceremonies, and sal seeds and fruit are a source of lamp oil and vegetable fat.

[edit]Gallery

Sala trunk constricted

by a ficus tree at

Jayanti.

Old leaf at Jayanti.

Flowering canopy at

Jayanti.

Salabhanjika or "sal tree

maiden", Hoysalasculpture, Belur,Karnataka

[edit]See also

References

1. ^ Sacred trees

2. ^ Ayurveda Shaal

3. ^ Buddhistische Bilderwelt: Hans Wolfgang Schumann, Ein ikonographisches Handbuch des Mahayana-

und Tantrayana-Buddhismus. Eugen Diederichs Verlag. Cologne. ISBN 3-424-00897-4, ISBN 978-3-424-

00897-5

4. ^ Eckard Schleberger, Die indische Götterwelt. Gestalt, Ausdruck und Sinnbild Eugen Diederich Verlag.

Cologne. ISBN 3-424-00898-2, ISBN 978-3-424-00898-2

5. ^ Sala, Asvakarna

Ashton (1998). Shorea robusta. 2006. IUCN Red List of Threatened

Species. IUCN 2006. www.iucnredlist.org. Retrieved on 12 May 2006.http://zipcodezoo.com/Plants/S/Shorea_robusta/

Family Dipterocarpaceae

Trees , evergreen or semievergreen, rarely deciduous in dry season . Xylem with aromatic resin in intercellular resin canals. Branchlets with stipular scars , sometimes annular . Leaves simple , alternate; stipules persistent or caducous

Trees , evergreen or semievergreen, rarely deciduous in dry season . Xylem with aromatic resin in intercellular resin canals. Branchlets with stipular scars , sometimes annular . Leaves simple , alternate; stipules persistent or caducous , large or small; leaf blade with lateral veins pinnate, margin entire or sinuate-crenate. Inflorescences few- or many-flowered, terminal or axillary racemes or panicles; flowers usually sweetly scented; bracts usually fugacious and minute, rarely persistent and large. Inflorescences, calyces, petals, ovary, and other parts usually with stellate , squamate , fascicled or free-standing hairs . Flowers bisexual , actinomorphic , contorted. Calyx lobes 5, free or united at base , imbricatein bud if not united. Petals 5, adnate or connate at base. Stamens (10-) 15 to many, free from or connate to petals; filaments usually dilated at base; anthers 2-celled, with 2 pollen sacs per cell (Chinese species) ; connective appendagesaristate , filiform or stout. Ovary superior, rarely semi-inferior, slightly immersed in torus, usually 3-loculed, each locule 2-, rarely many ovuled; ovules pendulous, lateral or anatropous . Fruit usually nutlike, sometimes capsular and 3-valved, 1(to many) -seeded, with persistent, variously accrescent calyx of which 2 or more lobes are usually developed into lorate wings. Seed exalbuminous ; cotyledons fleshy , equal or unequal, applanate or folded or cerebriform , entire orlaciniate ; radicle directed toward hilum , usually included between cotyledons.About 17 genera and 550 species: tropical Africa, Asia, and South America (in Asia, most species and genera in NW Borneo) ; five genera and 12 species (one endemic, one introduced ) in China.[1]

Genus Shorea

Trees usually large, prominently buttressed . Bark usually fissured , flaky . Stipules caducous , large or small; leaf blade ± leathery, tertiary veins parallel, margin entire. Flowers in axillary or terminal lax cymose panicles; bracts persistent

Trees usually large, prominently buttressed . Bark usually fissured , flaky . Stipules caducous , large or small; leaf blade ± leathery, tertiary veins parallel, margin entire. Flowers in axillary or terminal lax cymose panicles; bracts persistent , caducous, or absent. Sepals with 3 outer larger than 2 inner. Petals white, yellow, or pink, usually pubescent . Stamens (12-) 15 or 20-100; anthers ovoid , oblong , or panduriform; connective subulate-cuspidate or stout, club-shaped; valvesequal or outer one slightly larger. Ovary ovoid, pubescent; style subulate ; stigma entire or 3-toothed. Fruit usually 1-seeded, closely surrounded by thickened bases of accrescent calyx segments; sepals developed into lorate wings, outer 3 much larger than 2 inner.

About 200 species: Bhutan, Cambodia, China, NE India, Indonesia, Laos, Malaysia, Myanmar, Nepal, Philippines, Thailand, Vietnam; two species in China.[2]

Physical Description

Species Shorea robusta

Trees to 40 m tall, ± tardily deciduous; trunk to 2 m in diam.; crown spreading . Bark gray to dark reddish brown, becoming fissured and flaky ; inner bark not laminated; wood hard; heartwood dark brown. Branchlets

Trees to 40 m tall, ± tardily deciduous; trunk to 2 m in diam.; crown spreading . Bark gray to dark reddish brown, becoming fissured and flaky ; inner bark not laminated; wood hard; heartwood dark brown. Branchlets densely buff scabrous-pubescent. Stipules fugacious , lanceolate, small, lepidote ; petiole 2-2.5 cm, buff scabrous-pubescent; leaf blade 10-40 × 5-24 cm, ovate to oblong , thinly leathery, midvein prominent abaxially and conspicuous adaxially, lateral veins ca. 12 pairs prominent abaxially, tertiary veins densely scalariform , glabrous , base obtuse to cordate, apex acuminate. Flowers subsessile , on panicles to 25 cm; branches racemose, secund ; bracts caducous , minute. Petals strongly contorted, creamy-yellow or sometimes with a medium pink stripe , 1-1.5 cm × ca. 5 mm, linear . Sepals ovate, to 2 mm in bud, subequal , densely buff pubescent . Stamens many; anthers panduriform, setose toward apex; connective appendagesshort, stout, exceeding anther apex, sparsely setose. Ovary ovoid , densely buff pubescent. Fruit sepals unequal, spatulate , sparsely pubescent, 3 longer to 8 × 1.5 cm, 2 smaller to 3.5 × 0.5 cm; nut ovoid, ca. 5 × 12 mm. Fl. Feb-May, fr. May-Jul. [source] This is a rare species in China. [source]

Habitat

Gregarious in savanna woodlands; below 800 m [3]. 

Taxonomy[ Back to top ]

Domain: Eukaryota ( ) - Whittaker & Margulis,1978 - eukaryotes

Kingdom: Plantae ( ) - Haeckel, 1866 - Plants

Subkingdom: Viridaeplantae ( ) - Cavalier-Smith, 1981

Phylum: Tracheophyta ( ) - Sinnott, 1935 Ex Cavalier-Smith, 1998 - Vascular Plants

Subphylum: Euphyllophytina ( )

Infraphylum: Radiatopses ( ) - Kenrick & Crane, 1997

Class: Magnoliopsida ( ) - Brongniart, 1843 - Dicotyledons

Subclass: Dilleniidae ( ) - Takhtajan, 1967

Superorder: Malvanae ( ) - Takhtajan, 1967

Order: Clusiales ( ) - Dumortier, 1829

Family: Dipterocarpaceae ( )

Tribe: Shoreae ( )

Genus: Shorea ( ) - Roxburgh ex C. F. Gaertner, Suppl. Carp. 47.

1805. - Shorea

Specific epithet: robusta - C. F. Gaertner, Suppl. Carp. 48. 1805.

Botanical name: - Shorea robusta C. F. Gaertn.

Notes

Name Status: Accepted Name . Latest taxonomic scrutiny: 15-Mar-2000

Place of publication : Suppl. carp . 1:48, t. 186, fig. 1. 1805

Name verified on 10-Apr-1995 by ARS Systematic Botanists. Last updated: 22-May-1997

Similar Species[ Back to top ]

Members of the genus Shorea

ZipcodeZoo has pages for 295 species, subspecies, varieties, forms, and cultivars in this genus. Here are just 100 of them:

S. acuminata   (Dark Red Meranti)  · S. acuminatissima   (Yellow Meranti)  · S. acuta · S. affinis · S. agami   (White Meranti)  · S. agamii · S. agamii agamii   (White Meranti)  · S. agsaboensis · S. alba · S. albida   (Light Red Meranti)  · S. almon

S. acuminata   (Dark Red Meranti)  · S. acuminatissima   (Yellow Meranti)  · S. acuta · S. affinis · S. agami   (White Meranti)  · S. agamii · S. agamii agamii   (White Meranti)  · S. agsaboensis · S. alba · S. albida   (Light Red Meranti)  · S. almon   (Light Red Meranti)  · S. alutacea · S. amplexicaulis · S. andulensis   (Light Red Meranti)  · S. angustifolia · S. angustiloba · S. aptera   (Borneo Tallow Tree)  · S. argentea · S. argentifolia   (Dark Red Meranti)  · S. asahi · S. asahii · S. assamica   (White Meranti)  ·S. assamica assamica   (White Meranti)  · S. assamica globifera · S. assamica koordersii · S. assamica philippinensis   (White Meranti)  · S. astrosticta · S. astylosa · S. atrinervosa   (Yellow Balau)  · S. attopoensis · S. auriculata · S. bailloni · S. bakeriana · S. bakoensis · S. balangeran   (Red Balau)  · S. balanocarpoides   (White Meranti)  · S. barbata · S. beccariana · S. beccarii · S. belangeran · S. bentongensis   (White Meranti)  · S. biawak · S. bisophylla · S. blumutensis   (Yellow Meranti)  · S. brachyptera · S. bracteata · S. bracteolata   (White Meranti)  · S. brevipetiolaris · S. brunnescens · S. buchananii · S. bullata   (Dark Red Meranti)  · S. calcicola · S. cambodiana · S. camphorifera · S. cara · S. carapae · S. chaiana · S. chinensis · S. chrysophylla · S. ciliata · S. cinerea · S. cochinchinensis · S. collaris · S. collina   (Red Balau)  · S. compressa · S. confusa · S. congestiflora · S. conica · S. contorta   (Light- Red Meranti) · S. cordata · S. cordifolia · S. coriacea · S. costata · S. crassa · S. crassifolia · S. cristata · S. curtisii   (Dark Red Meranti)  · S. cuspidata · S. dasyphylla · S. dealbata   (White Meranti)  · S. dispar · S. disticha · S. dolichocarpa · S. domatiosa · S. dyeri · S. dyerii · S. elliptica   (Dark Red Meranti)  ·S.

exelliptica · S. eximia · S. faguetiana   (Yellow Meranti)  · S. faguetioides · S. falcata · S. falcifera · S. falciferoides · S. falciferoides falciferoides · S. fallax · S. farinosa · S. ferruginea · S. flava · S. flaviflora   (Dark Red Meranti)

Further Reading[ Back to top ]

A dictionary of the flowering plants and ferns, by J. C. Willis. CambridgeThe University Press, 1919 ENG url p. 581.

A manual of dangerous insects likely to be introduced in the United States through importations. Ed. by W. Dwight Pierce, entomologist, southern field crop insect investigations. Washington: Govt. print. off., 1918. ENG url p. 193, p. 194.

Agricultural news. Bridgetown, Barbados, Bowen & sons;1902-22. ENG url p. 233. American forestry. Washington, D.C.: American Forestry Association, 1910-1923. ENG url p. 77. An introduction to the natural system of botany: or, A systematic view of the organisation, natural affinities,

and geographical distribution, of the whole vegetable kingdom: together with the uses of the most important species in medicine, the art by J London: Longman, Rees, Orme, Brown, and Green, 1830. ENG url p. 370, p. 42.

An outline of plant geography, by Douglas Houghton Campbell. New York, The Macmillan company, 1926. ENG url p. 184, p. 389.

Annales de la Soci�t� entomologique de France. Paris: La Soci�t�, FRE url p. 481. Annales du Muse colonial de Marseille. Marseille: Muse colonial, 1907-1954. FRE url p. 135. Annals of applied biology. [Wellesbourne, Warwick, etc.]Association of Applied Biologists [etc.]

ENG url p. 225, p. 228, p. 229, p. 230, p. 235, p. 246, p. 250, p. 251, p. 252, p. 254, p. 255, p. 258, p. 259, p. 260, p. 261, p. 268.

Archiv f�r Naturgeschichte. Berlin: Nicolai, 1835- GER url p. 325. Ashton, P. 1996. Annotations to: Conservation status listing for Dipterocarpaceae. Ashton, P.S. 1990. Annotations to: conservation status listings for Dipterocarpaceae. Balakrishna, P. 1993. Annotations to WCMC printout entitled 'Unresolved India - 534 records'. Includes letter

to Kerry S. Walter dated 9 Aug 1993 and annotations to list dated 21 Sep 93. Baquar, S.R. 1995. Trees of Pakistan. Pakistan: Royal Book Company. Birds of the plains, by Douglas Dewar with sixteen illustrations from photographs of living birds, by Captain

F. D. S. Fayrer, I. M. S. London, J. Lane;1909 [1908] ENG url p. 248. Botanische Jahrbucher fur Systematik, Pflanzengeschichte und Pflanzengeographie. 2 1881 Stuttgart [etc.]

Schweizerbart [etc.] GER url p. 451. Botanisches Zentralblatt; referierendes Organ f�r das Gesamtgebiet der Botanik. Jena [etc.]G. Fischer

[etc.] GER url p. 118, p. 127, p. 134, p. 157, p. 239, p. 249, p. 256, p. 28, p. 32, p. 378, p. 424, p. 62, p. 63. Bulletin de la Soci�t� entomologique de France. Paris: La Soci�t�, 1896- FRE url p. 302. Bulletin of the British Museum (Natural History). London: BM(NH) ENG url p. 109. Burma, its people and productions; or, Notes on the fauna, flora, and minerals of Tenasserim, Pegu, and

Burma. Hertford: S. Austin, 1882-1883. ENG url p. 77. Carnegie Institution of Washington publication. Washington, Carnegie Institution of Washington, 1902-

ENG url p. 12, p. 12, p. 16, p. 171, p. 24, p. 250, p. 386. Catalogue of Chinese rubbings from Field Museum / researched by Hoshien Tchen, M. Kenneth Starr;

prepared by Alice K. Schneider; photographs by Herta Newton, and Field Museum Division of Photography; edited by Hartmut Walravens. Chicago, Ill.: Field Museum of Natural History, 1981. ENG url p. 397.

Catalogue of the books, manuscripts, maps and drawings in the British Museum (Natural History) London: BM(NH), 1903-1940. ENG url p. 1239.

Coleoptera: Lamellicornia. London, Taylor and Francis, 1910-[50] ENG url p. 317. Conspectus regni vegetabilis: secundum characteres morphologicos praesertim carpicos in classes ordines

et familias digesti, adjectis exemplis nominibusque plantarum usui medico technico et oeconomico inservientium = Uebersicht de zun�chst als Leitfaden N�rnberg: Joh. Leonhard Schrag, 1835. LAT url p. 60.

Der Tropenpflanzer; zeitschrift fr tropische landwirtschaft. Berlin. GER url p. 411, p. 463. Die Rohstoffe des Pflanzenreichs: versuch einer Technischen Rohstofflehre des Pflanzenreiches / Unter

Mitwirkung von Max Bemberger, von Julius von Wiesner. Leipzig: W. Engelmann, 1914. GER url p. 227, p. 327, p. 757.

Distributional notes on Nepal birds [by] Robert L. Fleming and Melvin A. Traylor. [Chicago]Field Museum of Natural History, 1968. ENG url p. 193.

Edinburgh New Philosophical Journal. url p. 314, p. 86. Edinburgh journal of science. Edinburgh. url p. 317, p. 70. Einleitung in das nat�rliche System der Botanik; oder, Systematische Uebersicht der Organisation,

nat�rlichen Verwandtschaften und geographischen Verbreitung des ganzen Pflanzenreichs, nebst Angabe des Nutzens der wichtigsten Arten in der Heilkunde, Weimar, Verlage des Landes-Industrie-Comptoirs, 1833. GER url p. 520.

Entomologische litteraturbl�tter, Repertorium der neuesten arbeiten auf dem gesammtgebiet der entomologie. Berlin. GER url p. 62.

Exotic microlepidoptera, v. 1-5, Mar. 1912-Nov. 1937. [London]Printed by Taylor and Francis, 1912-1937. ENG url p. 96.

Experiment station record. Washington: G.P.O., 1889-1946. ENG url p. 1238, p. 1320, p. 144, p. 144, p. 147, p. 153, p. 239, p. 332, p. 345, p. 347, p. 392, p. 448, p. 449, p. 45, p. 453, p. 47, p. 504, p. 523, p. 543, p. 555, p. 559, p. 6, p. 839, p.843, p. 844, p. 895, p. 989, p. 990, p. 993.

Flora Indica, or, Descriptions of Indian plants / by the late William Roxburgh. Serampore: Printed for W. Thacker, 1832. ENG url p. 615.

Forestry quarterly. Ithaca, N.Y. [etc.] ENG url p. 130, p. 275, p. 98. Great Basin naturalist memoirs. [Provo, Utah]Brigham Young University, 1976-1992. ENG url p. 1196,

p. 199, p. 594, p. 607, p. 676, p. 684, p. 687, p. 826. Grierson, A.J.C. and Long, D.G. 1983. Flora of Bhutan: including a record of plants from Sikkim. Royal

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H. Haessel, 1879. GER url p. 124. Handw�rterbuch der naturwissenschaften / hrsg. von prof. dr. E. Korschelt (zoologie) prof. dr. G. Linck

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Insects abroad. Being a popular account of foreign insects, their structure, habits, and transformations. By Rev. J.G. Wood. Illustrated with six hundred figures, by E.A. Smith and J.B Zwecker, engraved by G. Pearson. London, Longmans, Green and Co., 1874. ENG url p. 486.

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McGuffin, M. et al., eds. 2000. Herbs of commerce, ed. 2. (Herbs Commerce ed2)

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Natural science. London: Macmillan & Co., [1892-1899] ENG url p. 14. Phytochemie, von Friedrich Rochleder. Leipzig, W. Engelmann, 1854. GER url p. 83. Plant-geography upon a physiological basis. Oxford: Clarendon Press, 1903. ENG url p. 382, p. 836, p. 837. Rao, Y.S. 1992. Forest news. Tigerpaper 4: 1-2. Rehm, S. 1994. Multilingual dictionary of agronomic plants. (Dict Rehm) Report of the Agricultural Research Institute and College, Pusa. Calcutta: Supt. Govt. Print., India, 1909-

1916. ENG url p. 53. Report of the proceedings of the third entomological meeting: held at Pusa on the 3rd to 15th February 1919

/ edited by T. Bainbrigge Fletcher. Calcutta: Supt. of Government Printing, 1920. ENG url p. 215, p. 320. Report of the proceedings of the Entomological meeting Calcutta;Superintendent government printing, India,

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Natural History, 1982. ENG url p. 25, p. 26. Textbook of theoretical botany, by R. C. McLean and W. R. Ivimey-Cook. London, Longmans, Green[1951-

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p. 352. Zeitschrift fr wissenschaftliche Mikroskopie und fr mikroskopische Technik. Stuttgart [etc.]S. Hirzel [etc.]

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113-131.

Notes[ Back to top ]

Contributors

Ashton, P. 1998. In IUCN 2008. 2008 IUCN Red List of Threatened Species. IUCNRedList.org. Downloaded July 19, 2008.

Ashton, P. 1998. Shorea robusta. In: IUCN 2006. 2006 IUCN Red List of Threatened Species. www.iucnredlist.org . Downloaded on 20 October 2006.

Bisby, F.A., Y.R. Roskov, M.A. Ruggiero, T.M. Orrell, L.E. Paglinawan, P.W. Brewer, N. Bailly, J. van Hertum, eds (2007). Species 2000 & ITIS Catalogue of Life: 2007 Annual Checklist. Species 2000: Reading, U.K.

Brands, S.J. (comp.) 1989-2007. Systema Naturae 2000. The Taxonomicon. Universal Taxonomic Services, Amsterdam, The Netherlands. Accessed March 24, 2007.

"Shorea robusta". in Flora of China Vol. 13 Page 52. Published by Missouri Botanical Garden Press. Online at EFloras.org.

The International Plant Names Index. Accessed Jan 19, 2007. USDA, ARS, National Genetic Resources Program. Germplasm Resources Information Network -

(GRIN) [Online Database]. National Germplasm Resources Laboratory, Beltsville, Maryland. URL (April 30, 2008)

USDA, NRCS. 2005. The PLANTS Database, Version 3.5 (http://plants.usda.gov). National Plant Data Center, Baton Rouge, LA 70874-4490 USA.

Identifiers

Biodiversity Heritage Library NamebankID: 2670738 Catalogue of Life Accepted Name Code: ITS-506787 Globally Unique Identifier: urn:lsid:ipni.org:names:321427-1 GRIN Nomen Number: 33870 Integrated Taxonomic Information System (ITIS) Taxonomic Serial Number (TSN) : 506787 International Plant Names Index (IPNI) ID: 321426-1 IUCN ID: 32097 U.S.D.A. Plant Symbol : SHRO3 Zipcode Zoo Species Identifier : 61262

Footnotes

1. Xi-wen Li, Jie Li & Peter S. Ashton "Dipterocarpaceae". in Flora of China Vol. 13 Page 48. Published by Science

Press (Beijing) and Missouri Botanical Garden Press. Online at EFloras.org. [back]

2. "Shorea". in Flora of China Vol. 13 Page 48, 51, 52. Published by Science Press (Beijing) and Missouri Botanical

Garden Press. Online at EFloras.org. [back]

3. "Shorea robusta". in Flora of China Vol. 13 Page 52. Missouri Botanical Garden Press. Online at EFloras.org.

[back]

©2004-2009 the BayScience Foundation, Inc. All rights reserved.

Indian medicinal plants: a compendium of 500 species, Volume 5

 By P. K. Warrier, V. P. K. Nambiar, C. Ramankutty, R. Vasudevan Nair pg no124-128

ORIGINAL ARTICLEYear : 2006  |  Volume : 60  |  Issue : 9  |  Page : 361-370 

Antioxidative and hypocholesterolemic effect of Lactobacillus casei ssp casei (biodefensive properties of lactobacilli)

Suman Kapila, Vibha, PR SinhaAnimal Biochemistry Division, National Dairy Research Institute, Karnal, India

Correspondence Address:Suman KapilaAnimal Biochemistry Division, National Dairy Research Institute, Karnal India

DOI: 10.4103/0019-5359.27220

PMID: 16940685

 ¤ Abstract  

BACKGROUND: A positive correlation between an individual's cholesterol level and development of CHD has been suggested. Low levels of high-density lipoprotein cholesterol (HDL-C) and high levels of low-density lipoprotein cholesterol (LDL-C) are important risk factors and oxidation of LDL has been implicated as an initiator of atherosclerosis. AIM: Attempts are being made worldwide for the search of effective antioxidants that can prevent oxidation of LDL. Role of fermented milk and culture containing dairy products as effective antioxidants and their potential hypocholesterolemic effect is the focus of research. Keeping this in view, the various lactobacilli cultures were screened for their in vitro antioxidative activity. Lactobacillus casei ssp casei showing maximum antioxidative activity was selected for carrying out in vivo studies. SETTINGS AND DESIGN: Six groups of Wistar albino rats were fed on diets containing 20% fresh or oxidized soybean oil supplemented with 5% lyophilized culture or fermented milk prepared using L. casei ssp casei for a period of 90 days. The plasma was separated in different lipoprotein fractions and analyzed for cholesterol content and thiobarbituric acid reactive substances (TBARS). RESULTS: The cholesterol levels were less in plasma of groups fed on fermented milk by 2-11% and by 15-25% in groups fed on lyophilized culture as compared to group fed on skim milk. The levels of TBARS were lower in the LDL fraction of plasma in rats fed on fermented milk or culture than the control group fed on skim milk. CONCLUSIONS: The results depict the cholesterol-lowering and antioxidative potential of Lactobacillus casei ssp casei for their application as dietary adjunct.

Keywords: Hyperlipidemia, lactobacilli, lipoproteins, TBARS, vitamin E

How to cite this article:Kapila S, Vibha, Sinha PR. Antioxidative and hypocholesterolemic effect of Lactobacillus casei ssp casei (biodefensive properties of lactobacilli). Indian J Med Sci 2006;60:361-70

How to cite this URL:Kapila S, Vibha, Sinha PR. Antioxidative and hypocholesterolemic effect of Lactobacillus casei ssp casei Kapila S, Vibha, Sinha PR. Antioxidative and hypocholesterolemic effect of Lactobacillus

casei ssp casei (biodefensive properties of lactobacilli). Indian J Med Sci [serial online] 2006 [cited 2010 Nov 4];60:361-70. Available from: http://www.indianjmedsci.org/text.asp?2006/60/9/361/27220

The enormous potential of good quality food in promoting and maintaining health has set the tone of research in the area of food for health worldwide. The scientific validation of many conventional fermented foods like yoghurt has served as a health link between user and the producer. The lactobacilli are important inhabitants of the intestinal tract of man and animals. Considerable evidence has implicated lactobacilli in a number of potentially beneficial roles, viz, immunostimulation, pathogen exclusion, production of bioactive materials, anticarcinogenic activity, deconjugation of bile acids, etc. In the face of growing opportunities for functional foods, dietary adjuncts and health-related products, it is prudent to understand the activities of various lactobacilli under in vivoconditions.

Atherosclerosis is primarily a disease of the large arteries and is the major cause of heart disease, stroke and death, both in developed and developing countries.[1] Although epidemiological studies have revealed several risk factors associated with atherosclerosis, including hyperlipidemia, the incidence of atherosclerotic heart disease is increased in patients with hypercholesterolemia. Regulation of the serum cholesterol level is important to prevent atherosclerosis, as it has been shown that atherosclerosis could be suppressed by controlling the level of cholesterol in the serum. The hypocholesterolemic effects of lactobacilli have been reported.[2],[3],[4] The oxidation and oxidative processes of LDL are considered important to the pathogenesis of atherosclerosis.[5],[6] Therefore, by preventing the oxidation of LDL, it may be possible to reduce the incidence of atherosclerosis. It has not been confirmed whether lactobacilli actually prevent the oxidation of LDL, although few workers have reported these bacteria to have antioxidative properties.[7],[8] Such properties would suggest that the oxidation of LDL could be inhibited by the consumption of lactobacilli. Keeping this in view, the present investigation was undertaken with the objective to determine the cholesterol-lowering and preventive effect of Lactobacillus sp. on the oxidation of lipoproteins.

 ¤ Materials and methods  

1, 1-diphenyl-2-picrylhydrazyl (DPPH), vitamin E, thiobarbituric acid (TBA), Butylated Hydroxyl Toluene (BHT) and NADPH were procured from Sigma (St. Louis, MO, USA).The other chemicals used were from Sisco Research Laboratories, Hi-media and Loba (Mumbai, India).

Bacterial cultures 

Twelve strains of lactobacilli were screened for antioxidative activity. All the cultures were obtained from National Collection of Dairy Cultures (NCDC), National Dairy Research Institute, Karnal (Haryana), India. The list is as follows: L. acidophilus 14, L . acidophilus 15, Lactobacillus sp. L13, L. casei ssp. casei 19, L. casei ssp.casei 63, L. delbrueckii ssp. bulgaricus 4, L. delbrueckii ssp. bulgaricus 9, L. helveticus 6, L. fermentum 156, L. fermentum 155 , L. fermentum 141 and L. plantarum20. The cultures were maintained by subculturing fortnightly by inoculating into MRS broth[9] and incubating for 18 to 20 h at 37°C.

Detection of antioxidative strain 

For detection of antioxidative Lactobacillus strain, the method of Terahara was followed.[8] The Lactobacillus strains were cultured by inoculating in skim milk (10% v/v) at 37°C for 18 to 20 h.

The supernatants were extracted twice with ether (5 ml). The resulting ethereal extract was evaporated to dryness and dissolved in methanol (0.2 ml) and used as a sample for the detection of radical scavengers by TLC. The extract (2 ml) was spotted on TLC plate (Silica Gel G, E-Merck, Mumbai) using chloroform: methanol (8:2) as a developing solvent. The radical scavengers on the plate were visualized by DPPH, which got converted from purple to colorless upon reaction with radical scavengers.

Antioxidative assay 

Two TBA (thiobarbituric acid) methods, based on the in vitro microsome (MS) and linoleic acid peroxidation assay, were followed for the screening of probioticLactobacillus sp, exhibiting antioxidative activity.

MS-TBA assay 

Antioxidative activity of the intracellular cell-free extract (IE) of the Lactobacillus sp. was determined by in vitro MS-TBA assay.[10]

The intracellular extract was prepared by culturing lactobacilli for 18 h at 37°C in MRS broth. The cells were harvested by centrifugation at 12,000 xg for 10 min, followed by saline (0.9%) washing; 10 mg of wet cells were disintegrated by ultrasonic vibrations. The cell debris was removed by centrifugation at 12,000 xg for 10 min to obtain IE. The IE was used for TBA estimation. Rat liver MS was prepared by removing liver and working with ice-cold KCl (1.15%). The liver was homogenized in 4 ml of 5 mM Tris-maleate buffer (5 mM, pH 7.4) containing KCl (1.15%) by using homogenizer. The homogenate was centrifuged at 15,000 xg for 15 min. The supernatant was collected and recentrifuged at 100,000 xg for 1 h. The pellet obtained was washed once with Tris-maleate buffer (5 mM, pH 7.4). This was referred to as rat liver microsome (MS). The MS were suspended in KCl (1.15%) and the final concentration of the fraction was approximately 5 mg of protein/ml.

For rat liver MS-TBA, 40 ml of MS (5 mg protein/ml) was mixed with 840 ml of Tris-maleate buffer (50 mM, pH 7.4), 40 ml of FeSO 4 (1.25 mM), 40 ml of NADPH (1.5 mM) and 40 ml of IE solution of Lactobacillus and incubated at 37°C for 30 min. At the end of incubation, 50 ml BHT (0.2%), 300 ml TCA (20%) and 600 ml TBA (0.05 M) were added rapidly to the MS mixtures to terminate peroxidation and centrifuged at 1,200 xg for 20 min. The supernatant was collected and absorbance was measured at 534 and 570 nm. Percent inhibition of oxidation was defined as

Linoleic acid peroxidation assay 

The antioxidative activity of Lactobacillus sp. was assayed by using thiobarbituric acid (TBA) method based on inhibition of linoleic acid peroxidation by intracellular cell-free extract. Linoleic acid was chosen as the source of unsaturated fatty acid.[11] The TBA method was used for measurement of lipid peroxidation[12] and Fe-ascorbate system was used for the catalysis of oxidation.[13] 

One hundred µl of linoleic acid was emulsified with 0.2 ml of Tween 20 and 19.7 ml of distilled water. Phosphate buffer solution (0.02 M, pH 7.4) was mixed with 1 ml of linoleic acid emulsion, 0.2 ml of FeSO 4 (0.01%), 0.2 ml of ascorbate (0.01%) and 0.4 or 0.8 ml of intracellular cell-free extract and incubated at 37°C. Distilled water was substituted for intracellular cell-free extract in the blank sample. After 3 h of incubation, 2 ml of the reaction solution was mixed with 0.2 ml of TCA (4%), 2 ml of TBA (0.8%), 0.2 ml of butylated hydroxyl toluene (BHT, 0.4%) and incubated at 100°C for 30 min and cooled. The absorbance was measured at 534 nm and 570 nm. The percent inhibition of linoleic acid peroxidation was defined as described in MS-TBA assay.

In vivo studies 

Male albino rats of Wistar strain used in the experiment were procured from Small Animal House of Haryana Agricultural University (HAU), Hisar. They were housed in cages in an air-conditioned room at 24 ± 1°C as per the norms of Institutional Animal Ethics Committee. The animals were about 8 weeks old and their body weight ranged between 78.57 and 94.50 g. Forty-eight adult male rats were divided into six groups of eight rats each and maintained for a period of 90 days [Table - 1]. Composition of the diets has been described in [Table - 2]. Oxidized oil was prepared by heating fresh soybean oil at 60°C for 15 days (peroxide value 91 mEq).

Blood was withdrawn from the orbital venous plexus under anesthesia and collected in heparin-coated vials. Plasma was prepared from the blood by centrifugation (1,500 xg, 10 min).

Determination of vitamin E levels in plasma by HPLC 

The level of vitamin E in the plasma was determined according to the method of Chawla and Kaur.[15] Briefly, 0.5 ml of plasma was deproteinized with an equal volume of 95% ethanol containing 5% ascorbic acid. Three extractions were carried out using 2 ml petroleum ether each time, to maximally extract the vitamin and pooled in amber glass tube. HPLC analysis was carried out in reverse phase C18 Spherisorb 5 m 0.052, 4.6 x 250 mm column with 5 cm guard column (Waters, USA). Mobile phase used was methanol: H 2 O:96: 4. Elution was carried out at the rate of 1 ml/min and the vitamin E peak was detected with the help of UV detector (Dual and Absorbance detector) at 290 nm.

Ultracentrifugation of plasma for separation of lipoproteins 

A portion of plasma obtained after centrifugation of blood was recentrifuged at 15,000 xg for 10 min, for removing all cellular debris and supernatant was collected. The method of Diebr-Rotheneder et al[16] was followed for the separation of lipoproteins by a single 20-hour run with a discontinuous density gradient. 

Preparation of discontinuous density gradient vPreparation of discontinuous density gradient 

Plasma (up to 1.5 ml) adjusted to a density of 1.22 g/ml with solid KBr was layered at the bottom of a centrifuge tube (total volume 5.0 ml) and then overlaid with KBr density solutions of 1.08 g/ml (1.25 ml), 1.05 g/ml (1.25 ml) and 1.0 g/ml (to fill the tube). All the KBr density solutions (1.08 g/ml, 1.05 g/ml and 1.0 g/ml) contained EDTA (200 mg/ml). All were prepared in phosphate buffer (pH 7.4). All density solutions were purged with nitrogen before use.

Determination of cholesterol and TBARS 

Density gradient tubes were centrifuged in Hitachi Ultracentrifuge using rotor SW at 100,000 xg at 10°C for 20 h. After centrifugation, the separated lipoprotein fractions were distributed in 10 different tubes, each containing 500 ml aliquot and cholesterol and HDL cholesterol levels were estimated using Autopak Kit (Bayer Diagnostic, India). To 300 ml of lipoprotein fraction, added 3 ml of HCl (0.05 N) and 1 ml of TBA (0.67%) and the reaction mixture was heated for 30 min in a boiling water bath. After the mixture had been cooled in an ice bath, TBARS in the reaction mixture were extracted with 4 ml of n-butanol. The absorbance of butanol phase was measured at 535 nm. The levels of TBARS were expressed as malondialdehyde equivalents/mg of cholesterol.

Statistical analysis 

Each result is expressed as the mean + SEM. One-way ANOVA was used to examine the difference between groups.

 ¤ Results  

Radical scavenging (RS) activity 

Out of the 12 strains of lactobacilli screened, 8 strains of lactobacilli showed radical scavenging potential on TLC plates [Figure - 1]. Similar kind of detection method was followed by Terahara.[8] They showed that

L. delbrueckii ssp. bulgaricus 2003 exhibited radical scavenging potential. Likewise, DPPH method was used for screening of antioxidants in marine bacteria from fish and shellfish and 112 bacterial isolates producing antioxidants obtained.[17]

Measurement of radical-scavenging activity: MS-TBA assay

Antioxidative activity of the intracellular cell-free extract (IE) of the Lactobacillus sp. determined by in vitro MS-TBA assay - results are shown in [Table - 3].

Maximum antioxidative activity in terms of percent inhibition of oxidation was observed in L. casei ssp. casei 19 (76.82%), followed by L. acidophilus 14 (62.21%),Lactobacillus sp.L13 (59.25%), L. casei ssp. casei 63 (53.30%), L. helveticus 6 (52.70%) and

L. delbrueckii ssp. bulgaricus 4 (52.64%). All other remaining strains exhibited less than 50% activity.

Linoleic acid peroxidation assay 

The inhibition of linoleic peroxidation followed similar trend [Table - 3] as observed in MS-TBA assay, i.e., maximum in L. casei ssp. casei 19 (72.04%), followed by L. acidophilus 14 (51.74%), Lactobacillus sp. L13 (51.38%) and rest of all the strains showed less than 50% inhibition.

Likewise, the antioxidative activity of 570 strains of lactic acid bacteria by TBA assay was studied and observed to be as high as 91% activity in Lactobacillus sp. SBT-2028 and 77% in L. casei ssp. pseudoplantarum SBT 0624.[10] They have also reported 51 to 63% antioxidative activity in different strains of L. casei ssp. casei. Similarly, the radical-scavenging ability of yoghurt organisms based on linoleic peroxidation assay was examined. Streptococcus thermophilus and L. delbrueckiissp. bulgaricus demonstrated an antioxidative effect on the inhibition of linoleic acid peroxidation.[18] In another study, both intact cells and intracellular cell-free extract of B. longum and L. acidophilus (10 9sub cfu/ml) inhibited linoleic acid peroxidation by 28 to 48%.[19]

In vivo studies 

TherThere was no variation in the body weight among the rats of the six groups, indicating that the type of diet did not have any affect on the body weight. Among the lipid profile, there was no variation in the cholesterol levels in the VLDL fraction in groups fed fresh oil/oxidized oil supplemented with skim milk or fermented milk or culture. Whereas levels of LDL cholesterol were significantly ( P < 0.001) higher in groups fed on fresh oil or oxidized oil supplemented with skim milk in comparison to groups fed on fresh/oxidized oil supplemented with fermented milk or culture. In HDL fraction, levels of cholesterol were higher by 13-29% in groups fed on fresh oil/oxidized oil supplemented with fermented milk in comparison to groups supplemented with skim milk [Table - 4].

Levels of TBARS in VLDL of rats fed on fresh/oxidized oil supplemented with fermented milk or culture are shown in [Figure - 2]. Results indicated maximum levels of TBARS in groups FS (17.05 nmoles MDA/mg cholesterol) and OS (25 nmoles MDA/mg cholesterol) which were fed on 20% fresh or oxidized oil supplemented with skim milk, whereas groups fed on fermented milk or culture showed lower values of TBARS ranging from 10.54 to 19.60 nmoles MDA/mg cholesterol. Similarly in LDL fraction, TBARS levels were more in groups fed on fresh or oxidized oil supplemented with skim milk. Among oxidized oil groups, there was decline in TBARS by 55% in group fed on culture in comparison to skim milk fed group.

Among fresh oil group in LDL fraction, there was insignificant decline in TBARS in rats fed on fermented milk or culture when compared to control group fed on skim milk. However, in respect of HDL fraction, there were not many variations in the levels of TBARS between the different groups [Figure - 2].

The effect of feeding different experimental diets on vitamin E levels in plasma of rats has been shown in [Figure - 3]. The results clearly showed that vitamin E levels were less in groups fed oxidized oil as compared to fresh oil fed groups and supplementing diets with fermented milk or culture resulted in increased levels of vitamin E.

 ¤ Discussion  

Our aim was to check the antioxidative and hypocholesterolemic effect of lactobacilli. Results clearly depict the antioxidative nature of L . casei ssp casei under in vitro condition as observed in MS-TBA and linoleic acid peroxidation assay. The inhibition of peroxidation was more than 70% in both experiments. The culture ( L .casei ssp casei ) was used for carrying out in vivo studies in order to ascertain whether the similar preventive effect of culture on oxidation can be observed underin vivo condition. Therefore, in order to induce oxidative stress and hyperlipidemia, rats were fed diet containing 20% fresh or oxidized soybean oil. The effect of supplementing diet with fermented milk or culture on oxidative stress and cholesterol level was observed. The levels of LDL cholesterol were significantly lower in groups fed on fresh oil or oxidized oil supplemented with fermented milk or culture in comparison to groups fed on diet supplemented with skim milk. In addition, feeding of fermented milk resulted in increased HDL cholesterol level in experimental rats by 14-29%. Atherogenic index expressed as the ratio of LDL/HDL [Table - 4] was significantly lowered by 1.6 to 2.1 times in rats fed on fermented milk or culture. Similar observations have been made on feeding lactobacillus strain NTU101,102 Lactobacillus reutri and Lactobacillus gasseri to hamsters, mice and rats respectively.[4],[20],[21]The oxidation of LDL plays an important role in the pathogenesis of atherosclerosis and the antioxidants in the foods like tea, wine, vegetables inhibit this oxidation.[8],[22] Similarly in the present study, reduction in levels of TBARS in LDL fraction on consumption of L. casei ssp casei may exhibit prevention of oxidation of LDL. The effect was very prominent in oxidized oil group; there was significant (55%) reduction in TBARS levels in LDL fraction in group fed on culture in comparison to control, which were given skim milk. The levels of TBARS in the LDL fraction obtained from rats fed on oxidized soybean oil were higher than that in the rats fed on fresh soybean oil. Similar observation has been reported by other workers also.[23] Not only was there a reduction in TBARS levels, but levels of total plasma cholesterol were also less in groups fed on lyophilized culture, thereby exhibiting the hypocholesterolemic effect of L. casei ssp casei . Hypocholesterolemic effects of lactic cultures have been reported earlier by several workers.[4],[20],[21] Furthermore, the levels of vitamin E in the plasma were also affected by the diet; it was lower in rats, as expected, which were fed on oxidized oil than in the rats fed on fresh soybean oil and higher in the experimental rats fed on culture than in the rats fed on skim milk, because feeding of fat-rich diet increases the oxidative stress and thus increases the requirements of antioxidants like vitamin E. These results show that the antioxidative ability of the diet containing the L. casei ssp casei and its fermented product [Figure - 3] was stronger than that in the diet containing the skim milk powder. Therefore, the antioxidant present in the culture of L. casei ssp casei had an antioxidative effect similar to that of vitamin E. It is rather difficult to compare the antioxidative ability of lactobacilli with that of other antioxidants because of their different characteristics. In respect of antioxidant in the culture of L. casei ssp casei, further studies are still needed to prove its clear role in prevention of LDL oxidation and to determine its chemical nature and characteristics. Attempts are now being made to use this potential culture in combination with other cultures for preparation of dahi for human consumption and to carry out further clinical studies.

 ¤ References  

1. Lovegrove JA, Jackson KG. Coronary heart disease. In : Gibson GR and Williams CM (editors). Functional Foods: 2000. p. 97-139.       

2. Beena A, Prasad V. Effect of yogurt and bifidus yogurt fortified with skim milk powder, condensed whey and lactose-hydrolyzed condensed whey on serum cholesterol and triacylglycerol levels in rats. J Dairy Res 1997;64:453-7.      [PUBMED]  [FULLTEXT]

3. Ashar MN, Prajapati JB. Verification of hypocholesterolemic effect of fermented milk on human

subjects with different cholesterol levels. Folia Microbiol (Parha) 2000;45:263-8.     [PUBMED]  [FULLTEXT]

4. Chiu CH, Lu TY, Tseng YY, Pan TM. The effects of Lactobacillus-fermented milk on lipid metabolism in hamsters fed on high-cholesterol diet. Appl Microbiol Biotechnol 2006;71:238-45.     [PUBMED]  [FULLTEXT]

5. Liao DF, Jin ZG, Baas AS, Daum G, Gygi SP, Aebersold R, et al. Purification and identification of oxidative stress induced factors from vascular smooth muscle cells. J Biol Chem 2000;275:189-96.

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6. Ling WH, Cheng QX, Ma J, Wang T. Red and black rice decrease atherosclerotic plaque formation and increase antioxidant status in rabbits. J Nutr2001;131:1421-6.      [PUBMED]  [FULLTEXT]

7. Zommara M, Tachibana N, Sakono M, Suzuki Y, Odo T. Whey from cultured skim milk decrease serum cholesterol and increase antioxidant enzymes in liver and red blood cells in rats. Nutr Res 1996;16:293-302.       

8. Terahara M, Nishide S, Kaneko T. Preventive effect of Lactobacillus delbrueckii subsp. bulgaricus on the oxidation of LDL. Biosci Biotechnol Biochem2000;64:1868-73.     [PUBMED]  [FULLTEXT]

9. deMan JC, Rogosa M, Sharpe ME. A medium for the cultivation of lactobacilli. J Appl Bacteriol 1960;23:130-5.       

10. Kaizu H, Sasaki M, Nakajima H, Suzuki Y. Effect of antioxidative lactic acid bacteria on rats fed a diet deficient in vitamin E. J Dairy Sci 1993;76:2493-9.     [PUBMED]  [FULLTEXT]

11. Bertelsen G, Christophersen C, Nielsen PH, Madsen HL, Stadel P. Chromatographic isolation of antioxidants guided by a methyl linoleate assay. J Agric Food Chem 1995;43:1272-5.        

12. Halliwell B, Chirico S. Lipid peroxidation, its mechanism, measurement and significance. J Clin Nutr 1993;57:715S-25S.     [PUBMED]  [FULLTEXT]

13. Decker E, Faraji. Inhibition of lipid peroxidation by carnosene. J AOCS 1990;67:650-2.        

14. AOAC. Official methods of analysis, 14th ed. Association of Official Agric Chemists. Washington, DC; 1984. p. 988.       

15. Chawla R, Kaur H. A new HPLC technique developed for simultaneous estimation of β -carotene, a-tocopherol and retinol in blood plasma and feed samples. NDRI News 2002;4:2.        

16. Dieber-Rotheneder M, Puhl H, Waeg G, Striegl G, Estubauer H. Effect of oral supplementation with D-alpha-tocopherol on the vitamin E content of human low density lipoproteins and resistance to oxidation. J Lipid Res 1991;32:1325-32.        

17. Takao T, Kitatani F, Watanabe N, Yagi A, Sakata K. A simple screening method for antioxidants and isolation of several method for antioxidants and isolation of several antioxidants produced by marine from fish and shellfish. Biosci Biotechnol Biochem 1994;58:1780-3.       

18. Lin MY, Yen CL. Reactive oxygen species and lipid peroxidation product-scavenging ability of

yoghurt organism. J Dairy Sci 1999;82:1629-34.     [PUBMED]  [FULLTEXT]

19. Lin MY, Chang FJ. Antioxidative effect of intestinal bacteria Bifidobacterium longum ATCC15708 and Lactobacillus acidophilus ATCC4356. Dig Dis Sci 2000;45:1617-22.     [PUBMED]  [FULLTEXT]

20. Taranto MP, Medici M, Perdigon G, Ruiz Holgado AP, Valdez GF. Effect of Lactobacillus reuteri on prevention of in hypercholesterolemia in mice. J Dairy Sci 2000;83:401-3.     [PUBMED]  [FULLTEXT]

21. Usman, Hosono A. Effect of administration of Lactobacillus gasseri on serum lipids and fecal steroids in hypocholesterolemic rats. J Dairy Sci 2000;83:1705-11.     [PUBMED]  [FULLTEXT]

22. Yokozawa T, Dong E. Influence of green tea and its three major components upon low density lipoprotein oxidation. Exp Toxicol Pathol 1997;49:329-35.     [PUBMED]  [FULLTEXT]

23. Hayam I, Cogan U, Mokady S. Dietary oxidized oil and the activity of antioxidant enzymes and lipoprotein peroxidation in rats. Nutr Res 1995;15:1037-44.       

    Figures

[Figure - 1], [Figure - 2], [Figure - 3]

Antihyperlipidemic activity of isolated constituents from the fruits ofLagenaria siceraria in albino rats

DS Mohale1, AP Dewani2, AN Saoji1, CD Khadse3

1 Institute of Pharmaceutical Education and Research, Hinganghat Road, Wardha, India2 Sharad Pawar College of Pharmacy, Wanadongri Hingna Road, Nagpur, India3 P.W. College of pharmacy, Dhamangaon Road, Yavatmal, India

Click here for correspondence address and email  

       

    Abstract  

Hyperlipidemia is defined as increase in the lipid content (groups of fat or fat like substances along with their lipoprotein counterpart) in blood. Abundant evidence are there to proof the link between hyperlipidemia and atherosclerosis. Lagenaria siceraria commonly known as Bottle gourd, which is official in Ayurvedic Pharmacopoeia of India, and having composition of variety of essential phytoconstituents, so that the fruits are traditionally used for their cardioprotective, cardiotonic, general tonic, diuretic, aphrodisiac, antidote to certain poisons and scorpion strings, alternative purgative, and cooling effects. In the present study fruit juice was

obtained by crushing the fresh fruits of L. siceraria in the juicer and was subsequently dried in the oven at 40°-50°C. The parent dried juice extract was then fractionated by using the solvents according to polarity in ascending order i.e. by using chloroform: acetic acid, methanol, pyridine, and water. Each fraction was dried in oven at 40°-50°C. Thin layer chromatography (TLC) used active fraction obtained by column chromatography for further isolation. The solvent system developed on trial and error basis was n-butanol: methaol: water (6:2:2). Four spots were obtained and were named as LSN-I, LSN-II, LSN-III and LSN-IV. Isolated spots were collected by using preparative TLC the isolated compounds were tested for Antihyperlipidemic activity and compounds LSN-I, LSN-II, LSN-III has shown significant results. The study exhibited that elevated levels of blood cholesterol, triglycerides, LDL, were significantly reduced and decreased HDL was significantly increased by the administration of fractions of L. siceraria fruit juice.

Keywords: Atherosclerosis, hyperlipidemia, Lagenaria siceraria

How to cite this article:Mohale DS, Dewani AP, Saoji AN, Khadse CD. Antihyperlipidemic activity of isolated constituents from the fruits of Lagenaria siceraria in albino rats. Int J Green Pharm 2008;2:104-7How to cite this URL:Mohale DS, Dewani AP, Saoji AN, Khadse CD. Antihyperlipidemic activity of isolated constituents from the fruits of Lagenaria sicerariaMohale DS, Dewani AP, Saoji AN, Khadse CD. Antihyperlipidemic activity of isolated constituents from the fruits of Lagenaria siceraria in albino rats. Int J Green Pharm [serial online] 2008 [cited 2010 Nov 4];2:104-7. Available from: http://www.greenpharmacy.info/text.asp?2008/2/2/104/41181

    Introduction  

Hyperlipidemia is a highly predictive risk factor for atherosclerosis, coronary artery disease, and cerebral vascular diseases. [1] Atherosclerosis ( sclrero- hardening ) of arteries is a generalized disease of the arterial network known as a progressive and silent killer disease characterized by the formation of lesions called atherosclerosis plaques in the walls of large and or medium sized coronary arteries and which reduces blood flow to the myocardium - called coronary artery disease (CAD). [2] Abundant evidence links hyperlipidemia to atherosclerosis. Clinical trials showed conclusively that lowering serum cholesterol reduces morbidity and mortality from CAD in patients with established CAD and also reduces new CAD events and mortality in patients without established CAD. [3] Condiments, medicinal plants, fruits used in day-to-day preparation of food in Indian kitchens have been identified as hypolipidaemic in Ayurveda. [4] Lagenaria siceraria is commonly known as bottle gourd, (Calabash, Doodhi, and Lauki in Hindi) and Kadoo in Marathi) which is official in Ayurvedic Pharmacopoeia of India. [5] 

L. siceraria fruits are traditionally used for its cardioprotective, cardiotonic, general tonic, diuretic, aphrodisiac, antidote to certain poisons and scorpion stings, alternative purgative, and cooling effects. It cures pain, ulcers, and fever and is used for pectoral-cough, asthma and other bronchial disorders. [6],[7] 

    Materials and Methods  

Collection and Authentication of Plant Material

The fresh fruits of L. siceraria were collected in the months of August-December from the local market of Wardha,

Maharashtra state, India, and authenticated by the authority of the botany department, Nagpur University, Nagpur. A voucher specimen (specimen No. 9012) was submitted at Institute's herbarium department for future reference.

Extraction of Plant Material

The fruit juice was obtained by crushing the fresh fruits of L. siceraria in the mixer (juicer) and subsequently dried in the oven at 40-50°C.

Fractional Extraction of Parent Extract

The parent dried juice extract was then fractionated by using the solvents according to polarity in ascending order i.e. by using chloroform: acetic acid, methanol, pyridine, and water [Table 1]. Each fraction was dried in oven at 40-50C.

Column Chromatographic Isolation and Purification of Methanolic Extract

1. Column : Glass

2. Dimension : Length 45 cm, diameter 3 cm

3. Stationary phase : Silica gel

4. Sample : Methanolic extract

5. Mobile phase : Gradient elution

The Methanolic fraction of parent extract was subjected for isolation over the silica gel (mesh size- 100-200) column. Previously, the slurry of silica gel was prepared with the mobile phase. The column was washed with the mobile phase for sufficient period of time. Then Methanolic fraction was loaded over the silica gel. The mobile phase was passed continuously with constant flow rate (10 ml/min.). The fractions were collected at regular intervals of time, evaporated at temperature <40-50°C and subjected for evaluation of antihyperlipidaemic activity.

Isolation of Compounds by Thin Layer Chromatography (TLC)Active fraction obtained by column chromatography was used for further isolation by TLC. The solvent system developed on trial and error basis was n-butanol: methaol: water (6:2:2). Four spots were obtained and were named as LSN-I, LSN-II, LSN-III and LSN-IV. Isolated spots were collected by using preparative TLC.

Evaluation of Antihyperlipidaemic Activity of the Isolated Compound

Diagnostic kit

Diagnostic Kits used for estimation of Triglyceride, Cholesterol, and HDL-C were obtained from Merck Ltd.

Hyperlipidemia Inducer

Triton-X-100 was used for induction of Hyperlipidemia in experimental rats.

Animals and Treatments

Adult male albino rats, weighing 180-200 g, bred in the animal house of the Institute of Pharmaceutical Education and Research, Wardha, were used. The animals were housed in polypropylene cages in room temperature with a 12 h day-light cycle. During the whole experimental period, animals were fed with a balanced diet and water ad libitum. All the animal experiments were performed with prior approval from the Institutional Animal Ethical Committee (Registration No.535/0/a/CPCSEA/Jan 2002) of the Institute of Pharmaceutical Education and Research, Wardha, Nagpur University.

Induction of Hyperlipidemia

Hyperlipidemia was induced in Wistar albino rats by single intraperitoneal injection of freshly prepared solution of Triton-X-100 (100 mg/kg) in physiological saline solution after overnight fasting for 18 h. [8],[9] 

Dose Preparation and Administration of Extracts

The isolated compounds viz. LSN I, LSN II, LSN III, LSN IV of L. siceraria were dissolved in distilled water administered orally to the animals by gastric intubation, thrice a day with an interval of 3 h, just after administration of Triton-X-100 i.p. Protocol for Antihyperlipidaemic Activity

In the experiment a total number of 42 rats (36 hyperlipidemia rats, six normal) were used. The rats were divided into seven groups of six each.

Group I : Normal distilled water treated p.o. Group II : Triton-X-100 (100 mg/kg) i.p. Group III: Triton-X-100 (100 mg/kg) i.p. + Lovastatin (10 mg/kg) p.o Group IV: Triton-X-100 (100 mg/kg) i.p + LSN I p.o Group V : Triton-X-100 (100 mg/kg) i.p + LSN II p.o Group VI: Triton-X-100 (100 mg/kg) i.p + LSN III p.o Group VII: Triton-X-100 (100 mg/kg) i.p + LSN IV p.o 

Statistical Analysis

All the values are expressed in terms of Mean ± S.E. M. of ( n = 6)

The Triton control was compared with normal.

The experimental results were compared with Triton control.

    Discussion  

The study was undertaken in order to evaluate the antihyperlipidaemic activity of different isolated compounds of L. siceraria fruits. The compounds were isolated from the active methanolic fraction of parent extract of L. siceraria fruit juice using column chromatography and TLC. The isolated compounds were tested for antihyperlipidaemic activity and compounds LSN-I, LSN-II, LSN-III showed significant results [Figure 1],[Figure 2],[Figure 3],[Figure 4],[Figure 5] and [Table 2],[Table 3],[Table 4],[Table 5],[Table 6]. The study exhibited that elevated blood cholesterol, triglycerides,

LDL, and decreased HDL which occur in hyperlipidemia, was significantly reduced by the administration of fractions of L. siceraria fruit juice . This finding provides some biochemical basis for the use of fruit, fruit juice or fruit extracts in the management of patients with hyperlipidemia. This also helps to place the L. siceraria (Mol.) Stand fruit among the list of antihyperlipidaemic agents and having potential to be popularized as household remedy with preventive and curative effect against hyperlipidemia and its consequences.

     References  

1. Wang J. Multi-center clinical trial of the serum lipid lowering effects of a Monascus purpureus (red east) rice preparation from traditional Chinese medicine. Curr Ther Res 1997;58:12.  

1. Wang J. Multi-center clinical trial of the serum lipid lowering effects of a Monascus purpureus (red east) rice preparation from traditional Chinese medicine. Curr Ther Res 1997;58:12.       

2. Brown MS, Goldstein JL. Drugs used in the treatment of hyperlipiproteinemia, In Goodman and Gilman's, The pharmacological basis of therapeutics. 8 th ed. Maxwell MacMillan, International edition. New York: Bengmon Press; 1990. p. 874-96.       

3. Amundsen AL, Ose L, Nenseter MS, Ntanios FY. Plant sterol ester-enriched spread lowers plasma total and LDL cholesterol in children with familial hypercholesterolemia. Am J Clin Nutr 2002;76:338-44.       

4. Lal AA, Kumar T, Murthy PB, Pillai SK. Hypolipidemic effect of Coriandrum sativum in triton induced hyperlipidemic rats. Indian J Exp Biol 2004;42:909-12.       

5. Rahman AS. Bottle Gourd ( Lagenaria siceraria ) a vegetable for good health Natural Product Radiat 2003;2:249-56.       

6. Nadkarni KM, Nadkarni AK. Indian Materica Medica, Vol 1. Delhi: Popular Prakashan; 1996. p. 722-3.       

7. Sivarajan SS, Balchandra A. Ayurvedic drugs and their plant source. New Delhi: Oxford and IBH Publication Co. Pvt. Ltd; 1981. p. 176-7.       

8. Moss JN, Dajani EZ. Antihyperlipidemic agents. In: Turner RA, Hebben PA. Screening methods in Pharmacology. Vol 2. New York: Academic Press; 1971. p. 121-43.       

9. Vogel G, Vogel WH. Influence of lipid metabolism. In: Drug Discovery and Evaluation Pharmacological Assay. Springer-Verly: Berloin; 1997. p. 604-8.       

Correspondence Address:D S Mohale91, Vaishali Nagar, Behind Nandurkar College, Yavatmal - 445 001, MS India

DOI: 10.4103/0973-8258.41181

    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]  

    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]

This article has been cited by

1 Phytopharmacological Profile of Lagenaria siceraria: A Review

B.N. Shah, A.K. Seth, R.V. Desai

Asian Journal of Plant Sciences. 2010; 9(3): 152

[VIEW]

Antihyperlipidemic activity of Sphaeranthus indicus on atherogenic diet induced hyperlipidemia in rats

VV Pande1, Sonal Dubey21 Department of Pharmaceutical Analysis, Jaywantrao Sawant College of Pharmacy and Research, Pune - 28, India2 Department of Medicinal Chemistry, K.L.E. S. College of Pharmacy, Rajajinagar, Bangalore - 10, India

Click here for correspondence address and email

Date of Submission 08-May-2008

Date of Acceptance 26-Jun-2008

Date of Web Publication 6-Aug-2009

 

       

    Abstract  

The present study was designed to investigate the Antihyperlipidemic activity of alcoholic extract ofSphaeranthus indicus L. flower heads in atherogenic diet induced hyperlipidemia. Sphaeranthus indicus extract was administered in a dose of 500 mg/kg/day, p.o. for eight days. Marked decrease in body weight, total cholesterol, triglyceride, and low-density lipoprotein and very low density lipoprotein whereas significant increases in the level of high-density lipoprotein were obtained after treatment with Sphaeranthus indicus extract. The present work indicates that, Sphaeranthus indicus extract in a dose of 500 mg/kg/day effectively suppressed the atherogenic diet induced hyperlipidemia in rats, suggesting the potential protective role in atherosclerosis.

Keywords: Sphaeranthus indicus, atherogenic diet, hyperlipidemia

How to cite this article:Pande VV, Dubey S. Antihyperlipidemic activity of Sphaeranthus indicus

Pande VV, Dubey S. Antihyperlipidemic activity of Sphaeranthus indicus on atherogenic diet induced hyperlipidemia in rats. Int J Green Pharm 2009;3:159-61

How to cite this URL:Pande VV, Dubey S. Antihyperlipidemic activity of Sphaeranthus indicus

    Introduction  

Hyperlipidemia is the most prevalent indicator for susceptibility to atherosclerotic heart disease. It is characterized by abnormally elevated lipid (triglyceride and cholesterol) and lipoprotein (LDL-c, VLDL-c) levels in the blood. This is supported by an abundance of congruent result from genetic, epidemiological, experimental animal studies and clinical trials that the presence of high plasma lipid cholesterol increases the incidence of coronary heart diseases (CHD). Atherosclerosis is the preliminary lipid disorder that affects the arteries and many factors contributing to its etiology, among them diabetes, glucocorticoid, diet, psychological factors are the major one. A crucial step in the pathogenesis of atherosclerosis is believed to be oxidative modification of LDL-c. [1],[2],[3]

Medicinal plants are an indispensable part of the traditional medicine practiced all over the world due to low costs; easy access and ancestral experience. Traditional ayurvedic grantha like Bhavaprakash Nighanta described. Sphaeranthus indicus Linn. (Asteraceae ) commonly known as 'Gorakhmundi' is distributed throughout the plains in India in wet places. The plant is reported to be useful for epilepsy, anemia, diabetes, gout etc. Extract of flower contain the principal essential oil, alkaloid, tannin, glycoside, reducing sugar, semidrying fatty oil, albumin. [4] Although Tannins have been reported for their hypolipidaemic activity. [5]

All parts of the plant found medicinal uses. The juice of the plant is styptic and said to be useful in liver and gastric disorders. [6] The paste of the herb made with oil is applied in itch. [7] The herb has a bitter sharp flavor with bitter taste. [8] It increases the appetite, enriches the blood, cools the brain and gives luster to the eye. [9],[10]

Based on this information present study was designed to investigate the antihyperlipidemic effect of Sphaeranthus indicus extract (alcoholic), serum lipid and lipoprotein profile in atherogenic diet induced hyperlipidemia.

    Materials and Methods

Plant Materials and Chemicals

The flowers of Sphaeranthus indicus collected at Wardha (M.S.) were authenticated from Regional Research Institute, Kothrud, Pune. The plant specimen is available in Regional Research Institute. Specimen voucher No. is 812 for future reference. Lovastatin was obtained as gift sample from Dr. Reddy's Laboratories, Hyderabad. Diagnostic kits for estimation of cholesterol (Merck), Triglyceride (Merck) and High density lipoprotein (CDR Diagnostic) were purchased from Mediequip, Nagpur, India. Atherogenic diet was purchased from local market.

Plant Extracts

The flower heads of Sphaeranthus indicus were dried in shade, under normal environmental condition and then subjected to size reduction to get coarse powder. Such powdered material was charged into the Soxhlet apparatus, and extraction was carried out successively with the following solvents like Benzene, carbon tetrachloride, petroleum ether, chloroform, ethanol and water. Each time before extracting with the next solvent, the powdered material was air dried below 50° C and then each extract was concentrated by distilling off the solvent to obtain the crude residue. The drug was extracted with each solvent till complete extraction was effected (about 30 cycles). All the extracts were stored in desiccators over phosphorous pentoxide and self indicating silica gel G.

About 125 g of the powder was taken in soxhlet extractor and extracted with Benzene (2000 ml) for 72 hrs. The solvent recovered by distillation was concentrated to yield a residue. The process of extraction was repeated with used marc and same volume of Chloroform, petroleum ether, ethanol etc. The aqueous extract was prepared after ethanol extraction by same procedure and evaporated at 40° C to give dark brown color solid mass.

Experimental

Male albino rats (Wistar strain) weighing between 150-200 gm were maintained at 25 to 30° C and kept in well ventilated animal house under natural photoperiodic condition in large polypropylene cages and were fed standard rats chow and water ad libitum. The animal experiment was approved by animal ethical committee of institute. (650/02/c/CPCSEA)

Preparation of doses

Oral Administration of Extract

Dissolved 500 mg/kg, body weight of Sphaeranthus indicus extract in fresh distilled water and prepared suspension of extract was given orally with the help of baby feeding tube to albino rats.

Animals were divided into different groups with six animals in each group. Group I served as normal control and received standard diet throughout experimental period. Group II, III and IV received atherogenic diet (79% standard diet + 21% Butter fat) throughout the treatment period. Group III received alcoholic extract of Sphaeranthus indicus extract (500 mg/kg/day, p.o.). Group IV received Lovastatin (5mg/kg/day p.o.). Treatment periods for all these groups were eight days.

Biochemical assay

At the end of treatment period to all these groups, the animals were used for various biochemical parameters. Blood was collected by orbital plexus of rat under ether anesthesia and centrifuged by using table top centrifuge at 2000 rpm for 30 minute so at to get serum.

Serum total cholesterol, triglyceride was estimated by the method of CHOD-PAP and high density lipoprotein by the method of GPO-PAP. Low density and very low density cholesterol were calculated by using Friedwald formula and VLDL: TG/5 respectively. Atherogenic index and LDL: HDL ratio was calculated.Biochemical assay

At the end of treatment period to all these groups, the animals were used for various biochemical parameters. Blood was collected by orbital plexus of rat under ether anesthesia and centrifuged by using table top centrifuge at 2000 rpm for 30 minute so at to get serum.

Serum total cholesterol, triglyceride was estimated by the method of CHOD-PAP and high density lipoprotein by the method of GPO-PAP. Low density and very low density cholesterol were calculated by using Friedwald formula and VLDL: TG/5 respectively. Atherogenic index and LDL: HDL ratio was calculated.

Statistical analysis

One way analysis of variance (ANOVA) followed by Dunnets t -test was carried out and P < 0.05 was considered significant.

    Results and Discussion  

Rats fed with atherogenic diet for sixty days display increase in their body weight as compare to normal. Treatment with alcoholic extract of Sphaeranthus indicus at the dose of 500 mg/kg/day showed significant ( P < 0.05) decrease in body weight to 17.34% respectively as compared to control group (34.12%) [Table 1].

There was marked increase in the level of serum TC and LDL-c and decrease in the level of good cholesterol carrier, HDL in the animal treated with atherogenic diet. Elevated level of blood cholesterol especially LDL-c was the major risk factor for the coronary heart diseases (CHD) and HDL as cardio protective lipoprotein. Treatment with Sphaeranthus indicus extract (500 mg/kg/day, p.o.) significantly decreases the level of TC and LDL-c as compared to control [Table 2]. There was significant increase in the HDL as compared to control. This effect of Sphaeranthus indicus extract may be due to increased in the activity of Lecithin: Cholesterol acetyl transferase (LCAT) which incorporates free cholesterol free LDL into HDL and transferred back to VLDL and intermediate density lipoprotein.

Treatment with Sphaeranthus indicus extract (500 mg/kg/day, p.o.) showed marked reduction in TG level as compared to control. This effect might be due to increase in activity of the endothelium bound lipoprotein lipase which hydrolyses the triglyceride into fatty acid or may due to inhibition of lipolysis so that fatty acids do not get converted into triglyceride.

There was marked reduction in LDL: HDL-c ratio and atherogenic index. LDL: HDL-c ratio is effective predictor of coronary risk and atherogenic index is most important indicator of CHD at both high and low serum cholesterol level. In the present study extract reduced LDL: HDL-c ratio and atherogenic index.

From our study we can conclude that alcoholic extract of Sphaeranthus indicus showed significant Antihyperlipidemic activity.

     References  

1. Anuradha CV, Ravikumar P. Restoration on tissue antioxidants by fenugreek seeds (Trigonella Foenum Graecum) in alloxan-diabetic rats. Indian J Physiol Pharmacol 2001;45:408-20.     [PUBMED]  

1. Anuradha CV, Ravikumar P. Restoration on tissue antioxidants by fenugreek seeds (Trigonella Foenum

Graecum) in alloxan-diabetic rats. Indian J Physiol Pharmacol 2001;45:408-20.     [PUBMED]  

2. Bopanna KN. Antidiabetic and antihyperlipaemic effects of Neem seed kernel powder on alloxan diabetic rabbits. Indian Journal of Pharmacology, 1997;29:162-7.        

3. Bopanna KN, Bhagyalakshmi N, Rathod SP, Balaraman R, Kannan J. Cell Culture derived Hemidesmus indicus in the Prevention of Hyperchesterolemia in Normal and Hyperlipidemic Rats. Indian Journal of Pharmacology 1997;29:105-9.        

4. Chaddha YR. The Wealth of India. Vol 10. New Delhi: Published by the publications and information directorate CSIR; 1976. p. 364.       

5. Dastur JF. Medicinal Plants of India and Pakistan. Published by RJ Taraporewala and Company Ltd; 1986. p. 210.       

6. Dhar ml, Dhar MM, Dhawan BN, Mehrotra BN, Rag C. Screening of Indian plants for biological activity: I. Indian J Exp Biol 1968;6:232-47.       

7. Dhuley JN. Anti-oxidant effects of cinnamon (Cinnamomum verum) bark and greater cardamom (Amomum subulatum) seeds in rats fed high fat diet. Indian J Exp Biol 1999;37:238-42.     [PUBMED]  

8. Gupta MB, Nath R, Srivastava,N, Shankar K, Kishor K, Bhargava KP. Anti-inflammatory and antipyretic activities of beta-sitosterol. Planta Med 1980;39:157-63.       

9. Kirtikar KR, Basu BD. Indian Medicinal Plants. Vol 1 and 2. Allahabad, India: Published by Lalit Mohan Basu, Leader Road: 1987. p. 185-9.       

10. Purohit A. Hypolipidaemic effects of Neem leaves in cholesterol fed rabbits. Indian Drugs 1999;36;389-92.       

Correspondence Address:V V PandeDepartment of Pharmaceutical Analysis, Jaywantrao Sawant College of Pharmacy and Research, Pune - 28 India

DOI: 10.4103/0973-8258.54911

  

    Tables

  [Table 1], [Table 2]

Lipid abnormalities in streptozotocin-diabetes: Amelioration by Morus indica L. cv Suguna leaves

B Andallu1, AV Vinay Kumar2, N Ch. Varadacharyulu3

1 Sri Sathya Sai University, Anantapur - 515 001, Andhra Pradesh, India2 The Institute of Environmental and Human Health, Texas Tech University, USA3 Department of Biochemistry, Sri Krishnadevaraya University, Anantapur, India

Date of Submission 22-May-2008Date of Acceptance 29-May-2009Date of Web Publication 21-Jul-2009

Correspondence Address:B AndalluSri Sathya Sai University, Anantapur-515001, AP India

DOI: 10.4103/0973-3930.54289

PMID: 20165649

     Abstract  

Aim : To observe the influence of mulberry ( Morus indica L. cv Suguna) leaves on lipid abnormalities in STZ-diabetic rats. Materials and Methods: Treatment with dried mulberry leaf powder for a period of 8 weeks in hyperglycemic and hyperlipidemic STZ-diabetic rats. Results: Mulberry leaves regulated fasting blood glucose, ameliorated the abnormalities in lipid profile as indicated by significant ( P <0.01) decrease in serum triglycerides, phospholipids, cholesterol and plasma free fatty acids by 50, 6, 31 and 22% respectively in STZ- diabetic rats compared to diabetic control rats which had significantly ( P <0.01) raised levels of triglycerides, phospholipids, cholesterol and free fatty acids than the normal control rats. A marked increase in fecal bile acids (154%) was observed in mulberry treated diabetic rats compared to the diabetic control group indicating conversion of cholesterol to bile acids. In addition, mulberry supplementation significantly lowered LDL-C (67%) and VLDL-C (44%) levels and increased HDL-C (53%) and also decreased atherogenic index (58%) significantly when compared to the diabetic control group. Conclusion: Besides the diabetic rats, mulberry leaves affected lipid profile in

normal rats also indicating hypolipidemic effect as a result of the synergistic action of bioactive compounds.

Keywords: Atherogenic index, bile acids, cholesterol, hyperlipidemia, mulberry leaves, STZ- diabetic rats, triglycerides

How to cite this article:Andallu B, Vinay Kumar AV, Varadacharyulu NC. Lipid abnormalities in streptozotocin-diabetes: Amelioration by Morus indica L. cv Suguna leaves. Int J Diab Dev Ctries 2009;29:123-8

How to cite this URL:Andallu B, Vinay Kumar AV, Varadacharyulu NC. Lipid abnormalities in streptozotocin-diabetes: Amelioration by Morus indica

    Introduction

 

Diabetes mellitus is associated with a large number of lipid abnormalities. Emerging evidence confirms the pivotal role of hyperlipemia, mainly elevated blood cholesterol, particularly LDL cholesterol and VLDL cholesterol in the development of atherosclerosis-related disease. [1] Significant abnormalities in lipid metabolism and lipoproteins in diabetes are evident which in turn depend on the extent of insulin deficiency, insulin resistance, obesity, diet and the presence of concomitant primary and other secondary causes of hyperlipemia. In diabetic hyperlipemia, a series of bizarre lipoproteins and other lipids appear and interaction of this with oxidative stress and free radicals leads to enhanced lipid peroxidation in plasma, tissues and membranes, causing extensive tissue damage. It is well known that lipid peroxidation provides a continuous supply of free radicals that play an important role in etiopathogenesis of diabetes and its complications. [2] Various therapeutic methods used in diabetes treatment available today achieve transiently regulated euglycemia but fail to prevent lipid and lipoprotein alterations, ultimately, exposing the diabetic humans and animals to cardiovascular complications. [3] Moreover, many of these drugs exert various side/toxic [4] effects such as hepatotoxicity (troglitazone) or cardiac failure [rosiglitazone). [5] Concurrently, phytochemicals identified from traditional medicinal plants are presenting an exciting opportunity for the development of new types of therapeutics. This has accelerated the global effort to harness and harvest those medicinal plants that bear substantial amount of potential phytochemicals showing multiple beneficial effects in combating diabetes and the related complications without causing side effects. [6] In the recent years, search for natural dietary therapeutic methods for controlling diabetes are much active as diet plays a key role in the treatment of diabetes. [7] 

Mulberry has been explored as a medicinal plant and its medicinal properties are testified in various scriptures. It occupies an important position in the holistic system of Indian medicine 'Ayurveda' which has its roots in antiquity and has been practiced for centuries. The leaves of mulberry are nutritious, palatable, nontoxic and also enriched with different active principles. [8] Mulberry leaves ( Morus alba L.) have been used to cure "Xiao-ke" (diabetes) in

traditional Chinese medicine. [9] Mulberry leaves are used as traditional medicine with anti-inflammatory and antihyperglycemic actions. [10],[11] The antihyperglycemic effects of six N-containing sugars present in mulberry leaves were investigated in STZ-induced diabetic mice. [12] The root bark of Morus alba L. has been used as a blood pressure depressant in China and Japan from old times [13] but no reports are available on the hypolipidemic effect. Hence, the present research on mulberry opens up a new avenue to develop a novel therapy to combat diabetes and concomitant hyperlipidemia. 

    Materials and Methods  

Male albino rats (24) of Wistar strain with body weights ranging from 150-200g, procured from National Centre for Laboratory Animal Sciences, National Institute of Nutrition, Hyderabad were housed in individual cages in an air conditioned room (25 0 + 1 0 C) with light from 7 a.m to 7 p.m. The rats were allowed to acclimatize to the laboratory environment for 7 days, distributed into 4 groups according to the similar weights with six animals in each group as given below:

Group I - Normal control

Group II - Normal treated with mulberry leaf powder

Group III - Diabetic control

Group IV - Diabetic treated with mulberry leaf powder

The mulberry leaves ( Morus indica L.) were procured from the Regional Sericultural Research Station, Raptadu, Anantapur District, washed thoroughly, shade dried, powdered in an electric mixer and used in the experiment.

Animals of group III and IV were rendered diabetic by a single intra-peritoneal injection of streptozotocin (55 mg/kg). [14] Control rats (Group I and II) were injected with citrate buffer alone. After 72 hrs of injection, blood was drawn from retro-orbital plexus of conscious rats using heparinized capillaries and glucose was estimated by glucose oxidase method [15] using Span Diagnostic kit.

Group II and IV received experimental feed containing powdered mulberry leaves at 25% level (as per dose response) mixed with the standard feed (obtained from NCLAS, NIN, Hyderabad, A.P.). Group I and III received standard feed in powder form mixed with cellulose and protein to make it isocaloric to the experimental feed. All the groups of rats were maintained under standard housing conditions for a period of 8 weeks with free access to food and water. After 8 weeks of period, over-night fasted rats were sacrificed by exsanguination and blood was collected by cardiac puncture in vials for analytical procedures. Glucose, [15] cholesterol, [16] triglycerides, [17] phospholipids [18] and HDL cholesterol [19] in serum and free fatty acids [20] in plasma were estimated. LDL and VLDL

cholesterol [21] and atherogenic index [22] were calculated. Fecal bile acid content was estimated by the method of Scott. [23] Mean and standard error were calculated. [24] The data were statistically analysed by applying Duncan's Multiple Range Test[25] to assess the significant differences among the groups and values of P <0.05 were regarded as significant. These experiments were conducted as per CPCSEA guidelines and were approved by Institutional Ethical Committee.

    Results  

Mulberry supplementation significantly decreased fasting glucose (58%), lowered serum triglycerides, phospholipids, cholesterol and plasma free fatty acids by 50, 6, 31 and 22% respectively in diabetic rats compared to control rats which were characterized by significantly raised levels of fasting glucose (274%), triglycerides (144%), phospholipids (16%), cholesterol (42%) and free fatty acids (46%) compared to the normal control rats. A marked increase in fecal bile acids (154%) was recorded in mulberry fed diabetic rats compared to the diabetic control group which showed significant fall in fecal bile acids (22%) compared to the normal control rats [Table 1].

The values of different fractions of cholesterol viz., LDL, VLDL, HDL, and atherogenic index from different groups of rats are furnished in [Figure 1],[Figure 2],[Figure 3],[Figure 4]. Mulberry supplementation significantly lowered LDL-C (68%) and VLDL-C (43%) levels and increased HDL-C (54%) and also decreased atherogenic index (56%) significantly when compared to the diabetic control group which exhibited significant hike in the levels of LDL -C and VLDL-C and atherogenic index by 165, 138 and 138% respectively and decrease in the level of HDL-C by 42% compared to the control group as shown in the [Figure 1],[Figure 2],[Figure 3] and [Figure 4] respectively. Besides diabetic rats, mulberry supplementation influenced almost all the aforesaid parameters in normal rats. 

    Discussion  

Hyperlipidemia is a known complication of diabetes mellitus [26] and coexists with hyperglycemia and is characterized by increased levels of cholesterol, triglycerides and phospholipids, and also changes in lipoproteins. [27] Interest in the study of plasma lipids in diabetes arises from the widely acknowledged higher incidence of atherosclerotic disease which is a major cause of premature death in diabetic patients. [28] 

In the present study, serum triglycerides, phospholipids, cholesterol, LDL cholesterol, VLDL cholesterol and atherogenic index were significantly elevated while HDL cholesterol and fecal bile acids were significantly decreased in diabetic rats. Interestingly, the results further indicated that all these lipid and lipoprotein abnormalities were countered by mulberry leaves in diabetic rats. Glycemic control appears to be the major determinant of total and VLDL triglyceride concentrations. [29] 

Control of hyperlipidemia is a prerequisite for the prevention of diabetic microangiopathy (retinopathy, nephropathy and neuropathy) and macroangiopathy (ischemic heart disease), cerebral vascular disease (CVD) and arteriosclerosis obliterans in diabetes. [30] However, in certain cases of diabetes, treatment with insulin with normalization of plasma glucose levels did not restore the HDL-C concentrations to normal implying that, factors in addition to hyperglycemia are causing the lower HDL-C. [31] Synthesis of VLDL is promoted by an increase in the flux of free fatty acids in liver and ultimately the particles are converted to LDL. Studies revealed increased levels of VLDL as a consequence of decreased clearance and also over-production in type 1 DM subjects. The increased circulatory VLDL-C and the associated triglycerides due to defective clearance [32] of these particles from circulation is in agreement with earlier studies of Babu and Srinivasan [33] and others and these changes were attributed to the altered activity of lipoprotein lipase.

It seems that the changes in adipose tissue lipolysis or intrahepatic mechanisms involving other changes in fractional esterification of fatty acids are in the assembly or secretion of VLDL are responsible for the increase in triacylglycerol secretion rate. In vitro studies have shown a decrease in fractional catabolic rate for LDL from type 2 DM subjects and also evidence suggests that in vivo nonenzymatic glycosylation of LDL may result in decreased LDL clearance. [34] LDL cholesterol concentrations are strongly and positively related to atherosclerotic complications. [35] Apart from this, glycation induces compositional and structural changes in LDL. Glycated LDL interacts with platelets leading to the development of vascular complications in diabetes by altering platelet aggregation, platelet nitric oxide production, intracellular Ca 2+ concentration, activities of Na + - K + and Ca 2+ ATPases. [36] 

Increased glycation of apolipoproteins may play a role in the accelerated development of atherosclerosis in diabetes and altered activity of glycated LDL receptor contributes for hyperlipidemia. In addition, glycation of liporoteins may also generate free radicals increasing oxidative damage to the lipoproteins themselves. Glycoxidation and browning of sequestered lipoproteins may further enhance their atherogenicity. The more severely modified (glycoxidized) lipoproteins in vessel walls may behave as more potent antigens than less modified particles found in the plasma stimulating the in situ formation of atherogenic immune complexes. [37] 

The most characteristic lipid abnormality is hypertriglyceridemia with associated increase in plasma cholesterol. Elevated plasma triglyceride concentration is seen in type1 DM and type 2 DM either due to triglyceride over-production and /or underutilization. Lipoprotein lipase activity is markedly impaired, besides, a significant improvement in LDL internalization and degradation suggesting that chemical modification of LDL particle like nonenzymatic glycation of LDL itself might result in its increased incorporation in the arterial wall via a receptor independent pathway. Studies have strongly suggested an inverse relationship of HDL cholesterol with atherosclerosis to be independent of other lipid abnormalities. [38] Insulin has important effects on key steps in the metabolism of lipids and lipoproteins, and alterations in lipid metabolism are common in diabetic population. [28] 

HDL cholesterol, the smallest of the lipoprotein species containing approximately 20% cholesterol ester and very little triglyceride is strongly and independently related to CHD.

But, unlike LDL, the relationship is inverse, a low HDL level being an important predictor of CHD and high HDL level protecting against CHD. [39] A decrease in HDL turnover has been shown in diabetes. Some reports revealed that non-enzymatic glycosylation of HDL accelerates its catabolism in guinea pigs and the same mechanism might be responsible for the low levels of HDL in diabetic rats observed in the present study. [40] 

Other studies also revealed that glycated HDL clearance is accelerated from the circulation in contrast to the effect with glycated LDL whose catabolic rate is reduced. The accelerated clearance of HDL was seen even with mild glycation and was suggested as a contributing cause of low plasma levels of HDL in diabetic patients and therefore works as another factor underlying increased risk of atherosclerotic disease in diabetic patients. [41] 

In the present study, cholesterol, triglycerides and free fatty acids were brought down significantly by mulberry feeding in diabetic rats. This effect could be partly due to the control of hyperglycemia. Elevated LDL, VLDL and decreased HDL cholesterol concentrations in diabetic rats appear to be markedly altered favorably by mulberry supplementation. All the lipid abnormalities developed in STZ-diabetic rats were effectively countered by feeding mulberry leaf powder. Certain specific phytochemicals such as β sistosterol,[42] phenolics, [43] flavonoids, [44] saponins [45] and fiber [46] might be playing a role in rectifying the abnormalities. The precise mechanism underlying this effect appears to be complex. However, most of these compounds were reported to inhibit absorption of lipids from the intestines. At the same time, increased fecal bile acid excretion [47] in diabetic group treated with mulberry in the present study, reflects the conversion of cholesterol to bile acids and is a favorable feature reducing cholesterol induced risks probably by influencing either the activity or synthesis of the key enzyme, cholesterol 7α hydroxylase involved in the formation of bile acids from cholesterol. The components of mulberry leaves might also be influencing lipoprotein associated cholesterol fractions and probably the phytocomponents exert action similar to the drugs cholestyramine, mevanolin, lovastatin and simvastatin that are used for correcting the imbalance in plasma lipoproteins in diabetes. An in-depth study related to the activity of HMG COA reductase and cholesterol 7 α hydroxylase[48] in mulberry treated rats is warranted to understand the influence of the whole and individual mulberry leaf components in lowering lipid profile.

Consumption of plant material like mulberry leaves, containing antioxidants i.e. vitamin A, C, E, [49] carotenoids, [50] polyphenols [51] and phytonutrients [52] increases the antioxidant status in human blood and tissues and these compounds are capable of modulating LDL oxidation through several mechanisms. Recent studies have demonstrated that vitamin C is more potent in preventing LDL oxidation than vitamin E, and a combination of the two vitamins is even more effective than either alone. [53] Plasma levels of vitamin C and E were reported to be higher in European population with low incidence of cardiovascular diseases. Levy et al. [54] observed that dietary supplementation of natural β carotene normalized the elevated LDL cholesterol oxidation and thereby reduced the risk of development of atherosclerosis in diabetes. As β carotene is the chief constituent in mulberry leaves, it can be assumed that atherosclerotic preventive role was exerted by mulberry leaves by inhibiting LDL cholesterol oxidation. Doi et al. [55]reported the prevention of atherosclerosis by mulberry leaves. Quercetin an aglycone of isoquercetrin present in mulberry leaves inhibited the formation of conjugated dienes and TBARS by copper induced oxidative modification of rabbit and human

LDLs. Similarly mulberry leaf butanol extract (MLBE) and isoqercitrin also inhibited the oxidation of LDL suggesting that mu

lberry leaves can inhibit the oxidative modification of LDL. [55] 

From this experimental data, it is evident that mulberry leaves efficiently regulated blood glucose in diabetic rats and very efficiently ameliorated lipid abnormalities associated with diabetes in STZ-diabetic rats by virtue of various essential antioxidant, antidiabetic compounds and phytonutrients. The synergistic role played by these compounds is attributed to the protection of diabetic rats against lipid abnormalities. Further pharmacological and biochemical investigations are underway to elucidate the mechanism of the hypolipidemic effect of Morus indica L.cv Suguna leaves.

    Acknowledgements  

The University Grants Commission for the financial assistance, Sri Sathya Sai University for the facilities and Dr. G. Kesava Reddy, Former Associate Professor, Kansas University, USA for providing streptozotocin are gratefully acknowledged.

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Obesity-related cardiovascular risk factors after weight loss: a clinical trial comparing gastric bypass surgery and intensive lifestyle interventionD Hofsø,1 N Nordstrand,1 L K Johnson,1 T I Karlsen,1,2 H Hager,3 T Jenssen,4,5 J Bollerslev,6,7 K Godang,6 R Sandbu,1 J Røislien,1,8 and J Hjelmesæth1

1Department of Medicine, Morbid Obesity Centre, Vestfold Hospital Trust, PO Box 2168, 3103, Tønsberg, Norway

2Evjeklinikken AS, Evjemoen, 4735, Evje, Norway

3Department of Clinical Chemistry, Vestfold Hospital Trust, 3103, Tønsberg, Norway

4Institute of Clinical Medicine, University of Tromsø, 9037, Tromsø, Norway

5Section of Nephrology, Department of Medicine, Oslo University Hospital Rikshospitalet, 0027, Oslo, Norway

6Section of Endocrinology, Department of Medicine, Oslo University Hospital Rikshospitalet, 0027, Oslo, Norway

7Faculty of Medicine, University of Oslo, 0318, Oslo, Norway

8Department of Biostatistics, Institute of Basic Medical Sciences, University of Oslo, 0317, Oslo, Norway

(Correspondence should be addressed to D Hofsø Email: dag.hofso@siv.no)

Received August 12, 2010; Accepted August 26, 2010.

This is an Open Access article distributed under the terms of the European Journal of Endocrinology's Re-use Licence which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Objective

Weight reduction improves several obesity-related health conditions. We aimed to compare the effect of bariatric surgery and comprehensive lifestyle intervention on type 2 diabetes and obesity-related cardiovascular risk factors.

Design

One-year controlled clinical trial (ClinicalTrials.gov identifier NCT00273104).

Methods

Morbidly obese subjects (19–66 years, mean (S.D.) body mass index 45.1 kg/m2 (5.6), 103 women) were treated with either Roux-en-Y gastric bypass surgery (n=80) or intensive lifestyle intervention at a rehabilitation centre (n=66). The dropout rate within both groups was 5%.

Results

Among the 76 completers in the surgery group and the 63 completers in the lifestyle group, mean (S.D.) 1-year weight loss was 30% (8) and 8% (9) respectively. Beneficial effects on glucose metabolism, blood pressure, lipids and low-grade inflammation were observed in both groups. Remission rates of type 2 diabetes and hypertension were significantly higher in the surgery group than the lifestyle intervention group; 70 vs 33%, P=0.027, and 49 vs 23%, P=0.016. The improvements in glycaemic control and blood pressure were mediated by weight reduction. The surgery group experienced a significantly greater reduction in the prevalence of metabolic syndrome, albuminuria and electrocardiographic left ventricular hypertrophy than the lifestyle group. Gastrointestinal symptoms and symptomatic postprandial hypoglycaemia developed more frequently after gastric bypass surgery than after lifestyle intervention. There were no deaths.

Conclusions

Type 2 diabetes and obesity-related cardiovascular risk factors were improved after both treatment strategies. However, the improvements were greatest in those patients treated with gastric bypass surgery.

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IntroductionObesity (body mass index (BMI) ≥30 kg/m2) and its metabolic consequences, 

Obesity (body mass index (BMI) ≥30 kg/m2) and its metabolic consequences, hyperglycaemia and high blood pressure, are major risk factors of cardiovascular morbidity and mortality (1, 2). Alongside tobacco usage and physical inactivity they represent the five leading global risks to mortality (3). In addition, several other cardiovascular risk factors, such as metabolic syndrome, albuminuria, left ventricular hypertrophy and low-grade inflammation, are all closely associated with obesity (4–7).

As the prevalence of obesity, and especially extreme obesity, has dramatically increased in the last few decades (8), so too has the usage of bariatric surgery to treat morbid obesity (BMI≥40 kg/m2 or BMI ≥35 kg/m2 with at least one obesity-related comorbidity) (9). Currently, the most commonly performed bariatric procedure worldwide is the Roux-en-Y gastric bypass surgery (9). Several studies have documented how obesity surgery allows for large weight reduction and improvements in obesity-related conditions (10–15). Furthermore, comprehensive lifestyle intervention programmes have also demonstrated, although to a lesser extent, significant short-term weight reduction and improvements in cardiovascular risk factors in moderate to severely obese subjects (16–19). Importantly, increased physical activity, a pivotal component of all lifestyle intervention programmes, has been shown to have positive metabolic effects beyond weight reduction (20). However, only two controlled clinical trials have compared the effect of bariatric surgery and conventional therapy on the resolution of diabetes and cardiovascular risk factors (10, 11). In these studies, the average weight loss in the non-surgically treated groups was negligible. Not all morbidly obese subjects are suitable for bariatric surgery and therefore non-surgical treatment alternatives are needed.

The objective of this 1-year non-randomised controlled clinical trial was to compare the effect of Roux-en-Y gastric bypass surgery and a comprehensive lifestyle intervention programme on type 2 diabetes and obesity-related cardiovascular risk factors.

Study design and participants

This study was conducted at a public tertiary care centre at Vestfold Hospital Trust, Tønsberg, Norway.

Although preferable when conducting a clinical trial, we did not find randomisation to be appropriate. According to Norwegian guidelines, treatment seeking morbidly obese subjects should be offered either conservative or surgical therapy (21). We therefore considered it unethical to assign patients to surgery if they qualified for a lifestyle intervention programme and preferred this course of treatment to surgery. This stance also held vice versa.

In order to participate in the non-randomised controlled morbid obesity treatment, bariatric surgery versus intensive lifestyle intervention (MOBIL) study consecutive patients were pre-screened between December 2005 and May 2006 (Fig. 1). The MOBIL study aimed to address changes in several health outcomes related to obesity. Clinical and laboratory examinations were performed during pre-screening. Furthermore, patients who satisfied the criteria for bariatric surgery (22) and wanted either gastric bypass surgery or intensive lifestyle intervention were referred to a screening examination which included an oral glucose tolerance test, a somnography, pulmonary function tests, quality of life

questionnaires and a structured dietary interview. All patients underwent a thorough assessment 

conducted by a multidisciplinary team consisting of an internist, a dietician and, in cases of surgery, a surgeon before treatment. These health professionals provided complete information about the possible risks and benefits of an operation and encouraged patients to incorporate their own values and preferences into the decision-making process. Each patient and their physician agreed together upon the most appropriate choice of therapy. The elapsed period of time between the pre-screening examination and the screening examination was 18 weeks (11); this did not differ significantly between the study groups (P=0.063). By contrast, the time between the screening examination and either the date of surgery or the start of lifestyle intervention was significantly longer in the surgery group than in the lifestyle group, 65 weeks (14) versus 19 weeks (15) (P<0.001). One-year follow-up was completed by June 2009. This article reports changes in weight, glucose- and lipid metabolisms, blood pressure, albuminuria, left ventricular hypertrophy, low-grade inflammation, energy intake and physical activity.

Figure 1

The study was approved by the regional ethics committee of what was formerly known as the Southern Norway Regional Health Authority. The study is registered in the ClinicalTrials.gov-registry under the unique trial number NCT00273104. Written informed consent was provided by all the participants.

Intervention

Both treatment groups were seen by an internist half yearly and by a dietician when required. Changes in medications were made on an individual basis by both the patients' general practitioner and by hospital physicians.

Patients in the surgical group completed a low-calorie diet (3.3–3.8 MJ/day) in 3–6 weeks preceding surgery. A Roux-en-Y gastric bypass surgery was performed laparoscopically in 74 of the 76 surgically treated patients. The gastric pouch was about 25 ml, while the

intestinal limb lengths were measured as follows: alimentary limb, median 120 (range 80–250) cm; biliopancreatic limb, median 100 (range 50–170) cm; and common channel, variable length. The bariatric surgeons tended to choose longer limbs in the heaviest patients. After surgery, a standardised regimen of dietary supplements (23) and a proton pump inhibitor were prescribed to all patients. Patients with a high risk of venous embolism were prescribed low-molecular weight heparin. During follow-up, patients allocated to surgery were examined by a bariatric surgeon 6 weeks post surgery, while groups of patients were seen by a dietician quarterly. To optimise the result of the procedure patients were encouraged, both before and after the surgery, to normalise their eating behaviour and to increase their physical activity level.

The majority (59/63) of patients in the lifestyle group were referred to a rehabilitation centre specialising in the care of morbidly obese patients (Evjeklinikken). Using a cognitive approach the programme at this centre aimed to induce a weight loss of at least 10%. Each patient was motivated to increase their physical activity and to normalise their eating habits. The 1-year lifestyle programme comprises four stays at the rehabilitation centre lasting for either 1 week or 4 weeks (Fig. 2). The daily programme was divided between organised physical activity (3–4 h) and different psychosocially oriented interventions. The interventions involved individual consultations with a medical doctor, a nutritionist, a physiotherapist and a trained nurse. Those leading the counselling interviews were trained in motivational interviewing, a client-centred counselling style that aims to invoke behaviour change. The patients also took part in group sessions focusing on emotional aspects of sedentary behaviour as well as classroom lessons on topics related to nutrition, physical activity and co-morbidities. No special diet or weight-loss drugs were prescribed, but patients were encouraged to follow the guidelines of the Norwegian National Council of Nutrition (24), which recommend that the daily intake of protein, fat, carbohydrate and alcohol should account respectively for 10–20, <30, 50–60 and <5% of energy consumed. Outside of these stays, patients were contacted by phone once every 2 weeks. They were encouraged to self-monitor their eating habits and physical activities, as well as to visit their general practitioner for a consultation and weight control check once every 4 weeks. The remaining four participants were allocated to two rehabilitation centres with comparable intervention programmes. Fifty-four patients (86%) attended all scheduled centre visits.

Outcome variables

Demographic and clinical data were recorded on standardised forms. Height, weight and waist and hip circumferences were measured with patients in an upright position wearing light clothing and no shoes.

A 75 g oral glucose tolerance test was performed at 0800 h after an overnight fast. Type 2 diabetes was diagnosed in patients who used glucose-lowering agents or had fasting serum glucose ≥7.0 mmol/l and/or 2 h serum glucose ≥11.1 mmol/l (25). Remission of diabetes was defined as either partial (serum glucose levels below the diagnostic cut-off values and HbA1c <6.5%) or complete (fasting serum glucose <5.6

A 75 g oral glucose tolerance test was performed at 0800 h after an overnight fast. Type 2 diabetes was diagnosed in patients who used glucose-lowering agents or had fasting serum glucose ≥7.0 mmol/l and/or 2 h serum glucose ≥11.1 mmol/l (25). Remission of diabetes was defined as either partial (serum glucose levels below the diagnostic cut-off values and HbA1c <6.5%) or complete (fasting serum glucose <5.6 mmol/l, 2 h glucose <7.8 mmol/l and HbA1c <6.2%) in the absence of glucose-lowering agents (26). Combined remission rates (partial and complete) are presented unless otherwise specified.

Blood pressure was measured three times after at least 5 min rest. The average of the second and third measurements was registered. Patients using anti-hypertensive drugs, as well as those with systolic blood pressure ≥140 mmHg and/or diastolic blood pressure ≥90mmHg (27) were categorised as having hypertension. Remission of hypertension was defined as blood pressure below the diagnostic cut-off values in the absence of anti-hypertensive drugs.

Metabolic syndrome was defined according to the modified ATP III criteria (28). Albuminuria was defined as present if the albumin to creatinine ratio was ≥2.5 mg/mmol in men and ≥3.5mg/mmol in women (29). The product of QRS complex duration times Cornell voltage combination (RaVL+SV3, with 6 mm added in women) was used with a threshold value of 2440mm×ms to identify electrocardiographic left ventricular hypertrophy (30). Regression of electrocardiographic left ventricular hypertrophy estimated by the Cornell voltage-duration product is known to predict regression of echocardiographic left ventricular hypertrophy (31).

Dietary intake and physical activity during the preceding year were assessed through structured interviews performed by registered dieticians. Data were recorded on an optically readable food frequency questionnaire (Department of Nutrition, University of Oslo, Norway). Similar questionnaires have been validated using weighted records(32). Questionnaire data were scanned using Teleform 10.0 (Cambridge, UK). Dietary intake was calculated using a database assembled from official food composition tables (Norwegian Nutrition Council, 1995). Calculations were computer driven (Kostberegningssystem 6.0; University of Oslo, Norway). Time spent performing light (e.g. casual walking), moderate (e.g. brisk walking) and vigorous (e.g. jogging) intensity aerobic physical activities in periods of 10 min or more was recorded. Participants who performed 150 min or more per week of

moderately intense aerobic physical activities were considered to be physically active, as were those participants who performed 60 min or more per week of vigorously intense aerobic physical activities (33).

Perioperative (first 30 days) and late (after 30 days) complications were recorded in each patient's record file. In addition, all medical emergencies, hospitalisations and gastrointestinal side effects were reported on standardised self-report questionnaires. Reported symptomatic postprandial hypoglycaemia was documented by blood glucose <2.8mmol/l (34). Complications and medical emergencies not recorded in each patient's record file at our hospital were verified by reports from other institutions.

Laboratory analysesBlood samples were collected either in the fasting state or during the oral glucose tolerance test. Samples clotted 30 min at room temperature, and serum was separated by centrifugation. Analyses of blood lipids and glucose were performed by dry reagent slide technology on the Vitros 950 Analyzer until November 2006 and the Vitros FS 5.1 Analyzer (Ortho-Clinical Diagnostics, New York, NY, USA) thereafter. HbA1c was analysed using HPLC on Tosoh HLC-723 G7 (Tosoh Corporation, Tokyo, Japan).

Serum samples collected during the oral glucose tolerance test were either stored at −80 °C or analysed on the day of collection (glucose). Insulin, C-reactive protein and adiponectin were measured in stored serum obtained before the glucose ingestion. Insulin was analysed using an RIA (Millipore Corporation, Billerica, MA, USA), whereas C-reactive protein and adiponectin were analysed using enzyme immunoassays (R&D systems, Minneapolis, MN, USA). All samples were measured in duplicate. The intra- and inter-assay coefficients of variation were <10% for all assays.

Albumin and creatinine in urine were analysed using Konelab 60i (Thermo Electron Corporation, Helsinki, Finland) until August 2008 and Vitros FS 5.1 Chemistry System thereafter.

Statistical analysisThe sample size of the MOBIL study was calculated (80% statistical power, α-level of 0.05 and equal distribution to the treatment groups) based on anticipated remission rates of type 2 diabetes and obstructive sleep apnoea. Given remission rates of type 2 diabetes of 70% in the surgery group and 20% in the lifestyle group, at least 30 subjects with type 2 diabetes were required. Expecting a prevalence of type 2 diabetes of 25% and a dropout rate of 30% from the screening examinations, a minimum of 172 subjects were required for screening.

Data are presented as mean (S.D.) or number (%) unless otherwise specified. Skewed data were transformed using natural logarithms to approximate normality. Between-group

comparisons at baseline were analysed using independent samplest-test or Mann–Whitney U test for continuous variables and χ2 or Fisher's exact test for categorical variables. Within-group comparisons were performed using paired samples t-test or repeated measures ANOVA for continuous variables and McNemar test for dichotomised variables. Between-group changes in outcome variables were assessed using logistic and linear regression analyses, analysis of covariance, repeated measures ANOVA and Fisher's exact test. Furthermore, changes in categorical and continuous variables were adjusted for baseline differences. In addition, continuous variables were, in the entire study population, adjusted for gender, age, BMI at baseline and changes in relevant medication. Regression analyses were used to identify predictors of remission of diabetes and hypertension and to explore the independent effects of several variables on changes in HbA1c and blood pressure. For each variable, only subjects who had values available at both baseline and follow-up are presented and included in the analyses. There were <5% missing and/or excluded data unless otherwise noted. The significance level wasP<0.05. Statistical analyses were performed using SPSS 16.0 (SPSS Inc., Chicago, IL, USA).

  Other Sections ▼ Baseline characteristics of participants Patient flow throughout the study is shown in Fig. 1. Of the 146 patients included, 80

chose to have surgery and 66 chose to participate in a lifestyle intervention programme. The completion rate was 95%. Baseline characteristics of the participants who completed the study are shown in Table 1. No significant differences were found between the two study groups in terms of sex, ethnicity, obesity-related comorbidities or the usage of weight-loss drugs or statins. However, patients who chose gastric bypass surgery were on average 4 years younger and 12kg heavier than those in the lifestyle group.

Table 1

Participant characteristics at baseline. Data are given as mean (S.D.), median (range), or n (%). Differences between categorical data were determined using either χ2 or Fisher's exact test, whilst independent sample t-test or Mann–Whitney   (more ...)   

Weight reduction Weight changes in the two treatment groups during the study are shown in Fig. 3A.

Mean (S.D.) percentage 1-year weight reduction was 30% (8) in the surgery group and 8% (9) in the lifestyle group (within-groups both P<0.001 and between-groupsP<0.001). This corresponds to a mean (S.D.) loss of excess weight above 25kg/m2 of 67% (18) and 20% (23) (P<0.001) respectively.

Figure 3

Mean (95% CI) percentage weight change during follow-up (A) and distribution of 1-year changes in weight (B) within the surgery and lifestyle groups. Repeated measures ANOVA was used to compare the change in weight between the two study groups.

The cumulative distribution of percentage weigh The cumulative distribution of percentage weight change in the two treatment groups

is shown in Fig. 3B. Within the lifestyle group, 62% lost ≥5% of their initial weight, while 38% lost ≥10% of their initial weight. Within the surgery group, all subjects experienced a weight reduction >10% of their initial weight.

Changes in measures of obesity, glucose metabolism, blood pressure, lipids and inflammatory markers

With the exception of pulse pressure, changes in anthropometric measures, blood pressures and biochemical risk factors were significantly greater in the surgery group than in the intensive lifestyle group (Table 2). Both treatment groups experienced a significant reduction in all measures of obesity, glucose metabolism, blood pressure, total and low-density lipoprotein cholesterol, triglycerides and C-reactive protein during follow-up (all P≤0.034). Adiponectin increased significantly in both treatment groups (both P<0.001), whereas high-density lipoprotein cholesterol increased significantly only in the surgery group (P<0.001).

Table 2

Changes from baseline in various continuous variables. Unadjusted within-group changes are given as mean (S.D.). Adjusted between-group differences and corresponding P value were calculated with the use of analysis of covariance and presented as mean   (more ...)   

Subgroup analyses including subjects with ≥10% weight reduction showed that multi-adjusted (gender, age, BMI, baseline value and change in relevant medications) between-group changes in HbA1c and systolic and diastolic blood pressures did not differ significantly between the surgically and the conservatively treated groups (mean (95% CI) 0.0 (−0.2 to 0.2) %, P=0.965; −2 (−7 to 4) mmHg, P=0.536; and −3 (−7 to 1) mmHg, P=0.115, respectively).

Regression analyses, including percentage weight change, treatment choice, gender, age, BMI, baseline value and change in relevant medication as independent variables, showed that weight loss, but not treatment choice, was significantly associated with reduction in HbA1c and systolic blood pressure, but not diastolic blood pressure (R2=0.712, β=0.206, P=0.008; R2=0.580, β=0.313, P=0.001; andR2=0.423, β=0.140, P=0.210).

Regression analyses, including percentage weight change, treatment choice, gender, age, BMI, baseline value and change in relevant medication as independent variables, showed that weight loss, but not treatment choice, was significantly associated with reduction in HbA1c and systolic blood pressure, but not diastolic blood pressure (R2=0.712, β=0.206, P=0.008; R2=0.580, β=0.313, P=0.001; andR2=0.423, β=0.140, P=0.210).

All participants had 2 h glucose >2.8 mmol/l at baseline. In contrast, 2 (4%) patients in the lifestyle group and 15 (23%) patients in the surgery group had 2 h glucose <2.8 mmol/l after the intervention (P=0.003).

Type 2 diabetes and hypertension Among participants with type 2 diabetes, HbA1c was reduced from 6.6% (1.0) to

6.3% (0.9) in the lifestyle group and from 7.1% (1.5) to 5.8% (0.5) in the surgery group (adjusted between-group difference, P=0.003). Moreover, the hypertensive subgroups' systolic blood pressure declined from 144 mmHg (16) to 125mmHg (12)after the surgical procedure and from 143 mmHg (15) to 130mmHg (14) after the lifestyle intervention (adjusted between-group differences, P=0.061). Furthermore, the number of diabetic subjects using glucose-lowering agents dropped (from 11 to 6) in the surgery group and increased (from 6 to 10) in the lifestyle group (between-group difference, P=0.017). In contrast, the reduction in the number of hypertensive participants using anti-hypertensive drugs did not differ significantly between the surgery group (from 24 to 21) and the lifestyle group (from 30 to 25) (between-group difference, P=1.00).

The remission rates of type 2 diabetes and hypertension related to weight change after gastric bypass surgery and lifestyle intervention are shown in Fig. 4. The remission rates of both conditions were significantly higher after surgical treatment than after lifestyle intervention. Complete remission of type 2 diabetes was significantly more frequent in the surgery group than in the lifestyle group (11/14 vs 0/6, P=0.002).

Multiple regression analyses demonstrated that no usage of glucose-lowering agents and anti-hypertensive drugs at baseline were, independent of percentage weight change and treatment choice, associated with remission of type 2 diabetes (P=0.023) and hypertension (P<0.001). In addition, univariate linear regression analyses showed that greater reductions in HbA1c and systolic blood pressure were associated with surgical treatment (β=−0.408, P=0.011 and β=−0.187, P=0.096 respectively). Inclusion of percentage weight change in the regression analyses showed that weight loss mediated the effects of treatment choice on these outcomes (β=0.406, P=0.160 and β=0.142, P=0.410 respectively). Furthermore, weight loss was significantly associated with reductions in HbA1c and systolic blood pressure (β=0.926, P=0.002 and β=0.423, P=0.016 respectively).

Metabolic syndrome, albuminuria and left ventricular hypertrophy

The changes in the number of individuals with metabolic syndrome (−44 vs −14), albuminuria (−7 vs 3) and left ventricular hypertrophy (−10 vs −2) were significantly greater in the surgery group than in the lifestyle group (Fig. 5). The prevalence of metabolic syndrome reduced significantly in both treatment groups (both P≤0.001), while the prevalence of left ventricular hypertrophy was significantly reduced in only the surgery group (P=0.002). In contrast, the prevalence of albuminuria did not change significantly within either treatment group.

Figure 5

The prevalence of metabolic syndrome, albuminuria and left ventricular hypertrophy in the treatment groups at both baseline and 1-year follow-up. Between-group differences at 1 year were adjusted for differences in prevalence at baseline using logistic   (more ...)   

Lifestyle and medications

Although both treatment groups reported significantly lower energy intake at 1 year than at baseline (both P<0.001), the reduction was significantly greater in the surgery group than in the lifestyle group (Table 2). The number of subjects in the surgery group and lifestyle group which either moved from being inactive to active (12 vs 18), stayed inactive or active (57 vs 32) or moved from being active to inactive (4 vs 5) differed significantly between the groups (Fig. 6). Overall, there was a greater increase in the physical activity level of the lifestyle group than the surgery group. However, the median (range) time spent performing physical activities with moderate or vigorous intensity after the interventions did not differ significantly between the surgery and lifestyle groups, 20 (0–510 min) versus 65 (0–660 min), P=0.148.

Figure 6

Change in physical activity during 1-year follow-up. The proportion of participants who went from being physically active (≥150 min of moderate or ≥60 min of vigorous aerobic physical activity per week) to inactive (reduced)   (more ...)   

Usage of weight-loss medications was stopped in all surgically treated patients and started in only one patient in the lifestyle group. The change in the number of individuals using statins did not differ significantly between the surgery and the lifestyle (10 vs 3, P=0.114).

Last observation carried forward

In additional calculations, missing values were replaced by the last observed value of the respective variable (data not shown). These results did not alter the conclusions of the study.

Adverse events

Median (range) post-operative stay was 2 (1–9) days. Perioperative complications in the surgery group included one gastrojejunal anastomotic leakage, which was successfully re-operated on during the first post-operative day, one major bleeding at the site of the trocar insertion, which needed a blood transfusion, and two pneumonias treated effectively with antibiotics.

Late complications in the surgery group included four patients with symptomatic cholelithiasis (imaging verified), two patients with marginal ulcers, five patients with postprandial hypoglycaemia, one patient with a fracture of the fifth right proximal phalange and one patient with myocardial infarction. In the lifestyle group, one patient was diagnosed with breast cancer, one patient suffered a right ankle fracture that was treated with a stabilising cast and complicated by a deep venous thrombosis and one patient developed cholelithiasis. There were no deaths.

In total, 48% (33/69) of patients in the surgery group and 7% (4/59) of patients in the lifestyle group developed gastrointestinal symptoms, including abdominal pain, nausea, vomiting, diarrhoea and constipation (P<0.001).

  Other Sections ▼

DiscussionTo our knowledge, this is the first controlled, clinical trial that has sought to evaluate the effects of gastric bypass surgery and intensive lifestyle intervention on cardiovascular risk factors. When compared with the lifestyle group, the surgery group had higher remission

rates of type 2 diabetes and hypertension, as well as greater reductions in the prevalence of metabolic syndrome, albuminuria and left ventricular hypertrophy. Notably, intensive lifestyle intervention was also associated with favourable changes in measures of glucose metabolism, blood pressure, lipids and low-grade inflammation.

Strengths and weaknesses of the study

The strengths of this study include the prospective design, the fact that the control group obtained a significant weight loss from lifestyle intervention and the high participant completion rate. Limitations of the study include the lack of randomisation (addressed in ‘Subjects and methods’ section), a larger intervention delay in the surgery group and the short-term follow-up. Furthermore, the diagnoses of type 2 diabetes and hypertension were, in the absence of hypoglycaemic or anti-hypertensive drugs, based on only one measurement and not repeated measurements as recommended (25, 27). Finally, the majority of the study population was of Europoid origin, meaning that the results of this study cannot be generalised to include other ethnic groups.

Type 2 diabetes and hypertension

Both the case controlled Swedish Obese Subjects (SOS) study (10) and the Australian randomised controlled clinical trial (11) demonstrated that 2 year remission rates of type 2 diabetes were significantly higher in patients treated with bariatric surgery than in conservatively treated controls. The SOS study also reported higher remission rates of hypertension in the surgery group. Our study extends these findings by showing that morbidly obese patients treated with gastric bypass surgery were more likely to achieve remission of type 2 diabetes and hypertension than those who participated in a comprehensive lifestyle intervention programme. In addition to a shorter intervention period, our study differs from these previous trials in several ways. First, in contrast with our standardised and comprehensive lifestyle programme, the above two studies are notable for the fact that non-surgical treatment varied considerably and that weight loss was negligible. Second, our surgical procedure was gastric bypass, whereas purely restrictive, bariatric procedures were mainly implemented in the two other studies. Accordingly, the average 1-year weight reduction was more pronounced in our surgical group. Third, our definition of remission of type 2 diabetes differed slightly from the previous studies. Finally, the Australian study differed in the sense that it only included those patients who had type 2 diabetes of <2 years duration and a BMI of between 30 and 40 kg/m2. Nevertheless, despite these differences, the remission rate of diabetes in 

40 kg/m2. Nevertheless, despite these differences, the remission rate of diabetes in our surgical group was nearly identical with rates in these previous studies (70 vs 71 and 72%). Furthermore, while hypertension was resolved in approximately half the surgical patients in

our study, it did so in only one-third of the patients in the SOS study. Our study shows that weight reduction, and not treatment choice, predicted improvement in glycaemic control and systolic blood pressure. Furthermore, most of the beneficial metabolic effects were observed after a weight reduction of ≥10%. Accordingly, the metabolic effect of gastric bypass surgery seems to be mediated through weight reduction.

Despite extensive weight loss, type 2 diabetes and hypertension in a substantial number of surgically treated patients were not resolved. In contrast, remission of these conditions was observed in some lifestyle group patients despite only modest weight reduction. These findings might be explained by differences in the severity of the conditions and the possible beneficial effect of increased physical activity. Indeed, the absence of glucose-lowering drugs and anti-hypertensive medication independently predicted remission of both diabetes and hypertension. Furthermore, compared with subjects treated with surgery, a significantly higher proportion of the participants in the lifestyle group became physically active.

Both blood glucose and blood pressure are continuous risk factors for death and cardiovascular disease (35, 36). The observed decline in these measures in both intervention groups may therefore have positive health effects. Conversely, the five cases of symptomatic postprandial hypoglycaemia and the large proportion (23%) of patients with 2 h glucose <2.8 mmol/l in the gastric bypass group raise some concerns. Severe postprandial hypoglycaemia after Roux-en-Y gastric bypass surgery has been reported previously and post-surgical nesidioblastosis may contribute to this complication (37). Furthermore, severe and symptomatic hypoglycaemia in type 2 diabetic subjects has been shown to be associated with increased mortality (38). Consequently, it cannot be excluded that the reduction in 2 h glucose observed after Roux-en-Y gastric bypass surgery may also have negative long-term health effects.

Our study shows that weight reduction, and not treatment choice, predicted improvement in glycaemic control and systolic blood pressure. Furthermore, most of the beneficial metabolic effects were observed after a weight reduction of ≥10%. Accordingly, the metabolic effect of gastric bypass surgery seems to be mediated through weight reduction.

Despite extensive weight loss, type 2 diabetes and hypertension in a substantial number of surgically treated patients were not resolved. In contrast, remission of these conditions was observed in some lifestyle group patients despite only modest weight reduction. These findings might be explained by differences in the severity of the conditions and the possible beneficial effect of increased physical activity. Indeed, the absence of glucose-lowering drugs and anti-hypertensive medication independently predicted remission of both diabetes and hypertension. Furthermore, compared with subjects treated with surgery, a significantly higher proportion of the participants in the lifestyle group became physically active.

Both blood glucose and blood pressure are continuous risk factors for death and cardiovascular disease (35, 36). The observed decline in these measures in both intervention groups may therefore have positive health effects. Conversely, the five cases of symptomatic postprandial hypoglycaemia and the large proportion (23%) of patients with 2 h glucose <2.8 mmol/l in the gastric bypass group raise some concerns. Severe postprandial hypoglycaemia after Roux-en-Y gastric bypass surgery has been reported previously and post-surgical nesidioblastosis may contribute to this complication (37). Furthermore, severe and symptomatic hypoglycaemia in type 2 diabetic subjects has been shown to be associated with increased mortality (38). Consequently, it cannot be excluded that the reduction in 2 h glucose observed after Roux-en-Y gastric bypass surgery may also have negative long-term health effects.

Other cardiovascular risk factors

Surgical therapy was superior to lifestyle intervention both in terms of the resolution of metabolic syndrome, left ventricular hypertrophy and microalbuminuria, as well as with respect to improvements in inflammatory markers. In line with previous studies, we report that weight reduction was associated with resolution of metabolic syndrome (11, 17), beneficial changes in C-reactive protein and adiponectin levels(16) and reduction in left ventricular mass (12). Improvement in all renal parameters, including albuminuria, has been reported after gastric bypass surgery (13). Similarly, weight reduction after lifestyle intervention has been reported to resolve albuminuria(17). In contrast to this finding and closer to our own results, modest weight reduction in the intensive lifestyle group of the Diabetes Prevention Program did not reduce albumin excretion significantly (39).

Clinical and research implications of the work

In sum, we have shown that gastric bypass surgery is more effective than intensive lifestyle intervention in terms of improving type 2 diabetes and obesity-related cardiovascular risk factors. However, morbidly obese patients treated with lifestyle intervention also experienced significant and meaningful improvements in most cardiovascular risk factors, and significantly more patients in the lifestyle group than the surgery group became physically active. Gastric bypass surgery was associated with a significantly higher risk of gastrointestinal symptoms and complications as reported previously (40). Furthermore, although a specified set of dietary supplements seems to prevent vitamin deficiencies after gastric bypass surgery(23), several deficiencies may occur if an inadequate supplementation is prescribed(41). Finally, even though reduced overall mortality after bariatric surgery has been reported (42), it is still unclear whether short-term improvement in obesity-related cardiovascular risk factors translates into long-term reduced cardiovascular morbidity and mortality. Our results indicate that when treating morbidly

obese patients, gastric bypass surgery should not, despite its ability to improve risk factors, be considered the default course of treatment. Rather, both patient and physician should consider the possible side effects of this treatment, and, where appropriate, take up alternative conservative treatments. Indeed, intensive behavioural intervention has been shown to result in long-term weight reduction in some patients (43), while improved physical fitness is known to reduce all cause mortality (44). Moreover, it should be emphasised that if the success of bariatric surgery is to be optimised then behavioural changes are also necessary (45). Future studies comparing surgery and non-surgical treatment programmes should address the effect of these treatments on long-term cardiovascular morbidity and mortality.

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A

Release of Inflammatory Mediators by Human Adipose Tissue Is Enhanced in Obesity and Primarily by the Nonfat Cells: A Review

John N. Fain*

Department of Molecular Sciences, University of Tennessee Health Science Center, Memphis, TN 38163, USA

*John N. Fain: Email: jfain@uthsc.edu

Academic Editor: Giamila Fantuzzi

Received November 3, 2009; Revised January 27, 2010; Accepted February 23, 2010.

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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AbstractThis paper considers the role of putative adipokines that might be involved in the enhanced inflammatory response of human adipose tissue seen in obesity. Inflammatory adipokines [IL-6, IL-10, ACE, TGFβ1, TNFα, IL-1β, PAI-1, and IL-8] plus one anti-inflammatory [IL-10] adipokine were identified whose circulating levels as well as in vitro release by fat are enhanced in obesity and are primarily released by the nonfat cells of human adipose tissue. In contrast, the circulating levels of leptin and FABP-4 are also enhanced in obesity and they are primarily released by fat cells of human adipose tissue. The relative expression of adipokines and other proteins in human omental as compared to subcutaneous adipose tissue as well as their expression in the nonfat as compared to the fat cells of human omental adipose tissue is also reviewed. The conclusion is that the release of many inflammatory adipokines by adipose tissue is enhanced in obese humans.

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1. IntroductionThere is increasing evidence that obesity in humans is associated w

with low-level inflammation [1–6] that is often accompanied by hypertension and type 2 diabetes. Currently it is thought that the increase in visceral omental rather than abdominal subcutaneous adipose tissue best correlates with measures of insulin resistance [7] and cardiovascular disease [8–10]. However, the amount of visceral fat has an allometric relationship with total body fat content [11] which means that the increases in visceral fat mass seen in obesity reflect the initial ratio of visceral fat to total fat mass as well as the changes in total fat mass change. Thus during weight loss or gain there are concurrent changes in the amount of both subcutaneous and visceral fat.

The distribution of fat between premenopausal men and women is different with women having generalized lipid deposition as contrasted to men who tend to accumulate fat in the abdominal region resulting in a socalled “beer belly”. There are also sex differences in the ratio of visceral to abdominal subcutaneous fat mass between men and women [4]. The visceral fat mass of the women was approximately 50% of the abdominal subcutaneous fat mass while for the men it was 98% [4].

The measurement of abdominal subcutaneous and visceral fat mass can be done using either a computed tomography (CT) or MRI scan. Measurement of total body fat requires either a DXA scan or a bioelectrical impedance scale. In contrast, waist circumference is simply measured and provides as good if not better measure of the health risks of obesity than the more complex procedures [12, 13]. However, the use of BMI has the advantage of

comparing men and women on the same scale since it is an index of weight corrected for height.

This review will primarily discuss studies on the effects of obesity on circulating adipokines, the relative release of adipokines by the fat cells versus the nonfat cells of human adipose tissue, the effects of obesity on adipokine release by explants of human visceral omental adipose tissue, and the differences in gene expression between visceral and subcutaneous fat. The term adipokine, as used in this review, means any protein released by adipose tissue without regard to whether it is released by the fat or the other cells (nonfat cells) found in human adipose tissue.

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2. Effects of Obesity on Circulating Levels of AdipokinesAt least 24 adipokines have been reported whose circulating levels are elevated in obese humans (Table 1). Some of these putative adipokines such as CRP, haptoglobin, and amyloid A are actually acute phase proteins primarily released by the liver in response to the mild inflammatory response seen in human obesity. Most of the remaining 21 are inflammatory proteins such as IL-8, PAI-1, MCP-1, IL-6, IL-1Ra, TNFα, sTNF RII, and IL-18 but the source of the elevated circulating levels in obesity is unclear. Their elevations could result from release by tissues other than fat. In contrast, leptin levels are elevated in obesity and the current paradigm is that it is released by fat cells in adipose tissue. However, in mice it has been shown that activated T cells and other lymphocytes can also release leptin under inflammatory conditions [14, 15].

Table 1

Comparison of release of 37 adipokines by fat cells as compared to the other cells in human adipose tissue ranked by fat cell release along with the effect of obesity on their circulating levels in humans.

The circulating levels of zinc-α2-glycoprotein (ZAG) have been reported to be unaltered in obesity [17], but the level of ZAG gene expression in human adipose tissue is reduced in obesity [69, 70]. This illustrates the problem that changes in circulating levels of adipokines do not necessarily reflect changes in their release by or correlate with their mRNA levels in adipose tissue. Most of the adipokines are also cytokines and are released primarily by cells other than fat cells in human adipose tissue (Figure 1). Furthermore, circulating levels of all adipokines are also regulated by their release from other tissues as well as their degradation. For others such as interleukin 1β (IL-1β), no reports have been published indicating that IL-1β is elevated in the circulation of obese humans. However, IL-1β is an important regulator of the inflammatory response in human adipose tissue. It may well be a

paracrine regulator that acts locally and never reaches the blood in mild inflammatory conditions such as obesity. The same may apply to PGE2, which is the primary product of the cyclooxygenase-2 (COX-2) enzyme.

Some of the adipokines may actually have anti-inflammatory effects and circulate at higher levels in obesity as part of a homeostatic mechanism to counteract the effects of the inflammatory mediators. Probably interleukin 10 (IL-10) is such a molecule [71] and there is some evidence that interleukin 6 (IL-6) has dual effects since it has been claimed that it enhances insulin action in muscle [72]. Interestingly there is also evidence that administration of a meal enhanced release of IL-6 by human adipose tissue perfused in situ [73]. It is as yet unclear whether IL-6 is enhancing or inhibiting insulin action but the traditional view is that IL-6 inhibits insulin action [74].

While 24 putative adipokines are listed in Table 1   whose circulating levels are elevated in obesity there are only two out of 37, adiponectin and glutathione peroxidase 3 (GPX-3), whose circulating levels have been reported to be lower in human obesity. The current paradigm is that circulating levels of adiponectin are reduced in obesity [25, 33, 34]. However, the finding that circulating GPX-3 is also lower [35], if confirmed, suggests that GPX-3 may also be important. GPX-3 is unique among the five known isoforms of this enzyme since it is the only one that is secreted by cells [75]. It is a selenocysteine-containing protein with antioxidant properties. The circulating levels of GPX-3 and selenium have also been reported to be lower in patients with coronary artery disease than in age-matched controls [76].

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3. The Relative Release of Adipokines by the Nonfat versus the Fat Cells of Human Adipose Tissue

It has often been assumed that release of an adipokine by adipose tissue is due to the fat cells. This originated with the finding by Rodbell [77] that lipoprotein lipase [LPL] is localized in the fat cells of rat adipose tissue. It was in order to solve the problem of the localization of LPL that Rodbell [78] developed the collagenase procedure for separation of insulin-responsive fat cells from the nonfat cells in rat adipose tissue. However, Cleland et al. [79] found that most of the aromatase activity in human adipose tissue, that is responsible for estrogen formation from androstenedione, was localized in the nonfat cells and most of the IL-6 release by human adipose tissue was by the nonfat cells [80]. Fain et al. [62] subsequently reported on the relative release of 11 adipokines by the nonfat as compared to the fat cells of human omental and abdominal subcutaneous adipose tissue during an in vitro incubation. Leptin was found to be released exclusively by the fat cells, while TNFα,

hepatocyte growth factor (HGF), IL-10, IL-1β, PGE2IL-1β, PGE2, IL-6, vascular endothelial growth factor (VEGF) and interleukin 8 (IL-8) were primarily released by the nonfat cells.

In vitro, the relative release of adipokines by fat cells as compared to nonfat cells derived from human adipose tissue over a 48 hours incubation indicates that the highest release by fat cells was of fatty acid binding protein 4 (FABP-4) followed by IL-8 (Table 1). The high value for IL-8 release over 48 hours is primarily due to upregulation, since the rate of release over 48 hours derived from release during the first 40 minutes was only 2% of the 48 hours release value for both fat cell and nonfat cells [61]. Adipokine release was up-regulated to the same extent in both types of cells of either omental or subcutaneous fat [61].

The question arises as to how well in vitro release of adipokines over the first 48 hours of primary culture by human fat cells and nonfat cells reflects the in vivo situation. That cannot be determined because it takes a two-hour digestion to separate fat cell from nonfat cells and during that time there is upregulation of the mRNAs for inflammatory cytokines such as IL-8 and IL-6 [81]. However, what can be measured is the level of gene expression in the nonfat cells versus the fat cells at the start of the incubation which can be compared to release over 48 hours. These data are shown in Figure 1for 30 of the 37 adipokines shown in Table 1. There was an excellent correlation (Pearson correlation coefficient of 0.8) between release of adipokines over 48 hours by fat cells as % of that by nonfat cells and the initial ratio of the mRNA for the adipokine in fat cells versus nonfat cells. The data also demonstrate that leptin release is exclusively by the fat cells of omental adipose tissue, which also contained 28-fold more leptin mRNA than the nonfat cells (Figure 1

 Release of LPL was also primarily by the human fat cells and in agreement with the 79-fold greater amount of its mRNA found in fat cells as compared to nonfat cells.

Adiponectin has generally been considered to be an adipokine released exclusively by fat cells but while the ratio for mRNA expression in fat cells as compared to nonfat cells was 42-X the release of adiponectin accounted for only 40% of total release. Fain et al. [82] suggested that immature fat cells or other cells in the nonfat cell fractions of human adipose tissue also release adiponectin. Alternatively, the release could be due to adiponectin taken up by nonfat cells in vivo and then released during the 48 hours incubation. The release of amyloid A by human fat cells as % of that by nonfat cells was actually higher than that of adiponectin and its mRNA content in fat cell was 34-fold greater than that in nonfat cells. However, amyloid, like adiponectin, release appears to be about the same by nonfat as by fat cells. While leptin, LPL, amyloid A, and adiponectin are adipokines predominantly expressed in fat cells at ratios 30 to 80-fold greater than in nonfat cells (Figure 1), the

question of whether there is appreciable amyloid and adiponectin synthesis by the nonfat cells of adipose tissue remains to be established.

There are four other possible candidates for the designation of adipokines preferentially released by fat cells, since the ratios of their mRNAs in fat cells to nonfat cells ranged from 5 for FABP-4, 8 for ZAG, and 9 for adipsin/complement D as well as GPX3. However, release by fat cells accounted for less than half of their total release.

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4. Relative Expression of 100 Genes in Fat Cells versus the Nonfat Cells of Human Omental Adipose Tissue

Tab

Table 2   shows the relative gene expression in fat cells versus nonfat cells of 100 proteins, as determined by qRTPCR [83]. These proteins were chosen because they are important in inflammation or obesity, regulatory proteins or proteins enriched in fat cells.

Of the proteins whose gene expression is shown in Table 2   almost one-third (30) were significantly enriched in fat cells (shown in Bold), 29 were distributed equally (shown in italic) and 41 were significantly enriched in nonfat cells of human omental adipose tissue (shown in normal text). Thirty of these proteins are the adipokines whose release by adipose tissue was examined in the studies shown in Table 1   and Figure 1.

Of special interest was the finding that 11β HSD1, UCP-2, cyclic AMP phosphodiesterase 3B, AQP7, angiotensinogen, GPX-3, the insulin receptor, and NQO1 are preferentially localized in fat cells [83]. Interestingly ZAG, TLR4, cytochrome C oxidase, Akt2, adrenomedullin, and UCP-1 were also expressed at levels 4 to 8-fold greater in fat cells than in nonfat cells [Table 2]. The higher expression of ZAG in human fat cells than in nonfat cells confirms the report by Bao et al. [84].

An elevated expression in fat cells was seen for both cytochrome C oxidase, which is a marker for mitochondria, and Akt2, which is the isoform of Akt involved in insulin-stimulated glucose uptake into fat cells [85]. The enhanced expression of the mitochondrial protein UCP-1 in visceral omental fat cells was unexpected since it is thought of as a marker for brown fat cells. However, Sacks et al. [86] found far higher expression of UCP-1 in visceral epicardial fat as compared to subcutaneous fat. The 

increased expression of cytochrome C oxidase in fat cells as compared to nonfat cells of omental fat suggests that fat cells are relatively enriched in mitochondria. Deveaud et al. [87] have shown that cytochrome C oxidase is enriched in visceral epididymal fat of rats as compared to subcutaneous inguinal fat.

The circulating levels of adrenomedullin are elevated in human obesity [88, 89]. Furthermore, adrenomedullin is secreted by fat cells [90, 91] but it is unclear whether more adrenomedullin is secreted by fat cells than by the nonfat cells of human adipose tissue [88–91].

The proteins whose gene expression was predominantly in the nonfat cells included all the classical inflammatory proteins such as MCP-1, TGFβ1, IL-6, IL-8, COX-2, PAI-1, IL-1β, IL-8, and TNFα (Table 2). Other putative adipokines, such as vaspin, endothelin-1, omentin/intelectin, lipocalin-2, RANTES, and visfatin were also enriched in the nonfat cells. Vaspin is an adipose tissue-derived serpin whose gene expression in human visceral fat positively correlated with obesity [92]. Circulating levels of omentin/intelectin are lower in obesity [93] but the meaning of this is unclear.

The ratio of gene expression in fat cells to nonfat cells ranged from 0.06 to 128 (Table 2). However, if in vitro differentiated human omental adipocytes were compared to omental preadipocytes the ratios ranged from 0.001 to over a million for adiponectin [82, 83]. Clearly there is more expression of fat cell specific proteins in freshly isolated nonfat cells than in preadipocytes obtained by culturing the nonfat cells of human omental fat. This difference may be accounted for, in part, by the presence of small fat cells without enough fat to float, since isolated fat cells are operationally defined as cells containing enough lipid to float in isotonic incubation buffer. Another possibility is incomplete digestion of adipose tissue leaving some fat cells entrapped in the undigested tissue matrix. However, if this is the case these cells secrete very little leptin since its release by the nonfat cell fraction is less than 5% of that by isolated fat cells (Figure 1) and we could find no detectable fat in the nonfat cells [67].

OOne problem in comparing gene distribution between fat and nonfat cells is the possibility of preferential lysis of extremely large fat cells during the collagenase digestion of fat from extremely obese humans. The isolation of human fat cells is an art requiring particular batches of collagenase for optimal yield of responsive cells, gentle incubation conditions and an optimal ratio of collagenase to tissue [62, 67]. Fain et al. [67] calculated that there was a 23% greater loss of fat cells during digestion than of nonfat cells during the digestion of fat from extremely obese humans. The fat cells lost during digestion may well be the largest fat cells that release more inflammatory adipokines and leptin than the smaller cells. A further problem is the up-regulation of inflammatory response genes during the 2 hours required for collagenase digestion but this affects both fat cells and nonfat cells to the same extent [61] and thus has minimal effects on the ratios of mRNA expression in fat to nonfat cells.

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5. Comparison of mRNA Expression in Isolated Omental Fat Cells versus In Vitro Differentiated Adipocytes

Many studies on the relative gene expression of proteins in fat cells have utilized adipocytes differentiated in vitro such as murine 3T3L1 cells, but far fewer studies have appeared using human cell lines. The term fat cells is operationally defined as those cells that float and are isolated by collagenase digestion of human omental adipose tissue from women undergoing bariatric surgery. Adipocytes are those fat cells derived from the adipose tissue of the same group of women that underwent differentiation in vitro in the presence of insulin, dexamethasone, a methyl xanthine, and a thiazolidinedione.

In the data shown in Figure 2the mRNA content of freshly isolated omental fat cells versus in vitro differentiated adipocytes was compared using total RNA as the recovery standard as suggested by Bustin [94] since the expression of cyclophilin A used as the recovery standard differed significantly between fat cells and in vitro differentiated adipocytes. The data indicate that many proteins are expressed at far higher levels in adipocytes than in freshly isolated fat cells. Some proteins that are expressed at higher levels in adipocytes than in fat cells are not enriched in freshly isolated fat cells as compared to nonfat cells (Figure 2). These are shown in red and are: butyryl cholinesterase, haptoglobin, apelin, PGC1α (peroxisome proliferator activator receptor-γ coactivator 1α), ATR1 (angiotensin II receptor 1), αl glycoprotein, endocannabinoid receptor 1, endothelin-1, and omentin/intelectin.

Five mRNAs were found at comparable levels in adipocytes as compared to fat cells. These were the β1 adrenergic receptor, 25-hydroxyvitamin D3 1α hydroxylase, VEGF-a, ZAG, and lipin-1. Three genes were expressed at lower levels in adipocytes than in fat cells: adipsin, insulin receptor, and CIDEA. The data suggest that the one or more of the added factors required for differentiation of preadipocyes to adipocytes induce the expression of many proteins that are not induced in vivo and decrease the expression of others such as CIDEA and the insulin receptor. Clearly the use of human adipocytes differentiated in vivo from preadipocytes does not result in a pattern of gene expression comparable to that seen in intact fat from obese women.

Studies using freshly isolated explants preserve the cross talk between the various types of cells in fat. However, since the primary effect of obesity is to increase adipose tissue mass, it is difficult to know how to express data obtained by primary culture of human fat explants. How do you compare total release by adipose tissue from humans with 20 kg of fat as compared to those with 40 kg? In the studies shown in Figure 3release in vitro over a 48 hours incubation of omental and subcutaneous fat from each woman per kg of fat was multiplied by the total fat content. The women were then divided by tertiles based on body fat content.

There was enhanced release of endothelin-1, lipocalin-2, visfatin, GPX-3, and FABP-4 by the most obese women as compared to that by women in the bottom tertile (Figure 3). For ZAG we found no effect of obesity since total release was not significantly higher in women in the highest tertile but they had 124% more fat than women in the lowest tertile. Therefore there was actually decreased release per g of adipose tissue. This is in agreement with reports that gene expression of ZAG in fat is reduced in human obesity [69, 70]. There was enhanced total release of intercellular cell adhesion molecule 1 (ICAM-1), CD14, and LPL but not of osteoprotegerin, RANTES or amyloid A [42].

Another way to examine the effect of obesity is to correlate total release with the total fat mass of each woman. That resulted in a correlation coefficient for lactate release of 0.81 and for IL-8 release of 0.85 based on total release plotted against the fat mass of each woman (Figure 4). A positive correlation indicates that the more fat you have the greater the total amount of lactate or IL-8, if release per g of fat remains the same. In contrast, total amyloid and VEGF release did not correlate with total fat mass indicating that their release per g of tissue was less but the total release by fat remained constant.

Figure 4

Correlation between total release of IL-8, VEGF, Amyloid A, and lactate by adipose tissue and total fat mass. The total release was calculated by averaging release over 48 hours per kg by explants of visceral omental and subcutaneous adipose tissue from   (more ...)   

Data for 24 other adipokines are summarized in Table 3, along with those for lactate, amyloid A, and VEGF and IL-8 release shown in Figure 3. Adipokines that showed no correlation, that is, those whose total release actually decreased in obesity, were MCP-1, interleukin 1 receptor antagonist 1 (IL1-Ra), adipsin, osteoprotegerin, RANTES, ZAG, cathepsin S, vascular cell adhesion cell molecule 1 (VCAM-1) and NGFβ in addition to VEGF and amyloid A. A number of inflammatory adipokines had a significant correlation between total release and total fat mass besides IL-8 and these included, IL-10, transforming growth factor β1 (TGFβ1), visfatin, IL-1β, IL-6, CD14, endothelin-1, ICAM-1, TNFα, lipocalin-2, PAI-1, and angiotensin 1 converting enzyme (ACE) that are primarily released by the nonfat cells. There was also a significant correlation between total release and fat mass for FABP-4, GPX-3, and LPL.

A problem complicating release studies by human fat is that incubation in vitro induced an inflammatory response as judged by enhanced mRNA accumulation over the 48 hours incubation for IL-8, IL-10, TGFβ1, visfatin, IL-1β, IL-6, ICAM-1, TNFα, lipocalin-2, PAI-1 and ACE (Table 3). Interestingly, an increase in mRNA expression over 48 hours was seen for

MCP-1, osteoprotegerin, and NGFβ whose total release was not enhanced by obesity. Furthermore there was no significant change in the mRNA expression over 48 hours of CD14, endothelin-1 or ACE while there was a marked decrease in FABP-4, GPX-3, and LPL mRNA but enhanced release in obesity. These data suggest that the in vitro inflammatory response does not mimic completely the effect of obesity.

In conclusion, adipose tissue from extremely obese women, when incubated in vitro, releases more of a host of adipokines such as IL-8, IL-10, TGFβ1, visfatin, IL-1β, IL-6, ICAM-1, TNFα, lipocalin-2, PAI-1, and ACE than does tissue from women with a lesser amount of fat. While TNFα appears to be important it is one adipokine whose mRNA and release goes up transiently during in vitro incubation of adipose tissue, but unlike other members of the inflammatory cascade its release and gene expression return to near basal values by 48 hours [61, 96].

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7. Which Cells in the Nonfat Cell Fraction Derived from Human Adipose Tissue Are Responsible for Release of Inflammatory

Adipokines?Hellman et al. [97] reported in 1963 that obesity in the obese-hyperglycemic mouse resulted in greater accumulation of mast cells in white adipose tissue. They also pointed out that the relative nitrogen content per gram of the epididymal fat pad of the obese-hyperglycemic mouse was unchanged despite the marked reduction in the number of fat cells per g of tissue. Almost 40 years later Xu et al. [98] extended this to show that the expression of genes enriched in murine macrophages such as MCP-1, TNFα, CD68, and F4/80 was elevated in obese mice. They also demonstrated that all of these genes were preferentially expressed in the nonfat cells of murine white fat [98]. Weisberg et al. [99] independently published similar findings and emphasized that the size of fat cells positively correlated with the percentage of macrophages in murine adipose tissue.

Subsequently it was demonstrated that HAM56+ macrophage accumulation in visceral omental and subcutaneous fat depots of humans also positively correlated with the diameters of the fat cells in each depot. However, at any fat cell size there were more macrophages in omental than subcutaneous fat despite the fact that the average diameter of subcutaneous fat cells was 40% greater than that of omental fat cells [100]. The use of HAM56 as the macrophage marker is important since in humans CD68 [101, 102], CD14 [102], or F4-80 [102] are much less specific macrophage markers than in mice. Similar results are shown in Table 2   in that the gene expression of both CD14 and CD68 was not significantly differ

different between the fat cells and nonfat cells isolated from human omental adipose tissue.

The current paradigm is that obesity results in accumulation of macrophages in adipose tissue and these are primarily responsible for the release of inflammatory mediators [98–100]. A relevant question is whether macrophages are the only mononuclear phagocytes found in adipose tissue and whether they account for all of the adipokine release by nonfat cells. The potential contribution of the other nonfat cells in human adipose tissue such as the endothelial cells of the blood vessels, the smooth muscle cells and fibroblasts as well as other mononuclear phagocytes has not been carefully examined.

Why do macrophages localize in the white adipose tissue of obese animals? Whether enhanced lysis/death of large fat cells is the primary trigger that accounts for inflammation is unknown as well as what signal results in greater macrophage accumulation in adipose tissue. One of the functions of macrophages is to aid in the clearing of dead cells. Cinti et al. [103] suggested that macrophages are localized selectively to sites of necrotic-like cell death where they appear as crown-like structures when viewed in tissue sections. They also suggested that fat cell hypertrophy per se promotes cell death resulting in macrophage accumulation and aggregation around dead cells. The current paradigm is that the larger the fat cell the more likely it is to undergo cell death. However, a consistent finding is that human visceral omental fat cells are smaller than subcutaneous fat cells from the same individual but the macrophage accumulation is greatest in omental fat so something besides fat cell size is important [104]. Furthermore, thiazolidinediones appear to selectively enhance the breakdown of large fat cells in visceral omental fat resulting in smaller more insulin-sensitive fat cells [105]. The net effect of thiazolidinediones is to preferentially enhance deposition of fat in subcutaneous adipose tissue while decreasing that in visceral fat [105].

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8. The Relative Expression of mRNAs in Human Epicardial, Substernal, Omental, Mesenteric, and Subcutaneous Adipose

TissuesCurrently it is thought that it is the increases in visceral (intraperitoneal) rather than subcutaneous (extraperitoneal) adipose tissue is linked to the enhanced risk of diabetes, hypertension and cardiovascular disease in obesity [7–10]. Exactly how 

visceral adipose tissue is linked to this is unclear. It could be due to greater release of inflammatory factors by visceral fat or fatty acids and adipokines released by visceral adipose tissue that are preferentially delivered to the liver through the hepatic portal system.

The visceral fat is composed of omental and to a lesser extent mesenteric adipose tissue. The search for a major biochemical difference between these two types of 

PubMed articles by these authors

Fain, J. PubMed related articles

Review Release of

interleukins and other

visceral fat and abdominal subcutaneous fat of extremely obese women has turned up some interesting differences in gene expression (Table 4).

Table 4

Comparison of mRNAs in human mesenteric and subcutaneous as compared to omental adipose tissue from extremely obese women.

The gene expression of UCP-1, omentin, and haptoglobin in subcutaneous fat was less than 10% of that in omental fat. The data on UCP-1 confirm the initial report by Esterbauer et al. [107] that UCP-1 expression in subcutaneous fat was 12% of that in omental fat. However, the amount of UCP-1 gene expression, which is related to thermogenesis, in epicardial fat of humans is at least 9-fold greater than that in omental fat [106]. Sacks et al. [86] have postulated that the epicardial fat, which is located on the outside of the heart, serves to defend the myocardium against hypothermia.

Another protein whose gene expression was quite low (about 1%) in subcutaneous as compared to omental fat was omentin/intelectin (Table 4). It is also expressed at 3-fold higher levels in epicardial fat than in omental fat [108]. Its preferential expression in intraperitoneal adipose tissue probably reflects the fact that the blood vessels in these tissues arise from endothelial cells of the gut during development [108]. Unlike UCP-1, which is preferentially expressed in fat cells of omental fat (Table 2), omentin/intelectin is primarily found in the endothelial cells of the blood vessels [108].

It is unclear why haptoglobin is expressed at such low levels in subcutaneous fat but its expression is also low in mesenteric fat (Table 4). In contrast UCP-1 is found at the same level of expression in mesenteric fat as in omental fat while omentin/intelectin is found at far lower levels in mesenteric than in subcutaneous fat. As for the low level of expression of ATR2 in subcutaneous fat that is probably due to overexpression of ATR1 in subcutaneous fat.

Comparison of mesenteric with omental fat indicates that they have more in common with each other than with subcutaneous fat. This is especially true with regard to expression of UCP-1, prostaglandin D2 synthase, angiotensinogen, ZAG, NFκB1, ATR2, RBP-4, IL-6, and osteopontin.

However, MCP-1, IL-1β, adrenomedullin, PPARγ, and PAI-1 were expressed at significantly lower levels in mesenteric than in omental fat while their expression in subcutaneous fat was the same as or higher than that in omental fat. At this time these are simply lists of similarities and differences between omental and 

inflammatory cytokines by human adipose tissue is enhanced in obesity and primarily due to the nonfat cells.[Vitam Horm. 2006]

Release in vitro of adipsin, vascular cell adhesion molecule 1, angiotensin 1-converting enzyme, and soluble tumor necrosis factor receptor 2 by human omental adipose tissue as well as by the nonfat cells and adipocytes.[Metabolism. 2007]

Comparison of messenger RNA distribution for 60 proteins in fat cells vs the nonfat cells of human omental adipose tissue.[Metabolism. 2008]

Subcutaneous and visceral adipose tissue gene expression of serum adipokines that predict type 2 diabetes.[Obesity (Silver Spring). 2010]Review Adipokines:

inflammation and the pleiotropic role of white adipose tissue.[Br J Nutr. 2004]

mesenteric fat indicating that they are different tissues. It is also not yet established whether the differences in mRNA expressionbetween omental and mesenteric fat are in the fat or the nonfat cells. Furthermore we know almost nothing about the physiological differences in the metabolism and adipokine release of these two kinds of intraperitoneal fat.

There have been many studies comparing the differences in response of isolated fat cells derived from omental as compared to subcutaneous fat and pieces of adipose tissue from these depots [109, 110]. However, the data are confusing since the results have been almost as varied as the number of reports. This is especially true for PAI-1 gene expression and protein release. Some reported greater in vitro release of PAI-1 by omental than by subcutaneous fat [10], others no difference in gene expression or protein content between omental and subcutaneous [111] while yet another group reported greater release by subcutaneous than omental adipose tissue from extremely obese humans [112]. This is a common occurrence in studies comparing omental versus subcutaneous fat of humans and it is unclear why such variable results are obtained.

The picture with regard to leptin gene expression and release is equally controversial. While some groups have reported greater expression and secretion by subcutaneous as compared to omental fat [113, 114] another group reported no difference [110] and a similar finding is in Table 4.

TNFα is one adipokine that is expressed (Table 4) and released to the same extent by human omental and subcutaneous adipose tissue [96, 115]. Another inflammatory adipokine is lL-6 that is released at higher levels by omental adipose tissue than by subcutaneous adipose tissue [62, 80] but the gene expression of IL-6 was higher in freshly isolated subcutaneous adipose tissue (Table 4).

Lipolysis is reported to be greater in adipoctyes derived from subcutaneous than from visceral adipose tissue and attributed to the greater size of the subcutaneous adipocyes [116]. However, similar levels of expression for hormone sensitive lipase (HSL) and perilipin have been reported in subcutaneous as compared to omental adipose tissue (Table 4, [117, 118]).

Giorgino et al. [109] have reviewed the evidence that fat cells isolated from omental fat are more insulin-responsive than those from subcutaneous human fat. Higher levels of insulin receptor expression have also been seen in omental as compared to subcutaneous adipose tissue [117, Table 4].

The visceral fat is composed of the intraperitoneal omental and mesenteric in the 

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peritoneal cavity as well as the intrathoracic fat depots of the substernal and epicardial fat. The latter two fat depots differ in that the epicardial surrounds the heart while the substernal fat body is a separate tissue within the thoracic cavity. Gene expression in substernal can be compared to that of epicardial fat to distinguish possible differences between these two intrathoracic depots. Fain et al. [106] found that of 45 mRNAs all except five were expressed in substernal fat at levels within 0.4 to 1.6-fold of that in epicardial fat. These were haptoglobin (21-fold greater), prostaglandin D2 synthase (6-fold greater), nerve growth factor (5-fold greater), VEGFR/FLT1 (5-fold greater) and α1 glycoprotein (2-fold greater) with greater expression in epicardial as compared to substernal fat. UCP-1 is also expressed at in epicardial fat at 5-fold higher amounts than in substernal fat [86]. Of these only UCP-1 is expressed at greater levels in fat cells than in the nonfat cells of human omental adipose tissue (Table 2). These data are compatible with the hypothesis that the fat cells in epicardial fat have a unique function as a brown fat-like tissue and could be involved in thermogenesis.

Epicardial fat has been postulated to be an inflammatory organ releasing adipokines that contributes to coronary artery disease because of the unique anatomical relationship between this fat and the coronary arteries [119]. However, when the gene expression of IL-6, IL-1β, PAI-1 or cyclooxygenase-2 was compared in epicardial fat of patients undergoing coronary artery bypass surgery to that in obese individuals undergoing gastric bypass surgery their expression in epicardial fat was less than 25% of that in omental fat [106]. It could be argued that this was because the bypass patients differed in other aspects, which they did, but the expression of 20 other protein ranged from 0.4 to 1.3 in omental fat to that seen in epicardial fat. In contrast, significantly higher amounts (1.6 to 2-fold greater) of the insulin receptor, ZAG, leptin, angiotensinogen and LPL were expressed in epicardial fat as compared to that in omental fat [106]. The significance of these differences between epicardial and omental fat remains unclear but do not suggest that epicardial fat is more inflamed than omental fat.

In conclusion, the reported differences in gene expression, hormonal sensitivity, and release of adipokines by visceral as compared to subcutaneous adipose tissue have been almost as varied as the number of reports [109, 110]. Furthermore, they provide few clues that can explain the putative harmful effect of enhanced accumulation of visceral fat. The fat cells found in visceral fat are smaller than those of subcutaneous fat from obese individuals but is that due to greater breakdown of large fat cells in visceral fat?

There are clear differences between mesenteric and omental fat but again it is 

unclear what they represent. Comparisons of visceral omental versus subcutaneous fat are probably influenced by the degree of obesity and this was demonstrated for PPARγ where the ratio in visceral to subcutaneous was around 0.2 at a body mass index of 20 but increased to about 1.2 in individuals with a body mass index of 50 [117]. Future studies will require the development of procedures to accurately assess the gene expression and release of adipokines by the different human adipose tissue depots under more physiological and reproducible conditions.

Recently the microRNA (miRNA) profiles of human omental and subcutaneous have been compared in humans without or with diabetes [120]. The expression of 155 miRNAs was examined and some differences were found that were said to correlate with fat cell phenotype, obesity, and glucose metabolism [120]. However, no miRNA was found exclusively in one fat depot versus the other suggesting a common developmental profile [120].

I conclude that the gene expression profile of omental fat clearly differs from that of subcutaneous fat for some proteins. However, none of these differences appear to explain the putative harmful effects of visceral obesity. Furthermore, there is scant agreement in the literature with respect to most proteins. This is possibly due to small sample sizes, sex differences, age differences, the extent of obesity, and the disease status of the humans from whom fat samples were obtained. For ethical reasons samples of omental and subcutaneous fat cannot be obtained from healthy donors. Most samples of human omental fat have been obtained from individuals undergoing gallstone, gynecological, or bariatric surgery. While individuals healthy enough to undergo bariatric surgery are extremely obese, the normal weight individuals always have some underlying disease process that could affect gene expression and adipokine release.

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9. What Is the Link between TLR-4, Enlarged Fat Cells, and the Inflammatory Response Seen in

Obese HumansRecently the toll-like receptor 4 (TLR-4), that plays an important role in innate immunity through its ability to recognize bacterial lipopolysaccharides, has been postulated to play a role in the obesity-induced inflammatory response [95, 121, 122]. A loss-of-function mutation in TLR-4 prevents diet-induced obesity in mice and the development of insulin resistance [95, 121]. In macrophages and cultured adipocytes potent inducers of TLR-4 gene expression are bacterial 

lipopolysaccharides resulting in the release of inflammatory adipokines [123, 124]. In a monocyte/macrophage cell line (RAW 264.7) saturated, but not unsaturated fatty acids, induced the expression of COX-2 expression via TLR-4 [123]. Schaeffler et al. [122] reported that saturated fatty acids could induce the secretion of MCP-1 and other inflammatory adipokines in murine 3T3L1 adipokines through a pathway involving TLR-4.

Lin et al. [124] originally suggested that a fully intact pathway of innate immunity was present in rodent adipocytes that could be activated by bacterial lipopolysaccharides. Subsequently, functional TLR-4 has been found in human fat cells [125, 126] and the data in Table 2      indicates that in human omental fat the gene expression of TLR-4 is 5-fold greater in fat cells than in the nonfat cells. Zha et al. [127] reported that in vitro differentiated adipocytes had more TLR-4 mRNA than did preadipocytes and that TNFa secretion was induced by free fatty acids. My laboratory has similar findings in that the TLR-4 mRNA expression in human omental adipocytes differentiated in vitro was also 5-fold higher than that in preadipocytes (John N. Fain, unpublished experiments). In omental adipose tissue explants incubated for 48 hours TLR-4 gene expression was down regulated by about 70% but this was blocked in the presence of dexamethasone [128]. This may reflect a down-regulation of TLR-4 secondary to the 90 to 700-fold activation of the expression of inflammatory cytokines such as I-8, IL-6 and IL-1β that was markedly inhibited by dexamethasone [128].

It has been suggested that the hypertrophied fat cells seen in extreme obesity release large amounts of saturated fatty acids secondary to macrophage-induced lipolysis occurring in fat cells [129]. There is evidence in rodent adipocytes that bacterial lipopolysaccharides can stimulate lipolysis via TLR-4 [130]. However, addition of bacterial lipopolysaccharides to explants of human adipose tissue incubated for 48 hours enhanced release of IL-1β, IL-6, and IL-8 by 50% to 70% under conditions where there was no significant increase in lipolysis (John N. Fain, unpublished experiments). Possibly breakdown of hypertrophied fat cells could be the primary trigger for the inflammatory response via activation of TLR-4 by fatty acids in neighboring intact fat cells resulting in the release of inflammatory adipokines that cause monocyte recruitment into the adipose tissue and insulin-resistance. However, this hypothesis is probably an over-simplification since thiazolidinediones appear to enhance the breakdown of large fat cells and the accumulation of small fat cells but this is associated with enhanced insulin sensitivity [105].

It was surprising to find TLR-4, whose function has traditionally been thought of as 

being involved in pathogen-associated molecular recognition by immune cells, expressed at higher levels in fat cells than in nonfat cells in human fat cells. The physiological function, if any, of this enhanced expression remains to be elucidated. Another unanswered question is what is the primary trigger that results in the accumulation of activated macrophages in the adipose tissue of extremely obese humans?

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10. Hypoxia as the Primary Trigger of the Inflammatory Response

This hypothesis was originally proposed in 2004 by Trayhurn and Wood [1] and discussed in recent articles [131–134]. The best evidence for the “hypoxia hypothesis” is the evidence that adipose tissue is poorly oxygenated in the obese [134, 135]. The mechanisms involved are not understood beyond the accepted paradigm that HIF1α activation occurs resulting in activation of NFκB leading to increased gene transcription of inflammatory adipokines. Yin et al. [133] recently suggested that hypoxia in adipose tissue activates lipolysis and inhibits fatty acid uptake by adipocytes leading to activation of an inflammatory response via TLR-4. There is no evidence that activation of lipolysis per se induces an inflammatory response in human fat. Fain et al. [136] reported that growth hormone in the presence of dexamethasone, but not in its absence, stimulated lipolysis by explants of human omental adipose tissue over a 48 hours incubation but this was not accompanied by an increase in IL-8 gene expression or release.

Another problem is that while there is evidence that the adipose tissue from the ob/ob mouse is hypoxic in comparison to fat from obese mice, there was no increase in expression of VEGF while there was of hypoxia response genes such as HIF-1α, IL-6, Il-1β, and TNFα [134]. A similar finding has been reported by Halberg et al. [137] and remains to be explained since the current paradigm is that hypoxic tissues release VEGF that leads to increased tissue vascularization. However, the hypothesis may be incorrect or angiogenesis may also require other, as yet unknown, factors.

An attractive hypothesis is that as fat cells expand there is insufficient neovascularization to keep the cells from becoming hypoxic. This results in activation of HIF1α and a variety of responses including increased formation of inflammatory adipokines as well as activation of collagen synthesis and crosslinking of collagen involving lysyl oxidase [137]. There is global upregulation of extracellular matrix formation that hampers oxygen access to the cells and the 

increased stress resulting from expansion of the fat cells resulted in rupture of very large cells [137]. The fatty acids resulting from breakdown of triacylglycerols released by ruptured fat cells could activate macrophages as well as intact fat cells.

Alternatively, hypoxia leads to the death of large fat cells and macrophages are drawn to areas of recent cell death by mediators still to be described that are released after cell death, as suggested by Rausch et al. [135]. It may well be that visceral omental fat cells are more liable to lysis which explains why these fat cells are smaller than those found in subcutaneous adipose tissue. Furthermore it is commonly accepted, but may be an over-simplification, that visceral adipose tissue has more macrophages than subcutaneous adipose tissue and releases more inflammatory adipokines. Explants, but not isolated fat cells, of omental adipose tissue have been shown to release more PGE2, PAI-1, IL-6, and VEGF than abdominal subcutaneous adipose tissue on a per g basis [62]. Similar results have been reported for IL-8 content of and release by visceral omental as compared to subcutaneous human adipose tissue [138].

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11. SummaryThe data in Figure 5summarizes the relative release of selected adipokines by fat cells and nonfat cells of human adipose tissue. Of the adipokines shown in the figure only leptin, FABP-4, GPX-3, and adiponectin are expressed at 5 to 80-fold higher levels in fat cells than the other cells present in human fat and primarily released by fat cells. Adiponectin and GPX-3 are listed in blue because their circulating levels are lower in obesity.

Figure 5

The relationship between adipokine release and paracrine signaling in human adipose tissue. The adipokines are divided into those released by fat cells [leptin, FABP-4, adiponectin, and GPX-3] and those by nonfat cells in adipose tissue [IL-6, IL-8, IL-10,   (more ...)   

The adipokines with black lettering are those whose circulating levels are enhanced in obesity and whose total release by adipose tissue explants is enhanced in obesity: IL-6, IL-10, ACE, TGFβ1, ICAM-1, TNFα, IL-1β, PAI-1, and IL-8 that are released by nonfat cells. However IL-10 may be an anti-inflammatory adipokine primarily released by the nonfat cells, whose circulating levels as well as in vitro release are elevated in obesity. The release of leptin and FABP-4 by fat cells 

is also enhanced in human obesity. It should be understood that most of these adipokines act locally and whether the changes in circulating levels of adipokines seen in obesity reflect release by adipose or other tissues remains to be established.

Omentin/intelectin is a novel adipokine preferentially found in visceral fat depots, especially human epicardial fat whose site of origin is the endothelial cells of blood vessels. For this reason it is listed in Figure 5as being derived from the endothelial cells in the vessel wall. In conclusion, most of adipokines whose circulating levels are elevated in obesity and whose release by human adipose tissue is enhanced in obesity are inflammatory adipokines primarily derived from the nonfat cells of human adipose and other tissues.

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Articles from Mediators of Inflammation are provided here courtesy of

Hindawi Publishing Corporation

Adipocyte extracellular matrix composition, dynamics and role in obesityEdwin C. M. Mariman  and Ping Wang

Department of Human Biology, NUTRIM School for Nutrition, Toxicology and Metabolism, Maastricht University, P.O. Box 616, 6200 MD Maastricht, The Netherlands

Edwin C. M. Mariman, Phone: +31-43-3882896, Fax: +31-43-3670976, Email: e.mariman@hb.unimaas.nl.

Corresponding author.

Received November 20, 2009; Revised January 5, 2010; Accepted January 7, 2010.

The central role of the adipose tissue in lipid metabolism places specific demands on the cell structure of adipocytes. The protein composition and dynamics of the extracellular matrix (ECM) is of crucial importance for the functioning of those cells. Adipogenesis is a bi-phasic process in which the ECM develops from a fibrillar to a laminar structure as cells move from the commitment phase to the growth phase characterized by storage of vast amounts of triglycerides. Mature adipocytes appear to spend a lot of energy on the maintenance of the ECM. ECM remodeling is mediated by a balanced complement of constructive and destructive enzymes together with their enhancers and inhibitors. ECM remodeling is an energy costing process regulated by insulin, by the energy metabolism,

and by mechanical forces. In the obese, overgrowth of adipocytes may lead to instability of the ECM, possibly mediated by hypoxia.The global rise of overweight and obesity with the risk for complications like type II diabetes and cardiovascular disorders has intensified the attention on the metabolic and physiologic role of the adipose tissue. The major functions of adipose tissue are:

site for energy storage endocrine/paracrine regulator of energy metabolism thermal insulation of the body shock cushion to protect organs from mechanical damage

Those functions place specific demands on the adipose tissue with respect to its structure and composition. Here, we review various aspects of the adipocyte extracellular matrix (ECM) including its components, development, regulation, and relation to obesity.Adipose tissue is histologically categorized as a type of loose connective tissue and, as such, collagen contributes considerably to the non-cell mass of this tissue. At least partly, this collagen is produced by the adipocytes, but cells of the stromal vascular fraction containing preadipocytes, capillary endothelial cells, infiltrated monocytes/macrophages, and a population of multipotent stem cells can also contribute to it. It was reported that each adipocyte is surrounded by a thick ECM referred to as basal lamina containing collagen IV as a major component [1]. This characteristic is shared with cells of bone and cartilage, which is not surprising since adipocytes, osteoblasts, and chondrocytes are all of mesenchymal origin. However, while a strong ECM is the principal functional entity of bone and cartilage, the basal lamina of adipose tissue may be more of a necessity for survival of the adipocytes. Mature human adipocytes store triglycerides in a single fat droplet that almost entirely fills the cell volume. Since only a lipid monolayer forms the boundary between stored fat and cytosol, mechanical stress on such an organelle may easily lead to disruption. Transfer of mechanical stress from the outside to the inside of the cell can b

 be decreased by the strong external skeleton. In addition, a strong extracellular scaffold can by linking the basal lamina of adipocytes diminish locally experienced mechanical stress by spreading forces over a larger area of the tissue. A specific requirement of the ECM for cell survival indicates that the development of preadipocytes into fat-storing adipocytes should be accompanied by specific changes in the make-up of the ECM.

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Adipocyte ECM protein compositionSince the function of the ECM will depend on, and thus will be reflected by, its molecular make-up, it is important to know which proteins have been identified so far as part of the

adipocyte ECM. Overall, the ECM of adipocytes seems composed of the same proteins as found in other cell types. It is more the relative quantity in which those factors are combined that determines the cell-specificity of the ECM. Furthermore, this composition is related to the developmental stage, viability, and subtype of the adipocytes. The two main classes of ECM proteins are proteoglycans (Fig. 1) and fibrous proteins to which most published information is referring.

Fig. 1

Visualization of the ECM of 3T3-L1 preadipocyte (a) and adipocytes (b) by immunohistological staining of heparin sulfate proteoglycan:green heparin sulfate proteoglycans stained by antibody (10E4 epitope; Seikagaku, Tokyo, Japan); red nuclear stained   (more ...)   

Early studies on the protein composition of the ECM have used immunological 

techniques with or without protein labeling. In this way, Aratani and Kitagawa [2] demonstrated the presence of collagen IV, laminin complexes, and nidogen (entactin) in the ECM of mouse 3T3-L1 adipocytes. In differentiated bovine intramuscular preadipocytes (BIP), Nakajima et al. [3] showed the presence of type I–VI collagens, laminin and fibronectin, whereas based on staining, type II collagen was found to have an extremely low abundance. With the emerging of proteomics techniques, several studies of the adipocytes secretome have been performed. From those, an impression can be obtained on the protein composition of the adipocyte ECM. Based on current literature [2–13], a list of core proteins annotated to be part of the adipocyte ECM is shown in Table 1. This leaves out those proteins that are presently described as secreted or membrane proteins, some of which might interact with the ECM. Although there are a number of microarray studies on adipocytes, we have exclusively focused on proteomics data to avoid possible discrepancies between mRNA and protein levels. Altogether, 20 subunits of 12 different types of collagen have been identified from rodent cells, whereas subunits of collagens II, XI, and XXIII were not found with human visceral adipocytes. This might suggest species- or fat depot-specific differences. However, the failure to detect a protein cannot be taken as proof of its absence, because the chance of detection depends on the biochemical characteristics of a protein, on experimental conditions, and on employed proteomics techniques [14].

Compared to other ECM components, collagen VI seems more specific for adipocytes. It is highly enriched in adipocytes, and there is evidence of its contribution to the pathology of obesity-related diseases [15, 16]. The presence of all three subunits, α1(VI), α2(VI), and α3(VI), is required for the stable formation of collagen VI [17]. Using the yeast two-hybrid

system, collagen VI was shown to be able to strongly bind to collagen IV, an important component of the basement membrane. Both collagens are classified as non-fibrillar types and their interaction has been suggested to mediate anchoring of the basement membrane to cells. Collagen VI has also been shown to interact with other matrix proteins like proteoglycans and fibronectin. One study reported on the composition of adiposomes from mouse 3T3-L1 cells, the vesicles by which adipocytes are supposed to perform part of their endocrine function with the secretion of adipokines [8]. ECM proteins may become part of those vesicles as contaminants during their transportation or experimental preparation. On the other hand, the presence of collagens may be natural, because procollagens are packed into vesicles and secreted as such into the extracellular space. Remarkably, only collagen types I and VI were detected in adiposomes. It suggests that these collagens are specifically involved in the secretion of adipokines through thothose vesicles

The developmental dynamics of the ECM proteins has mainly been studied in vitro using differentiation of mouse 3T3-L1 preadipocytes. Aratani and Kitagawa [2] found a strong up-regulation of collagen IV, nidogen-1 (entactin), and various laminin complexes during the first few days of a 6-day differentiation period indicating the transition from a fibrillar to a laminar make-up.

Studies on the adipocyte secretome during adipogenesis also reveal useful information on the ECM components. Notably, in vitro cell culture medium contains not only intact or detached ECM components but also processing byproducts and degradation products of the ECM. Therefore, the amount of ECM proteins in the secretome may reflect both synthesis and degradation. Our group observed different patterns of expression for the various collagens detected in the medium during a 12-day period of differentiation and growth of 3T3-L1 cells. In the early phase of differentiation, there occurred a decrease of collagens type I and type III, whereas at a later stage, their level returned to that of day 0. The same pattern was found for the C-terminal processing peptides of those collagens, suggesting that the observed changes related to the synthesis of collagen. In parallel with this decrease of fibrillar collagens, an increased liability of the cells to detach from the culture dish was observed around day 4. An opposite pattern was seen for the components of collagen VI. They initially increased and later declined, but remained at a higher level compared to day 0. Collagen IV, the major collagen of the basal lamina, and collagen V gradually increased during differentiation.

In the study of Molina et al. [9] with a 7-day differentiation period of 3T3-L1 cells, the vast majority of secreted proteins were seen to be down-regulated at day 1. However, most of the matrix components were around basal level at day 1 and pe

peaked at day 3, including laminins, nidogens, biglycan, lumican, SPARC, and EMILIN1. In accordance with our observation, the same peaking behavior was observed for the components of collagen VI. Contrastingly, in this study, collagen IV subunit COL4A2 showed a similar peaking pattern as the other collagens ending at 50% of their basal level on day 7.

The variation in the results of the above studies may be ascribed to differences in protein preparation methods and in the ways that the data are quantified. In keeping with the biphasic pattern of collagen synthesis during differentiation of mouse preadipocytes, Nakajima et al. [18] reported the remodeling of collagen V and VI matrices using immunohistochemical staining of BIP cells with distinct ECM networks at days 4 and 10 of differentiation. What can be concluded is that, in the first days after the induction of adipogenesis, ECM dynamics reaches one of its highest levels with increasing production of components of the basal lamina. In line with the biphasic production of ECM components, the two phases of adipogenesis seem to be an early phase of commitment and differentiation, and a subsequent phase of cell growth characterized by massive fat storage.

When in 1974 Green and Meuth [19] reported the selection of the 3T3-L1 cell-line from Swiss mouse embryos, they measured proline hydroxylation and from that calculated the extent of collagen synthesis. The high level of collagen synthesis confirmed the fibroblast origin of those cells. During this assay, an interesting comparison was made between 3T3-L1 and another clone, 3T3-M2. The latter did not store fat and at the same time had a 50% lower rate of proline hydroxylation. This raises the question, whether collagen synthesis is a prerequisite for differentiation 

and fat storage. Experimental evidence exists in support of such a control mechanism.

Using ethyl-3,4-dihydroxybenzoate (EDHB) as a specific inhibitor of collagen synthesis, it has been shown that, in TA1 cells, preadipocytes derived from mouse 10T1/2 embryonic cells, differentiation and triglyceride accumulation were blocked in a dose-dependent manner [20]. This effect was only obtained when EDHB was added to preadipocytes but not when added to already differentiating cells. This suggests that collagen synthesis may serve different purposes during the early and late stages of adipocytes development. Early collagen synthesis seems a permissive requirement for the onset of differentiation, whereas late collagen synthesis may be more related to specific functions of the adipocytes and the adipose tissue, such as providing stability to the fat-storing cells. Such a requirement of collagen synthesis for cell differentiation may well be a general phenomenon and is not confined to preadipocytes [21].

Also in BIP cells, both collagen synthesis and triglyceride accumulation could be inhibited in a dose-dependent manner by EDHB [22]. However, a stage-dependency was not observed.

Looking in detail at collagens I–VI, the synthesis of all collagens except type III collagen was significantly affected with the most dramatic effects on collagens IV–VI. Interestingly, based on measurements of triglyceride accumulation, the inhibitory effect of EDHB on BIP cells could be reversed to a significant extent by culturing EDHB-treated cells in wells coated with collagen V or VI. This indicates that particularly the synthesis of collagens V and VI is important for the collagen-dependent initiation of preadipocyte differentiation and triglyceride storage.

and fat storage. Experimental evidence exists in support of such a control mechanism.

Using ethyl-3,4-dihydroxybenzoate (EDHB) as a specific inhibitor of collagen synthesis, it has been shown that, in TA1 cells, preadipocytes derived from mouse 10T1/2 embryonic cells, differentiation and triglyceride accumulation were blocked in a dose-dependent manner [20]. This effect was only obtained when EDHB was added to preadipocytes but not when added to already differentiating cells. This suggests that collagen synthesis may serve different purposes during the early and late stages of adipocytes development. Early collagen synthesis seems a permissive requirement for the onset of differentiation, whereas late collagen synthesis may be more related to specific functions of the adipocytes and the adipose tissue, such as providing stability to the fat-storing cells. Such a requirement of collagen synthesis for cell differentiation may well be a general phenomenon and is not confined to preadipocytes [21].

Also in BIP cells, both collagen synthesis and triglyceride accumulation could be inhibited in a dose-dependent manner by EDHB [22]. However, a stage-dependency was not observed. Looking in detail at collagens I–VI, the synthesis of all collagens except type III collagen was significantly affected with the most dramatic effects on collagens IV–VI. Interestingly, based on measurements of triglyceride accumulation, the inhibitory effect of EDHB on BIP cells could be reversed to a significant extent by culturing EDHB-treated cells in wells coated with collagen V or VI. This indicates that particularly the synthesis of collagens V and VI is important for the collagen-dependent initiation of preadipocyte differentiation and triglyceride storage.

The importance of the ECM for the function and survival of the adipocytes was demonstrated when we investigated protein-dynamics in mature non-dividing 3T3-L1 adipocytes by stable isotope protein labeling [23]. Among the limited number of examined proteins in this study there were three ECM components, COL1A1, COL1A2, and calreticulin. Both collagens showed the highest rate of labelling among all investigated

proteins. Moreover, the calculated isomer-exchange rate for COL1A1 and COL1A2 was 2:1, exactly the ratio by which they constitute the triplex collagen type I fibers indicating that the synthesis of the collagen I subunits is coordinated. Lower, but still considerable, labeling was seen for calreticulin and for the ECM processing enzyme protein disulfide isomerase (PDIA1 and PDIA3). What can be learned from this is that there is a constant turnover of the adipocyte ECM even in mature cells. Apparently adipocytes spend a lot of attention and, likely, a lot of metabolic energy on the maintenance of their ECM. It should be kept in mind, however, that the high collagen synthesis may be an effect of two-dimensional cell culture and may not accurately reflect the in vivo situation.

The relatively high degree of collagen replacement in mature adipocytes suggests that the ECM is under constant turnover, which is mediated by enzymes promoting construction of the ECM and enzymes involved in its degradation. Accordingly, for mature adipocytes, there has to be a balance between those processes, whereas during preadipocyte differentiation this balance is shifted towards the constructive factors. In Table 2, processing enzymes are listed that have been detected at the protein level. The constructive enzymes fall into two classes, the intracellular enzymes involved in processing of precursors of ECM-proteins and the extracellular inhibitors of the degrading enzymes. The degrading enzymes by themselves belong to either of two systems [24], the fibrinolytic system and the matrix metalloproteinases (MMPs). Although adipocytes express these enzymes, the contribution from the stromal vascular fraction can be significant [25].

The intracellular maturation of newly synthesized ECM-proteins takes place in the ER. There, the proteins undergo biochemical modification of amino acid side-chains. The ECM proteins then undergo processing by proteolytic cleavage before they are assembled into the ECM network. For collagen subunits, this can involve proline- and lysine-hydroxylation and glycosylation and removal of the N- and C-terminal peptides by procollagen-N- and -C-collagenase, respectively. After processing, proteins are self-assembled into monomers depending on the type of collagens and are then secreted. Experiments have shown how crucial can be the role of intracellular processing enzymes. Ibrahim et al. [20] demonstrated the inhibitory effect of EDHB on collagen protein synthesis, but at the same time reported that early administration of EDHB did not influence the mRNA level of collagen 6A2. Further, Nakajima et al. [22] showed that EDHB inhibits the assembly of subunits into collagen molecules, which is in keeping with EDHB influencing a post-transcriptional process of collagen formation. EDHB is a structural analog of ascorbate and α-ketoglutarate (2-oxoglutarate), which are both essential cofactors of the enzyme prolyl hydroxylase (P4HA1). Apparently, this enzyme activity is crucial for collagen formation and preadipocytes differentiation. Notably, EDHB is not specific for this particular enzyme but is an inhibitor for the whole family of prolyl hydroxylases [26].

Extracellular procollagen processing involves the cleaving-off of both N-terminal and C-terminal peptides [27]. Procollagen I/II amino propeptide-processing enzyme (ADAMTS-2) cleaves the N-terminal propeptide from the subunits of collagens I and II: COL1A1, COL1A2, and COL2A1. Cleavage takes place after assembly of two 1A1- and one 1A2-chains into a collagen type I monomer before those monomers multimerize into fibrils. The process requires the native conformation of the procollagens in which the N-terminus can adopt a hairpin structure. During the reaction, an intermediate is formed that has lost the N-propeptide from one 1A1-chain and from the 1A2-chain, but still contains the propeptide on the other 1A1-chain. Conformational changes in the N-terminus are suspected to be involved in determining this defined sequential cleavage of the propeptides [28]. Mutations in ADAMTS-2 can give rise to Ehlers–Danlos syndrome type VIIC in humans, a defect of the cartilage. Procollagen amino propeptide-processing activity has also been observed for gene family members of ADAMTS-2. ADAMTS-3 can process procollagen II but not procollagen I. Both N-proteinases lack the activity to process collagen III. Collagen I processing activity has also been observed for ADAMTS-14.

Collagen fibril formation also requires that the C-terminal peptides are removed from the subunits by proteolytic cleavage [27]. For collagens I–III this is done by the enzyme procollagen-C-proteinase, also referred to as mammalian tolloid protein or bone morphogenic protein 1 (BMP1). In contrast to ADAMTS-2, native collagen conformation is not a prerequisite for processing of the C-terminus. In addition to collagens, this enzyme processes the precursors of laminin 5 and biglycan, and of the modifying enzyme lysyl oxidase (LOX). Via alternative splicing, different isoforms of the C-proteinase can be formed [27].

Plasmin is the active component of the fibrinolytic system. It is able to cleave not only fibrin and von Willibrand factor but also some adipocyte ECM components: fibronectin, thrombospondin, and laminin. The activity of plasmin is regulated by several other proteins including the urokinase-type (u-PA) and the tissue-type (t-PA) plasminogen activators and plasminogen activator inhibitors (PAIs) (Fig. 2). The effect of the fibrinolytic system on adipose tissue has been studied in the mouse by abolishing or over-expressing one of those genes, but it is difficult to fit all observations into a uniform effect-model ([24] and references therein). The various findings suggest that this system affects angiogenesis rather than directly influencing ECM remodeling and adipogenesis. Sufficient blood flow by angiogenesis favors adipose tissue formation. Nevertheless, PAI-1 seems able to prevent the degradation of the ECM. Pharmacological inhibition of PAI-1 in different studies with mice on a high fat diet leads to reduced body weight, weight of fat depots, and adipocyte volume [29, 30]. Here, a higher blood vessel density in adipose tissue was observed, but this may be the consequence of the reduced volume of the fat pads.

More than 20 different members of the MMP family have been identified. Although MMPs collectively degrade ECM proteins, according to the UniProt database there is some form of substrate specificity (Table 3). Changing MMP expression in the mouse can influence adiposity. Inactivation of MMP3 on a high-fat diet results in increased development of adipose tissue with a hyperplastic as well as a hypertrophic response [31]. Similar observations were made after inactivation of MMP9 and MMP11 [24, 32]. Contrasting observations have been made with pharmacological inhibition of MMPs in mice on a high-fat diet. Co-inhibition of MMPs 2, 9, and 14 did not influence adipose tissue development [33], whereas the general inhibition of MMP activity led to a reduction in adipose tissue weight [34]. These sometimes controversial results reflect the complexity of MMP activity that depends on relative concentrations of different MMPs, their substrate specificity, and the activity of their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), and perhaps several others [24, 35].

No information can be obtained from the Uniprot database about an MMP that has collagen VI as a substrate, but recent investigations have provided information on this. Studies on breast cancer have indicated that adipocytes may support cancer cells. In this context, a role for collagen VI has been put forward [15]. Also, it was observed that MMP11 is associated with tumor invasion and poor prognosis. Further studies showed that COL6A3 is a substrate of MMP11 [36]. Moreover, MMP11 appears to be a strong negative regulator of adipogenesis [37] in line with the above-mentioned observations in MMP11 deficient mice.

All MMPs are secreted as inactive zymogens and need proteolytic cleavage to become activated by other MMPs or plasmin. The MMP most often reported in proteomics studies is MMP2 cleaving the basal lamina component collagen IV (Table 3). MMP2, as well as MMP13 (collagenase 3), is activated by MMP14 (MT1-MMP). Chun et al. [38] observed that adipocytes from MT1-MMP null-mice were 10×— smaller in diameter than wild-type adipocytes. Knock-out preadipocytes were able to differentiate on planar interfaces but, when cultured in a 3D matrix of collagen I fibrils, they failed to differentiate and did not accumulate triglycerides. The conclusion was that MT1-MMP is necessary for the modulation of rigid pericellular collagen to allow preadipocytes to grow out of the stroma. Observations on vascular smooth muscle cells indicate that MT1-MMP activity is also able to directly remodel the matrix by itself [39].

Four TIMPs (Table 4) have been detected up to now [40]. Like their counter players, the MMPs, TIMPs are regulated during adipogenesis [41]. In relation to adipose tissue development, TIMP1 has been studied the most [42]. A recent report suggests that, beyond direct adipose tissue ECM remodeling, TIMP1 may have an effect on adipose tissue development by acting in the hypothalamus to regulate food intake [43]. Interestingly, TIMP4 is specifically expressed in adipose tissue and adipocytes in humans according to

tissue-wide gene expression analysis of UniGene [44] and GNF Gene Atlas [45], and is highly regulated [46]. However, little is known about its function for ECM remodeling and adipose tissue development. Overall, it seems that the balance between TIMPs and MMPs, or their ratio, is more relevant than the activity of the individual groups of those processing enzymes.

A subfamily of metalloproteinases is the A Desintegrin And Metalloproteinase with Trombospondin motif (ADAMTS family). Members of this family have been found to have procollagen N-proteinase activity as mentioned above, but other members also may influence adipose tissue development. In the mouse, expression of ADAMTS-1, -4, -5, and -8 has been demonstrated in adipose tissue, and their expression level changes during the development of diet induced obesity. Expression seems highest in the stromal vascular fraction, which is in line with a suggested role in early steps of preadipocyte differentiation by the degradation of aggrecan, a chondroitin sulphate/keratan sulphate proteoglycan [47].

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Regulation of ECM dynamicsECM regulation and insulin

Some years ago, we studied the influence of insulin on the secretome of mature 3T3-L1 adipocytes [48]. Insulin induced a clear increase of the mature form of COL1A1, of a COL5A1 fragment, and of the C-terminal peptides of COL1A1, COL1A2, and COL3A1. A comparison with transcriptomics data showed that insulin did not up-regulate transcription of those genes. It was concluded that insulin acts at the level of post-transcriptional processing and secretion. In keeping with this, was the observed increase in relative abundance of PCOLCE, procollagen C-endopeptidase enhancer protein, that can activate the C-endopeptidase by 20-fold. Remarkably, we observed a significant up-regulation of the mRNA levels for various matrix protein processing enzymes including sulfatase 2 and prolyl hydroxylase, and a trend for increase of the mRNA for PCOLCE. Altogether, our data indicate that insulin up-regulates the transcription of genes for protein-modifying and -processing enzymes and in that way stimulates the formation of ECM-components and the ECM as a whole. This activity seems not confined to the ECM components, as a considerable increase of other secreted proteins was also observed without concomitant increase of their mRNA levels including adipsin and complement C3 [48]. In general, the observed effects of adding insulin to the cell culture medium were even stronger when the experiments were done in the presence of extra glucose. The experiments leading to the above effect of insulin activity mimic an acute increase in insulin on mature adipocytes. For preadipocytes in vitro, insulin is a potent stimulator of differentiation and triglyceride storage in line with its general anabolic role.

The detection of up-regulated expression of the gene for sulfatase 2 indicates that insulin changes the level of 6-O-sulfation of heparan sulfate proteoglycans (HSPGs) [48]. Wilsie et al. [49] detected a clear influence of HSPGs on lipid uptake in 3T3-L1 adipocytes. They propose a model in which HSPG serves as a binding site for lipid particles (apoE-VLDL). As such, a reaction center is created for the rapid hydrolysis of triglycerides from the particles by lipoprotein lipase and for the delivery of the fatty acids to the membrane bound fatty acid transporters. Alternatively, lipid particles are internalized together with HSPGs at which they have accumulated. In this way, the ECM plays a crucial role in lipid uptake which is in some way regulated by insulin.

ECM regulation and energy metabolism

When preadipocytes differentiate and cells begin to store fat, their ECM adopts a basal laminar structure. Uptake and storage of fatty acids depends on gene regulation through the activity of the nuclear receptor PPARγ in combination with another nuclear receptor, RXR. The parallel fat-storage and ECM development suggests that both processes might be concertedly regulated by PPARγ. However, not manygenes for ECM components or their processing enzymes have been shown to be direct targets of PPARγ. Suggestive results have been obtained for COL6A3 and thrombospondin 1 [50]. A specific responsive element for binding PPARγ exists in the promoter of the rodent gene for MMP1 [51]. When rosiglitazone, a strong agonist of PPARγ, was added to mature 3T3-L1 adipocytes, the cells lost 10% of their triglycerides. At the same time, a down-regulation was observed of the expression of genes for matrix proteins and various processing enzymes [52]. This suggests that reduction of the net triglyceride content of the cell maybe accompanied by reduction of the cell volume, while the building-up of the ECM is slowed down via transcriptional down-regulation. However, this effect seems not confined to ECM-proteins but also applies to other secreted proteins.

As mentioned above, an important way to control the turnover of the ECM in mature adipocytes is by regulating the processing enzymes for the synthesis and the breakdown of ECM proteins. An interesting protein complex in this respect is the enzyme prolyl-4-hydroxylase, which is involved in the modification of collagens necessary for their assembly into collagen monomers and fibrils. It is a tetramer of two alpha-1 chains (P4HA1) and two beta subunits, also referred to as protein disulfide isomerase (P4HB, PDI). The location of this complex is most likely the ER where the alpha-chains of the enzyme complex convert proline residues in collagens to 4-hydroxyproline. It requires vitamin C (ascorbic acid) as a cofactor supplying Fe2+for the reaction. In addition, the reaction requires O2

and α-ketoglutarate and converts them to CO2 and succinate, respectively. In principle, this conversion might be regarded as a shunt in the TCA cycle (Fig. 3) revealing that one proline

hydroxylation costs an energy equivalent of four ATPs (one GTP and one NADH). Apparently, collagen synthesis is a considerably energy-costing process. In fact, an exchange of metabolites between mitochondrion and ER as in the presumed shunt is not unlikely. For rat pancreatic islet mitochondria, it was shown that α-ketoglutarate is a metabolite of the cataplerosis [53]. Moreover, the mitochondria and ER can establish a direct interaction mediated by specific contact-proteins [54]. It was observed that pyruvate enhances the secretion of α-ketoglutarate from the mitochondria by fourfold [53]. In this respect, collagen synthesis might be influenced by the pyruvate concentration serving as a substrate for α-ketoglutarate synthesis and promoting its secretion from the mitochondria. Also, the efficacy of the reaction, and thereby of ECM and adipocyte development, seems to depend on the oxygen supply to the tissue. It is tempting to speculate that this enzyme complex is a site for oxygen sensing as has been shown for other members of the gene family, HIF-prolyl hydroxylase [55, 56]. Various regulatory aspects of the collagen proline hydroxylation are summarized in Fig. 3.

Fig. 3

Summary of the factors that influence the activity of the prolyl hydroxylase/protein disulfide isomerase (PH/PDI) enzyme complex

Protein disulfide isomerase (PDI), the molecular partner of prolyl hydroxylase, functions by creating and rearranging disulfide bonds during protein folding in the ER. In line with its catalytic properties, the activity of PDI seems to depend on the redox 

status in the cell [57, 58]. The folding of collagens into the proper structure needed for cleavage of the N-terminal peptide is probably just one of the numerous sites of action of this enzyme during formation of ECM components. Not only structural ECM components but also modifying enzymes like TIMP1 and TIMP2 rely for their activity on disulfide bonds. Through enzymes like PDI, the redox status of the cell and in particular the NADH/NAD+ ratio may have a strong influence on the turnover of the adipocyte ECM.

ECM regulation and mechanical forces

Although one of the functions of the ECM of adipocytes is the protection of the cell against disruption by mechanical forces, this aspect has not been much studied. When 3T3-L1 pre-adipocytes are subjected to mechanical stretching, this results in the inhibition of differentiation. A down-regulation of the expression of the PPARγ2 gene is observed, most likely mediated by the MAPK/ERK pathway [59]. It suggests that, when external forces are sensed, cells may spend their energy more on ECM remodeling, promoting survival than on

triglyceride storage. Stretching may also prevent the required change in morphology during differentiation. A relation between cell shape and the potential to differentiate was already demonstrated in 1983 by Spiegelman and Ginty [60]. Allowing 3T3-F442A cells to spread on a fibronectin-coated surface inhibited differentiation, whereas this effect was not seen when cells were allowed to maintain a spherical shape.

An interesting observation showing the involvement of the ECM in cell morphology development was made in MT1-MMP (MMP14) null mice [38]. Those mice are lipodystrophic, and their pre-adipocytes in an in vitro 3D-culture system adopt a different cell shape as cells from wild-type mice. A down-regulation of CREB and phosphorylated CREB was noticed. It seems as if the cells have lost their capacity to interact with surrounding collagen fibers and thereby lost the traction forces necessary to change shape. In summary, external mechanical forces on the cellinfluence its power to differentiate and function properly, whereas traction forces from the cell to the surroundings are necessary for cell morphology development. The ECM is an intermediate in both processes.

Other regulators of ECM

Besides insulin, other hormones, including leptin [61], angiotensin II [62], and estrogen [63], have been addressed to the development of the ECM in other organs but not in adipose tissue. Since adipose tissue expresses these hormones and also their receptors, it can be expected that the adipose ECM may also be under the regulation of these hormones, possibly via autocrine and paracrine action. While leptin and angiotensin II stimulate ECM production via TGF-β, estrogen protects ECM by reducing MMPs and increasing TIMPs.

The sympathetic nervous system can also be involved in the regulation of ECM dynamics. The study of vascular smooth muscle shows that stimulating α1- and β1/β2-adrenergic receptors induce and inhibit ECM production, respectively, via TGF-β [64]. Although it has not yet been addressed directly in adipose tissue, similar mechanisms may operate through its α2- and β-receptors that play a major role in the control of lipolysis.

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Adipocyte ECM, weight regulation and obesityOne of the main functions of adipose tissue is the temporary storage of fat as triglycerides. In case of a positive energy balance, preadipocytes develop into adipocytes, during which cell shape dramatically alters. This process depends on changes in the ECM. As outlined in the previous paragraphs, adipogenesis can be divided in roughly two phases: a first phase in which the cells are committed to triglyceride storage, and a second phase that relates more to the vast increase in the amount of triglycerides accompanied by rounding and growing of the cells.

If mature adipocytes take up more fat, the cell volume increases and so should the ECM [65]. In obese subjects, many of the adipocytes are hypertrophic and the question arises if and how the ECM can cope with this. It is tempting to propose that there is a physical limit to adipocyte growth and that this limit is determined by the ability of the cell to keep the ECM in such a condition that it can protect the cell against disruption. An emerging view is that hypertrophy of adipocytes prevents proper oxygen supply to the cell contents creating a state of hypoxia [66]. Lack of sufficient oxygen will induce apoptosis of some of the cells, which then attracts macrophages and generates a state of inflammation in the tissue that may be part of the pathogenesis of obesity-related disorders like type II diabetes. Not much is known about the mechanistic consequences of hypoxia, but the ECM may be involved. Hypoxia will undoubtedly induce a change in cellular redox status and, as mentioned above, this will lead to malfunctioning of ECM-protein processing enzymes like lysyl oxidase and prolyl-4-hydroxylase, the crucial enzyme for collagen synthesis. In obese insulin-resistant persons, proline hydroxylation may be inhibited even more because of a decreased supply of pyruvate as input for the TCA cycle. Under those conditions, one might expect a destabilization of the ECM with dramatic consequences for the ever-growing adipocytes. As such, the ECM may be an important player in the etiology of type II diabetes. Culturing immortalized or primary rodent adipocytes under high glucose/high insulin concentration or inducing insulin resistance in those cells by the addition of PUGNAc [67] leads to altered composition of the ECM with changes in the relative abundance of several ECM proteins (laminin β-1 chain, spondin 1, fibulin 2) and matrix processing enzymes (peptidyl–prolyl cis–trans isomerase B, MMP2, TIMP2) [11]. Khan et al. [16] also reported that the metabolic dysregulation of the body in the diabetic state is linked to changes of the adipocyte ECM. Allowing their stress-free expansion by knocking-out collagen VI improved whole body energy homeostasis.

Not much is known about the dynamics of the ECM during a negative energy balance. Yet this is important since, for the obese, the loss of fat mass is a preferred way to lower the risk for complications. When 3T3-L1 adipocytes are starved, they readily lose about 46% of their fat, but then this process comes to a stop, which can only be overcome by additional measures. Supplementation of the culture medium with TNFα leads to a further reduction of the fat content to 77% [68]. One could speculate that the loss of triglycerides decreases the volume of the cell creating stress between the cell contents and the surrounding ECM. This would require ththe ECM to remodel. However, shrinkage of the ECM may be hard or even impossible. In either case, stress will build up between cell and ECM, and this may inhibit lipolysis explaining why fat loss from adipocytes cannot reach completion. Indeed, 10% fat loss by rosiglitazone treatment was paralleled by transcriptional down-regulation of synthesis and processing of ECM proteins [52].

Inflammation is linked to obesity. Adipocytes in the obese increase their cytokine production and attract macrophages, which contribute to an enhanced systemic inflammation status. On the other hand, macrophages stimulate adipose tissue remodeling by macrophage-released MMP [69] and/or induced adipocytes-expressed MMPs [70], and may as such change adipocyte functioning.

Several other studies indicate a link between the ECM and weight regulation. Knocking-out collagen VI in the ob/ob mouse decreased body weight due to reduced fat mass at young age [16]. SPARC-null mice that exhibit increased adiposity [71] display a strong influence on the ECM conversion from a fibronectin-rich stroma to a laminin-rich basal lamina [72]. Human proof comes from drug treatment. HIV 1-infected patients receiving protease inhibitors as anti-viral therapy often develop peripheral fat wasting with difficulties in adipogenesis and fat accumulation [73]. This treatment was shown to change the activity of the endogenous proteases MMP2 and MMP9 as well as of other processing enzymes [74]. It suggests that adipogenesis and adipocyte stability may be affected by interfering with ECM dynamics. At the epidemiological level, increased plasma concentrations of MMP2 and MMP9 have been reported in obese adults [75], and increased levels of MMP9 and TIMP1 in obese children [76]. Plasma levels of TIMP1 have been found to be associated with adiposity in humans [42]. A genetic study with polymorphisms in the promoter region of the MMP1 gene among Korean subjects resulted in a significant association with BMI [77].

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SummaryAlthough many observations are from in vitro experiments, it seems reasonable tostate that two phases can be distinguished during adipogenesis. In the first phase, preadipocytes become committed to the storage of triglycerides. Synthesis of laminar collagens and of other components of the basal lamina is increased while the network of fibrillar collagens around the cell is maintained. In this phase, blocking prolyl-4-hydroxylase prevents differentiation and triglyceride storage. Mediated by a complex set of processing enzymes of which the activity is neatly regulated, the developing cell becomes embedded in the basal lamina. During the second phase, the adipocyte accumulates vast amounts of triglycerides accompanied by a change in cell shape and cell growth. Traction forces between cell and the surrounding meshwork mediated by the ECM are necessary for this process to occur. A situation is reached in which the fibrillar network functions as an outer skeleton protecting the adipocyte from mechanical disruption, and in which the laminar scaffold reduces external forces by spreading them over the tissue. Consequently, mature adipocytes spend considerable energy in maintaining and renewing their fibrillar network. ECM formation is stimulated by insulin through the transcriptional up-regulation of genes for processing enzymes. Further, being the source of pyruvate, glucose supports collagen synthesis via

stimulation of the prolyl-hydroxylase/PDI complex. Other control conditions are the redox status and the supply of oxygen. In obesity, hypoxia may negatively influence ECM maintenance leading to ECM instability, cell death, and the attraction of macrophages. Alternatively, hypertrophy of adipocytes may be directly linked to ECM instability. Loss of fat mass from adipocytes during calorie restriction seems limited. This may be the consequence of the stress build-up between the shrinking cell volume and the rigid ECM, but this hypothesis still needs experimental proof. It is clear, however, that the ECM plays an important and often ignored role in adipocyte development and function and as such in lipid metabolism, weight regulation, and obesity.

Acknowledgment

We thank Dr. J. Broers for his assistance in immunohistology of (pre)adipocytes.

Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

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Articles from Springer Open Choice are provided here

PubMed articles by these authors

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Springer

The Medical Risks of Obesity

Xavier Pi-Sunyer, MD1

1Division of Endocrinology, Diabetes and Nutrition, St. Luke's-Roosevelt Hospital Center, Columbia University, New York, NY

Correspondence: Xavier Pi-Sunyer, MD, Division of Endocrinology, Diabetes, and Nutrition, St. Luke's-Roosevelt Hospital Center, 1111 Amsterdam Avenue, Room 1020, New York, NY 10025. Tel: 212-523-4161 Fax: 212-523-4830 ; Email: fxpI@columbia.edu

Obesity is at epidemic proportions in the United States and in other developed and developing countries. The prevalence of obesity is increasing not only in adults, but especially among children and adolescents. In the United States in 2003 to 2004, 17.1% of children and adolescents were overweight, and 32.2% of adults were obese. Obesity is a significant risk factor for and contributor to increased morbidity and mortality, most importantly from cardiovascular disease (CVD) and diabetes, but also from cancer and chronic diseases, including osteoarthritis, liver and kidney disease, sleep apnea, and depression. The prevalence of obesity has increased steadily over the past 5 decades, and obesity may have a significant impact on quality-adjusted life years. Obesity is also strongly associated with an increased risk of all-cause mortality as well as cardiovascular and cancer mortality. Despite the substantial effects of obesity, weight loss can result in a significant reduction in risk for the majority of these comorbid conditions. Those comorbidities most closely linked to obesity must be identified to increase awareness of potential adverse outcomes. This will allow health care professionals to identify and implement appropriate interventions to reduce patient risk and mortality. A systematic search strategy was used to identify published literature between 1995 and 2008 that reported data from prospective longitudinal studies of obesity and comorbid medical conditions. This article will review evidence for significant associations of obesity with comorbidities to provide information useful for optimal patient management.Keywords: cancer, cardiovascular disease, diabetes, obesity, cornorbidity, mortality

Obesity, defined as a body mass index (BMI) ≥ 30 kg/m2,1,2 is a medical condition encountered daily by physicians throughout the United States. The prevalence of obesity is increasing and reaching epidemic proportions (Figure 1),3 although recent data suggest that the prevalence is leveling off among children and adolescents.4From 2003 to 2004 in the United States, 32.2% of adults were obese and 17.1% of children and adolescents were overweight.5

Figure 1

Trends in obesity among US adults over 17 years (1990–2007).3

A major concern for physicians who care for patients who are overweight or obese is the high risk of accompanying comorbid disorders, such as diabetes, cardiovascular disease (CVD), and cancer. An important need for primary care physicians is to identify these comorbidities and the resulting adverse outcomes. Awareness of the disorders with the strongest associations with obesity is important to allow early diagnosis and treatment of these conditions, and to identify the patients most likely to benefit from weight loss. This will allow early identification and assessment of risk so that appropriate interventions can be implemented to reduce risk and mortality.

This article will review significant associations of obesity with comorbidities to provide the clinician with the information necessary to offer optimal patient management. A systematic search strategy was used to identify English language articles cited in PubMed between 1995 and 2008 that reported data from prospective, longitudinal studies of obesity and comorbid medical conditions. Relevant citations were comprehensively reviewed to determine those that confirmed a significant relationship between obesity and comorbid conditions.

Numerous large, long-term epidemiological studies have shown that obesity is strongly associated with an increased risk of all-cause, cardiovascular, and cancer mortality (Figure 2).6 In the National Health and Nutrition Examination Study (NHANES) III, obesity was associated with an increased prevalence of type 2 diabetes, gallbladder disease, coronary heart disease (CHD), hypertension, osteoarthritis (OA), and high blood cholesterol among > 16 000 participants.7 Results from other studies have shown a strong association between obesity and the prevalence of comorbid illnesses, health complaints, and physical disability.8–11 A 10-year follow up from Nurses’ Health Study (N > 121000) and the Health Professionals Follow-up Study (N > 51000) evaluated the risk of diabetes, gallstones, and hypertension in obese (BMI ≥ 30 kg/m2) men and women compared with those with a normal BMI.12 The risk of diabetes, gallstones, and hypertension was increased in women, while the risk of diabetes, gallstones, hypertension, heart disease, and stroke was increased in men. Based on the available data, The Obesity Society concluded that obesity is causally associated with functional impairment and reduced quality of life, serious disease, and greater mortality.13

Figure 2

The association between obesity and comorbid conditions, which will be discussed in the section that follows, is illustrated in Figure 3, where chronic conditions such as kidney disease, OA, cancer, diabetes, sleep apnea, nonalcoholic fatty liver disease (NAFLD), hypertension, and most importantly, CVD, are directly related to obesity.11,12 Further, many of these comorbidities also are directly associated with CVD.

Figure 3

Association of obesity and important comorbidities.

Many of the epidemiological studies have been confirmed by observations that weight loss improves patient outcomes. The results from the Swedish Obesity Study showed that weight loss from bariatric surgery reduced most cardiovascular risk factors.11 An American Heart Association Committee also concluded that weight loss and physical activity could prevent and treat obesity-related CHD risk factors.14Data reporting that a specific outcome is improved by weight loss will also be reviewed.

  Other Sections ▼

Obesity and ComorbidityDiabetes

The long-term risk of type 2 diabetes increases significantly with increasing weight. In the Nurses’ Health Study, the effect of weight change on the risk for clinical diabetes was evaluated in 114281 women.15 After adjusting for age, body weight was the major risk factor for diabetes during a 14-year follow-up. Among women with a 5- to 7.9-kg weight gain, the relative risk for diabetes was 1.9 and for those with an 8- to 10.9-kg weight gain, the relative risk was 2.7. In contrast, a 5-kg weight loss resulted in a 50% reduction in the risk of diabetes.

Consistent with this observation, several studies have shown that weight loss is associated with a significant reduction in the risk of diabetes.16 In a prospective, 20-year study of 7176 British men, the rate of new diabetes was 11.4 per 1000 person-years among obese subjects versus 1.6 among normal-weight subjects (P < 0.0001), but the effect of weight change during a 5-year follow-up on the development of diabetes found a relative risk of

0.62 among those losing weight compared with 1.0 for stable weight and 1.76 among those gaining > 10% body weight (P < 0.0001). Similarly, a Health Technology Assessment that examined the effect of weight loss in patients with diabetes found significant improvement in the risk of developing didiabetes.17 Long-term weight loss was also associated with a reduction in the risk of type 2 diabetes in the Diabetes Prevention Program.18 Thus, despite the known risk of type 2 diabetes associated with obesity, weight loss has the potential to improve outcomes.

Weight loss was also associated with improved diabetes control in the Look AHEAD (Action for Health in Diabetes) study19 Look AHEAD is a randomized trial of intensive lifestyle intervention versus usual support and education in 5145 patients with type 2 diabetes andBMI > 25 kg/m2. The intensive group lost 8.6% of body weight compared with 0.7% in supportive group (P < 0.001). At 1 year, intensive intervention resulted in clinically significant weight loss in people with type 2 diabetes, which was associated with improved diabetes control and reduction in CVD risk factors and medication use.

Cardiovascular Disease

Obesity is an independent risk factor for CVD, defined as including CHD, myocardial infarction (MI), angina pectoris, congestive heart failure (CHF), stroke, hypertension, and atrial fibrillation.7,14 Overall, results from large prospective and observational studies confirm the marked adverse effects of obesity on CVD.

Numerous large-scale, long-term studies in the United States have investigated the role of obesity in CVD risk and on the development of CVD. The Multiethnic Study of Atherosclerosis evaluated the effects of obesity on CVD risk factors and on subclinical signs of CVD in 6814 participants who were free of CVD at baseline.20Hypertension and diabetes as well as subclinical cardiovascular findings were more prevalent in obese (BMI ≥ 30 kg/m2) than nonobese participants. In addition, data collected from the original cohort of 5209 participants of the Framingham Heart Study over 44 years were used to evaluate the effect of obesity (BMI ≥ 30 kg/m2) on the risk of CVD (angina, MI, CHD, or stroke), diabetes, hypertension, and hypercholesterolemia.21 During follow-up, the age-adjusted relative risk for CVD was 1.46 in men and 1.64 in women, and the age-adjusted relative risk for hypertension was even higher among obese men and women (2.21 and 2.75, respectively). In a separate analysis of the Framingham Heart Study, the lifetime risk of CVD was assessed among obese men and women with diabetes versus nonobese subjects.22 During a 30-year follow-up, the risk of CVD was 54.8% in normal-weight women versus 78.8% among obese women with diabetes and 78.6% versus 86.9% among normal and obese men with diabetes, respectively.22

Similar results have been obtained in studies performed outside of the United States. The International Day for the Evaluation of Abdominal Obesity (IDEA) study evaluated waist circumference, CVD, and diabetes mellitus in 168000 primary care patients in 63 countries.23 Overall, 24% of men and 27% of women were obese, and the risk of CVD and diabetes was strongly associated with BMI and waist circumference. Lastly, among 7176 British men followed for 20 years, the rate of major CVD was 24.9/1000 in obese (BMI ≥ 30 kg/m2) subjects versus 13.9/1000 among normal-weight (BMI < 25 kg/m2) subjects.16

Results from the Framingham Heart Study also showed that obesity increases the risk of atrial fibrillation.24 Among 5282 participants (of whom 55% were women) without atrial fibrillation at baseline, subjects were classified as normal (BMI < 25 kg/m2), overweight, and obese (BMI ≥ 30 kg/m2). During a mean follow-up of 13.7 years, a 4% increase in risk of atrial fibrillation/1-unit increase in BMI was observed in men and women after adjustment for cardiovascular risk factors. Compared with normal-weight individuals, in obese subjects the hazard ratio for atrial fibrillation was 1.52 for men and 1.46 for women.

AmoAmong 111847 patients with non–ST-segment myocardial infarction (NSTEMI) who were included in the CRUSADE registry,25 excess BMI was also strongly associated with an earlier age of first NSTEMI. The registry collected data from January 2001 to January 2007 in high-risk patients with unstable angina and NSTEMI. Extreme obesity (BMI > 40 kg/m2) had the strongest association with age at first MI after adjustment for baseline factors. After adjustment for baseline demographic data, cardiac risk factors, and medications, the first NSTEMI occurred 3.5, 6.8, 9.4, and 12 years (P < 0.0001) earlier with increasing adiposity (BMI 25.1–30 kg/m2, 30.1–35 kg/m2, 35.1–40 kg/m2, and > 40 kg/m2, respectively).

Hypertension, a risk factor for CVD, is related to obesity. An analysis from the Women's Health Study found a significant association between obesity, the development of hypertension, and diabetes.26 In this analysis of 38172 women who were free of diabetes and CVD at baseline with a mean 10.2 years of follow-up, the age-adjusted incidence rate/1000 of diabetes in obese women (BMI ≥ 30 kg/m2) was 7.58 among normotensive patients (120/75) versus 20.53 among hypertensive patients. Further, a significant association between BMI and hypertension was observed in a prospective study from Norway, the Nord-Trondelag Health Study.27Among> 15900 women and > 13800 men at least 20 years old without hypertension, diabetes, or CVD at baseline, the risk for hypertension was increased ≥ 1.4-fold among men and women whose BMI increased from baseline compared with those who maintained a stable BMI.

Metabolic Syndrome

A combination of commonly associated cardiovascular risk factors is known as metabolic syndrome (MetS). Metabolic syndrome represents a group of cardiometabolic risk factors

that include abdominal obesity combined with elevated blood pressure, fasting plasma glucose, and triglycerides, and reduced high-density lipoprotein cholesterol levels. Metabolic syndrome is associated with an increased risk of cardiovascular mortality.28 Guidelines for the diagnosis and management of MetS are available from a number of professional organizations including the American Heart Association and the International Union of Angiology.29,30

As discussed previously, abdominal obesity, a key part of the constellation of risk factors for MetS, is strongly associated with the risk of diabetes.31,32 An analysis of the associations between risk factors for MetS in 2735 participants from the Dallas Heart Study showed that higher BMI was significantly associated with MetS in both diabetic and nondiabetic patients.31 In a prospective cohort study that examined the association between MetS and type 2 diabetes among 4022 patients with atherosclerosis, abdominal obesity was the component most strongly associated with the risk of type 2 diabetes.32 Data from 9 European studies were examined to determine the association between MetS and abdominal adiposity in > 15000 men and women.28 The definition of MetS was satisfied in 41 % of men and 37.9% of women, and those with MetS were more often obese and had a higher prevalence of diabetes than nonobese participants. A prospective study of 3051 elderly men with diabetes or CHD also found that obesity and physical inactivity as well as cigarette smoking and high carbohydrate diet were significantly associated with a greater risk of MetS.33

In contrast, weight reduction alone or combined with lifestyle intervention is associated with a significant reduction in the prevalence of MetS.34,35 The prevalence of MetS and abdominal obesity was significantly reduced from 74% to 58% in a lifestyle intervention group versus to 67.7% in a standard care group (P = 0.025).34 In a separate study, a moderate 8-kg reduction in weight after 1 year resulted in a significant (P < 0.05) reduction in the prevalence ofMetS from 35% to 27%.35

Cancer

A number of large-scale, prospective studies have confirmed a significant association between obesity and cancer. The strongest association is between an elevated BMI and cancer risk. A prospective cohort study in the United States found a significant association between obesity and cancer.36 This prospective study involved > 900000 subjects from the American Cancer Prevention Study II who were free from cancer in 1982 and had a mean follow up of 16 years. Among those with a BMI ≥ 40 kg/m2, mortality from all causes of cancer was 52% higher in men and 62% higher in women compared with those with a normal BMI. Body mass index was also significantly associated with higher rates of death

due to cancer of the esophagus, colon and rectum, liver, gallbladder, pancreas, kidney, non-Hodgkin lymphoma, and multiple myeloma.

In the Million Women Study from the United Kingdom, increasing BMI was associated with a significant increase in risk for 10 out of 17 of the most common types of cancer.37 Over 1.2 million UK women, aged 50 to 64 years during 1996 to 2001, were recruited into the Million Women Study and followed for a mean of 5.4 years for cancer incidence and 7 years for cancer mortality. Increasing BMI was associated with an increased incidence of all cancers combined in addition to endometrial cancer, adenocarcinoma of the esophagus, kidney cancer, leukemia, multiple myeloma, pancreatic cancer, non-Hodgkin lymphoma, ovarian cancer, breast cancer in postmenopausal women, and colorectal cancer in premenopausal women (Figure 4).

A prospective study evaluated the effect of BMI and weight gain on prostate cancer incidence and mortality among 287700 men in the NIH-AARP Diet and Health Study.38 During a mean follow-up of 5 to 6 years, the relative risk for mortality from prostate cancer was 1.46 and 2.l2 for a BMI ≥ 30 kg/m2 and ≥ 35 kg/m2, respectively. In a separate study of 69991 men, the risk of high-grade nonmetastatic and metastatic prostate cancer was increased with obesity (1.2- and 1.5-fold, respectively), and the risk of high-grade nonmetastatic cancer was reduced to 0.58 with > 11-lb weight loss.39

In the Health Professionals Follow-up Study, a significant association between obesity and colon cancer was observed in men.40 This 18-year, prospective follow-up study of 46349 men who were cancer-free at baseline found a multivariate hazard ratio (HR) for colon cancer was increased at a BMI > 22.5 kg/m2, but was highest (HR, 2.29) at a BMI > 30 kg/m2. An estimated 30% of all colon cancer cases were attributed to overweight and obesity.

Pischon et al41,42 evaluated the association between the risk of colon and rectal cancer and renal cell carcinoma and body weight in the European Prospective Investigation into Cancer and Nutrition (EPIC) study. More than 368000 men and women who were cancer-free at baseline were followed for a mean of 6.1 years in the EPIC Study. Body weight and BMI (≥ 29.4 kg/m2) were significantly associated with the risk of colon cancer in men but not women (relative risk [RR], 1.55; P = 0.006). Among 348500 men and women with a 6-year follow-up, the RR for renal cell carcinoma associated with increased BMI in women was 2.25 (P = 0.009; BMI ≥ 29 kg/m2) but no significant increase was observed for men (RR, 1.22; P = 0.51).

Arthritis and Disability

Osteoarthritis has a major impact on patient mobility, disability, lost productivity, and patients may become disabled from OA early in life.43,44 Obesity is strongly associated with an increased risk of OA of the knee but only a moderate association with OA of the hip has been found.45 Because OA strongly impacts patient lifestyle and function, it is important to recognize this effect of obesity and the potential need for weight loss and rehabilitation.

The relationship between OA of the hip and knee and obesity was examined in The Rotterdam Study.45 Radiographic confirmation of OA was established in 3585 participants at baseline, and patients were followed for a mean of 6.6 years. A BMI > 27 kg/m2 was associated with a 3.3-fold greater risk of OA and progression of OA of the knee but not the hip. In a longitudinal study of 715 women in the Chingford population over 4 years, mean age 54 years at baseline, those in the top BMI tertile had an increased risk of knee OA compared with women in lower BMI tertiles.46 In The Framingham Heart Study, the effect of obesity on the increased risk of knee OA was determined in elderly patients without knee OA at baseline.47 Among 598 patients who developed OA over a 1O-year follow-up, the risk for OA was increased by 1.6 for each 5-unit increase of BMI.

The association between obesity and OA of the knee is thus widely recognized. A 

number of prospective studies have examined the relationship between obesity and disability in patients with knee OA.48–52 A prospective cohort study of 5784 participants at least 50 years old was conducted to examine the effect of obesity on knee pain and disability.49 Obesity accounted for a substantial proportion of severe disabling knee pain in this cohort, and the authors concluded that health interventions to avoid obesity would have a major impact on improving disability associated with knee OA. Another cross-sectional study of 3664 participants > 25 years old found that obesity was associated with a higher risk of OA of the hip or knee, chronic pain, and a mobility disability.52 In 56 obese adults,51 knee OA was significantly associated with reduced exercise capacity, ambulatory capacity, and quality of life.

Importantly, weight loss has been shown to significantly improve signs and symptoms of OA and improve disability and function in obese patients.53–58 A meta-regression analysis that included 4 trials including a total of 454 patients was conducted on the effect of weight loss on OA.53 Mean baseline BMI ranged from 29 to 36 kg/m2 in each of 5 intervention groups, and weight loss ranged from 1.7 to 6.7 kg over 6 weeks to 18 months. Modest weight loss (5.1%) improved physical disability among patients with knee OA. A randomized study of 87 obese (BMI ≥ 30 kg/m2) adults at least 60 years old with symptomatic knee OA was also undertaken to evaluate the effect of weight loss intervention.57 At 6 months, those randomized to intervention had lost a mean of 8.7% of body weight compared with no

weight loss in the control group. Functional status was significantly (P < 0.05) improved in the intervention group with greater improvements observed with more weight loss. Others have found significant improvements in function and pain with weight loss and/or exercise among patients with knee OA.55,56,58 Thus, recognition of the impact of obesity among patients with knee OA offers an opportunity to significantly improve associated disability and pain by encouraging weight loss.

Gallbladder Disease

Gallbladder disease is a common cause of hospitalization, especially among women, and has a considerable impact on health care costs.59 An epidemiologic study from the National Health Service in England and Scotland found a significant association between obesity and gallbladder disease among women.59 In this study, data for 1.3 million women (mean age, 56 years), representing 7.8 million person-years of follow-up, were evaluated. After adjusting for age, socioeconomic status, and other factors, women with higher BMI at study entry were more likely to be admitted and spend more days in the hospital for gallbladder disease. For each 1000 person-years of follow-up, women with BMI in the lowest BMI category (18.5–24.9 kg/m2) spent a mean of 16.5 days hospitalized versus 44 days for women in the obese category (BMI 30–39.9 kg/m2). Overall, 25% of hospital days for gallbladder disease were aattributed to obesity.

In a prospective evaluation from the Health Professionals Follow-up Study, the association between abdominal obesity and the incidence of symptomatic gallstone disease was determined in a cohort of 29 847 men who were free of prior gallstone disease and who provided complete data on waist and hip circumferences.60 Men with BMI ≥ 28.5 kg/m2 had a 2.49-fold greater risk of gallstones compared with men with a normal BMI (< 22.2 kg/m2) Similar findings were observed in the Swedish Twin Registry Study.61 The Swedish Twin Registry study assessed the effects of overweight and obesity (BMI > 30 kg/m2) on symptomatic gallstones in 58400 participants. Overweight and obesity were both associated with a significant increase in the risk of symptomatic gallstones (OR = 1.86 and 3.38, respectively).

Acute Pancreatitis

Acute pancreatitis is closely associated with obesity, and a number of studies have shown that obesity increases the severity of and mortality from acute pancreatitis.62–65 Obesity is a primary risk factor for local complications, organ failure, and death from acute pancreatitis.

In a meta-analysis of 5 studies including a total of 739 patients, obesity (BMI ≥ 30 kg/m2) was identified as a risk factor for the development of local and systemic complications in acute pancreatitis and was also associated with increased mortality.64 Among these patients

from the 5 studies, severe acute pancreatitis was significantly associated with obesity (OR 2.9, 95% CI 1.8–4.6). Among these obese patients, significantly more systemic (OR 2.3, 95% CI 1.4–3.8) and local complications occurred (OR 3.8, 95% CI 2.4–6.6), and mortality was higher (OR 2.1, 95% CI 1.0–4.8).

Nonalcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease (NAFLD) represents a spectrum of disorders that range from steatosis to nonalcoholic steatohepatitis and ultimately cirrhosis and hepatocellular carcinoma.66 Nonalcoholic fatty liver disease is associated with obesity, dyslipidemia, hypertension, and insulin resistance, components of the MetS that increase cardiovascular risk.67 It affects approximately 15%–30% of the general population, and has a prevalence of approximately 70% in people with type 2 diabetes.67

Studies have indentified obesity as a predictor of NAFLD.68–70 In a multivariate analysis among 832 Chilean participants, the primary variable associated with NAFLD was BMI > 26.9 kg/m2.68 The diagnosis of NAFLD was based on ultrasound and no history of alcohol abuse or hepatitis C infection. Multivariate analysis found that obesity was significantly and independently associated with NAFLD with odds ratio of 6.2. In a cross-sectional study of 326 Israelis who participated in a National Health survey, the prevalence of NAFLD was 30%; NAFLD was more common in men (38%) than in women (21%), and obesity (BMI ≥ 30 kg/m2) was independently associated with NAFLD (odds ratio 2.9).69 A study of 218 nonsmoking, healthy men found that 24 met criteria for NAFLD.70 Lack of fitness and BMI ≥ 30 kg/m2 were significantly (P < 0.001) and independently associated with NAFLD.

I

Increased physical activity and bariatric surgery in selected cases may be effective therapy for NAFLD.71,72 Although evidence is limited, weight loss may be beneficial for reducing the risk of NAFLD in obese patients.71

Pulmonary Complications

Obstructive sleep apnea (OSA) potentially results in a number of complications including pulmonary hypertension, right heart failure, drug-resistant hypertension, stroke, and arrhythmias.73–78 Obstructive sleep apnea is characterized by upper airway obstruction that occurs as repetitive episodes during sleep.74 Among the typical features of OSA are loud snoring, fragmented sleep, repetitive hypoxemia/hypercapnia, daytime sleepiness, and cardiovascular complications. Although the prevalence of OSA is 2% to 3% among middle-aged women and 4% to 5% among middle-aged men, the prevalence among obese patients is > 30% and among the morbidly obese ranges from 50% to 98%.75–77 Thus, obesity

is the most important risk factor for the development of OSA, where 60% to 90% of adults are overweight, and the relative risk in obese patients (BMI > 29 kg/m2) is ≥ 10.74

The independent association between sleep-disordered breathing and weight gain was evaluated in a population-based, prospective study of 690 randomly selected residents of Wisconsin.76 Participants had a mean age of 46 years, a mean baseline BMI of 29–30 kg/m2, and were evaluated twice at 4-year intervals. A 10% weight gain predicted a 32% increase in the apnea-hypopnea index and a 6-fold increase in the odds of developing moderate-to-severe sleep-disordered breathing.

Similarly, the effect of weight gain on sleep-disordered breathing was determined in a prospective study of 2968 men and women in the United States.77 Baseline mean BMI was approximately 29 kg/m2, mean age was 62 years, and participants were examined at baseline and 5 years. An increased number of respiratory events was aassociated with weight increases. Men with a 10-kg increase in weight had 5.21-fold increased risk of developing > 15 events/hour and women had a 2.5-fold increased risk.

A reduction in OSA was observed in The Swedish Obesity Study among patients with diabetes who lost weight.78 This study evaluated 1729 patients with a baseline BMI > 40 kg/m2 undergoing bariatric surgery and 1748 given conservative medical therapy as the control group. A significant (P < 0.001) reduction in symptoms of OSA was observed among the bariatric surgery group at 2 years including apnea (24% to 8%), snoring (44.5% to 10.8%), and daytime sleepiness (25.8% to 12.7%). Those with a mean 31% weight loss had a 2- to 13-fold decrease in the risk of developing new OSA, and those with OSA were 2.5 to 7 times less likely to report continuing OSA symptoms.

Depression

An association between obesity and major depressive disorder (MDD) has long been recognized although a causal association is uncertain. Importantly, many antidepressant drugs are associated with weight gain. The National Epidemiologic Survey on Alcohol and Related Conditions evaluated the relationship between BMI and psychiatric disorders in 41 654 respondents.79 Among participants, BMI was significantly associated with mood, anxiety, and personality disorders. The odds ratio for a psychiatric disorder was 1.21- to 2.08-fold greater among obese (BMI 30-39.9 kg/m2) and extremely obese (BMI ≥ 40 kg/m2) subjects, and the odds ratio for a lifetime prevalence of MDD was 1.53 and 2.02 among obese and extremely obese compared with normal weight subjects.

Others have found similar results. The 2006 Behavioral Risk Factor Surveillance System (N = 217 379) found that adults with current depression or a lifetime diagnosis of depression or anxiety were significantly more likely to have unhealthy behaviors including smoking,

obesity, physical inactivity, binge drinking, and heavy drinking.80 The adjusted odds ratio for depression and obesity (BMI ≥ 30 kg/m2) was 1.6 vs 1 for nonobese subjects, and the odds ratio increased with increasing severity of MDD. Among 4641 middle-aged women, MDD was strongly and consistently associated with obesity, lower physical activity, and among the obese, higher caloric intake.81 The prevalence of moderate or severe MDD increased from 6.5% with a BMI < 25 kg/m2 to 25.9% with a BMI > 35 kg/m2. The prevalence of obesity increased from 25.4% to 57.8% among those with no MDD versus those with moderate-to-severe MDD. The odds ratio for having MDD was 4.4 for a BMI of 30 to 35 kg/m2 and 4.95 for a BMI of ≥ 35 kg/m2. Using standard criteria for MDD, the odds ratios were 1.92 for a BMI of 25 to 30 kg/m2, 2.92 for 20–35 kg/m2, and 5.72 for a BMI of ≥ 35 kg/m2.

Despite the absence of a clear causal relationship between obesity and MDD, an awareness of this relationship and the opportunity to improve depression and quality of life by recommending appropriate weight loss interventions is needed.

  Other Sections The net impact of the increased burden of disease associated with obesity is

increased mortality, which is well established in this population. An extensive number of epidemiological studies have established a significant increase in cardiovascular and non cardiovascular mortality associated with obesity (Table 1). Overall, large-scale studies such the Nurses’ Health Study, NHANES, Women's Health Initiative Observational Study, American Cancer Society Prevention studies, and others have documented the adverse effects of obesity on mortality from CVD, cancer, and other comorbidities.6,82-89 An increase in years of life lost was found among obese versus nonobese subjects in an analysis of the NHANES database.90 Overall, years of life lost were 1 to 9 for those with low BMI (< 17–19 kg/m2) compared with 9 to 13 for those with a high BMI (≥ 35 kg/m2).

Importantly, studies of patients undergoing gastric bypass surgery for morbid obesity have demonstrated significant reductions in mortality with substantial weight loss.91,92 Adams91 reported a retrospective cohort study of mortality in 7925 surgical patients and 7925 severely obese control subjects who were matched for age, sex, and BMI. During a mean follow-up of 7.1 years, all-cause mortality decreased by 40% (57.1 to 37.6/10 000 patient-years), and mortality decreased by 56% for CAD, 92% for diabetes, and 60% for cancer (P < 0.01 for each). The Surgical Obesity Study was a prospective evaluation of gastric surgery (n = 20lO) or conventional treatment (n = 2037) of patients with morbid obesity.92 Overall mortality was reported after 10.9 years of follow-up, where average weight change was 2% in the control group and 14% to 25% in the surgery group depending on the procedure. The adjusted hazard ratio for mortality was 0.71 (P = 0.01) in the surgery group versus the control group.

  Other Sections ▼

SummaryObesity is at epidemic proportions in the United States and other devObesity is at epidemic proportions in the United States and other developed countries, but, importantly, even in developing countries. Large, high-quality longitudinal or prospective studies have confirmed that obesity is a significant risk factor for and contributor to increased morbidity and mortality, primarily from CVD and diabetes, but also from cancer and other acute and chronic diseases, including osteoarthritis, liver and kidney disease, sleep apnea, and depression (Figure 3). For the majority of these comorbid conditions, weight loss can result in a significant reduction in risk.

The economic costs of obesity are substantial. A model based on NHANES and the Framingham Heart Study was used to

assess the lifetime health and economic consequences of obesity.93 The analysis showed substantial effects on lifetime

health and economic consequences of obesity, and the authors suggested that significant benefits could be expected from

interventions to prevent or reduce obesity.93 Disease risks and costs increased with increasing BMI. For instance, the risk of

hypertension was 2-fold higher and diabetes was 3-fold higher among 45- to 54-year-old obese men compared with

nonobese men.93 Lifetime medical costs increased incrementally with increased BMI and age by approximately 2-fold for

each group. Obesity also has a significant impact on quality-adjusted life years and reduces years oflife.90,93,94 In an analysis

of quality-adjusted life years, obese men and women lost 1.9 million and 3.4 million quality-adjusted life years and

experienced lower health-related quality of life compared with normal weight subjects.94 A model of the economic costs of

obesity found a substantial impact could be reduced with effective measures to prevent weight gain.93

Recognition of the association between obesity and comorbidities is critical for patient diagnosis and management by

primary care physicians. Physicians need to be aware of comorbidities and their implications for outcomes and patient

management of the obese patient. Global efforts to control obesity and minimize factors that contribute to obesity are

essential to improving health status and life expectancy worldwide.

Acknowledgments

The author would like to acknowledge the assistance of Richard S. Perry, PharmD in the preparation of this manuscript,

which was funded by Amylin Pharmaceutics, Inc.

Footnotes

Conflict of Interest Statement

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Current Status of Medical and Surgical Therapy for ObesityEDWARD C. MUN, GEORGE L. BLACKBURN, and JEFFREY B. MATTHEWSDepartment of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MassachusettsThe incidence of obesity (especially childhood obesity)and its associated health-related problems havereached epidemic proportions in the United States. Recentinvestigations suggest that the causes of obesityinvolve a complex interplay of genetic, environmental,psychobehavioral, endocrine, metabolic, cultural, andsocioeconomic factors. Several genes and their proteinproducts, such as leptin, may be particularly importantin appetite and metabolic control, although the geneticsof human obesity appear to involve multiple genes andmetabolic pathways that require further elucidation. Severeobesity is frequently associated with signiÞcantcomorbid medical conditions, including coronary arterydisease, hypertension, type II diabetes mellitus, gallstones,nonalcoholic steatohepatitis, pulmonary hypertension,and sleep apnea. Long-term reduction of significantexcess weight in these patients may improve or

resolve many of these obesity-related health problems,although convincing evidence of long-term beneÞt islacking. Available treatments of obesity range from diet,exercise, behavioral modiÞcation, and pharmacotherapyto surgery, with varying risks and efÞcacy. Nonsurgicalmodalities, although less invasive, achieve only relativelyshort-term and limited weight loss in most patients.Currently, surgical therapy is the most effectivemodality in terms of extent and duration of weight reductionin selected patients with acceptable operativerisks. The most widely performed surgical procedure,Roux-en-Y gastric bypass, achieves permanent (followedup for more than 14 years) and signiÞcant weight loss(more than 50% of excess body weight) in more than90% of patients.

The notion that obesity is the result of a lack ofwillpower on the part of affected individuals issimplistic, unscientific, and counterproductive in combatingthis disease. It is increasingly clear that the regulationof body weight is dependent on multiple biologicfactors modified by various environmental and psychosocialfactors. The food intake of an average adultmatches energy expenditure within 0.17% per decade,1indicating the presence of a biologic regulatory systemwith remarkable precision in energy metabolism. Althoughthe influence of genetic factors on the expressionof obesity has been demonstrated repeatedly,2–4 the recentexplosion in the prevalence of obesity is most likelycaused by environmental and behavioral changes. Nevertheless,recent advances in the genetics of obesity andin energy metabolism should yield new insights intofundamental physiologic regulatory processes and maylead to more effective and specific therapies.The degree of obesity is most conveniently quantifiedby the body mass index (BMI) because of its ease ofcalculation and relatively accurate correlation with bodyfat content. BMI represents a ratio of weight and bodysurface area, expressed as weight (kilograms) divided bythe square of height (square meters). Conventional categoriesof relative body weight corresponding to BMI areshown in Table 1.5 Using these categories, more thanhalf of all adults6 and approximately a quarter of allchildren are overweight (BMI . 25) or obese (BMI .30),7 and 18% of all Americans are obese (BMI . 30).8,9Approximately 280,000 annual deaths are estimated tobe attributable to obesity in the United States.10 Four of5 obese people have at least one debilitating illnessassociated with the underlying obesity. In developedcountries, obesity is more common among those of lowersocioeconomic status. The prevalence of obesity is higher

in certain ethnic groups such as African Americans andMexican Americans. U.S. public health officials indicatethat obesity currently is not effectively addressed(Healthy People 2010). For example, in a recent community-based study over a 3-year period, more than half(53.7%) of the subjects gained weight, whereas only aquarter (24.5%) were able to avoid a weight gain, andsuccessful weight loss and maintenance were seen in lessthan 5% (4.6%).11 The urgency of the current weightproblem is also reflected by the most recent top 10leading health indicator list, in which the top 2 arephysical activity and obesity, which rank above tobaccouse. Many epidemiology studies have documented a closerelationship between increasing adiposity and deathAbbreviations used in this paper: BMI, body mass index; GBP, gastricbypass; LCD, low-calorie diet; LFD, lower-fat diet; NPY, neuropeptide Y;VBG, vertical banded gastroplasty; VLCD, very low-calorie diet.© 2001 by the American Gastroenterological Association0016-5085/01/$35.00doi:10.1053/gast.2001.22430v

Table 1. Classification of ObesityObesity class BMI (kg/m2)Underweight ,18.5Normal 18.5–24.9Overweight 25.0–29.9Obesity I 30.0–34.9II 35.0–39.9Extreme obesity III $40.0Data from US Department of Health and Human Services.5Figure 1. Mortality risk increases with obesity. Adapted and reprintedwith permission.133Figure 2. Effects of leptin on food intake. A decreased plasma levelof leptin activates NPY/agouti-related protein neurons in the arcuatenucleus of the hypothalamus, resulting in increased expression ofboth NPY and agouti-related protein. Increased NPY and agouti-relatedprotein release then stimulates food intake and subsequent weightgain. At the same time, pro-opiomelanocortin neurons in the sameregion of the hypothalamus are inhibited by leptin deficiency. A resultantdecrease in a–melanocyte-stimulating hormone expression andrelease leads to reduced activity in melanocortin-mediated anorexia,and thus increased food intake and weight gain.670 MUN ET AL. GASTROENTEROLOGY Vol. 120, No. 3What Is Morbid AboutMorbid Obesity?Severe obesity is associated with the developmentof a variety of medical conditions, thus the term morbidobesity. Premature mortality has been repeatedly observedin severely obese patients.16,33,34 Some of the contributingconditions include coronary artery disease, hypertension,type II diabetes mellitus, sleep apnea syndrome,obesity hypoventilation syndrome, and necrotizing panniculitis.Morbid obesity is also associated with numerousother disabling conditions such as chronic venousstasis disease, osteoarthritis, urinary incontinence, gastroesophagealreflux disease, fatty liver, cholelithiasis,idiopathic intracranial hypertension (pseudotumor cerebri),sex hormone dysfunction, and clinical depression.

Many of these comorbidities are closely related to theincreased intra-abdominal pressure and sagittal abdominalwall diameter.35 Increased risk is observed in obesepatients for the development of various cancers, includingbreast, colon, uterine, and prostate cancer. Table 2lists various obesity comorbidities encountered in theclinical setting.Does Weight Loss Reduce theMorbidity of Obesity?The purpose of treatment for obesity is to restorenormal metabolic and organ function. It should be rememberedthat the goal of any antiobesity therapy is notto reduce weight per se, but to reduce the disability andmorbidity, and thus to increase the quality of life. Therationale for treatment of obesity is not only the increasedmortality attributable to obesity, but also thenumerous lines of evidence suggesting that weight lossreduces risk factors for comorbid disease.5 A modest tomoderate loss of excess weight (the difference betweenactual weight and the ideal body weight for a givenheight) achieved by life-style changes has been shown toreduce blood pressure in overweight hypertensive36,37and nonhypertensive patients,38 to improve serum lipidprofile (reduced triglyceride and total and low-densitylipoprotein cholesterol levels, along with increased highdensitylipoprotein cholesterol levels),39,40 and to improveglucose tolerance and fasting glucose levels innormoglycemic and diabetic patients.41–43 Diet, exercise,and standard behavior modification have been the traditionalmethods, but these do not always work by themselves.Pharmacologic treatments have yielded mixedresults over the years, with some patients respondingwell but others not at all. Recently, a new generation ofdrugs promises hope for treatment of those with clinicallysignificant obesity, but to date these therapies havenot been shown to be effective for cases of severe obesity.Surgical treatments for obesity in general achieve moreprofound and long-lasting weight loss and improve orresolve most comorbidities of severe obesity. In additionto the improvements in hypertension,44,45 serum lipidlevels,46 and diabetes mellitus,47,48 weight reduction bysurgery improves respiratory insufficiency caused bysleep apnea and obesity hypoventilation syndrome,49,50reflux esophagitis,51,52 pseudotumor cerebri,53 and venousstasis ulcers.35 However, a more recent study bySjostrom et al.54 shows differential effects of long-term (8years) surgical weight loss on diabetes and hypertension.Although surgical weight loss was associated with sig-Table 2. Obesity ComorbiditiesCardiovascular systemCoronary artery disease

HypertensionCongestive heart failureCor pulmonale, pulmonary hypertensionDeep vein thrombosisPulmonary embolismRespiratory systemObstructive sleep apneaAsthmaObesity hypoventilation syndromeEndocrine systemType II diabetes mellitusGlucose intolerance, decreased insulin sensitivityDyslipidemia (hypercholesterolemia, hypertriglyceridemia)Amenorrhea, dysmenorrheaPolycystic ovary syndromeInfertilityHirsutismGynecomastiaBreast cancerGastrointestinal and abdominal wall systemGastroesophageal refluxNASH, fatty liverCholelithiasisColon cancerHernias (umbilical, epigastric, incisional, inguinal)Musculoskeletal systemDegenerative joint disease, osteoarthritisChronic low back painGenitourinary systemUrinary stress incontinenceHypogonadismUterine cancerProstate cancerIntegumentVenous stasis diseaseSuperficial thrombophlebitisCellulitis, panniculitis, candidiasisIncreased postoperative wound infectionPsychoneurologic systemClinical depressionMigraine headacheIdiopathic intracranial hypertension (pseudotumor cerebri)Cerebrovascular accident (stroke)February 2001 MEDICAL AND SURGICAL THERAPY FOR OBESITY 671nificant reduction in both diabetes and hypertension at 2years, by 8 years the incidence of hypertension in thesurgical group was the same as that in matched controls.Despite the clear benefits seen with surgical weightreduction, several key questions remain: Does the degreeof improvement of obesity comorbidities by surgery reducethe complications of those conditions, and therebytranslate into a longer life and improved quality of lifefor the patients? Is the degree of improvement offset bythe risk of surgical complications? What is the cost (orbenefit) of such treatment to society? The Swedish ObeseSubjects (SOS) study is one of the few longitudinalinvestigations attempting to answer these fundamentalquestions. Medical TherapyCapitalizing on the increased market for obesitytherapy, numerous commercial programs have sprung upacross the United States and formed a thriving multi–billion dollar industry. These nonsurgical programs are

classified into 4 basic approaches: diet, exercise, behaviormodification, and drugs. Accumulating data reveal theeffectiveness of these modalities in inducing modestweight loss in many participants; however, these approachesare usually effective only in the short run, andindefinite continuation of such treatments is usuallydifficult for patients to sustain. Studies are ongoing,particularly in search of safe and effective drug therapiesdirected against the molecular defects of obesity.Diet TherapyTheoretically, it should be a simple matter toachieve weight loss by dieting, producing an energydeficit in which intake is less than energy expenditure.As so many can attest by their own personal experience,this approach is far more difficult to put into action in anenvironment where delicious, high-calorie foods areabundant and easily obtained. This inherent difficulty iswell illustrated by the typical results of many diet programs,in which early weight loss is achieved by mostpatients, but the weight loss is not maintained over thelong term.A comprehensive and critical evaluation of multipledietary clinical trials was carried out by an expert panelconvened by the National Institutes of Health (NIH) in1998 in an effort to establish evidence-based guidelinesfor the treatment of obesity.5 Despite confounding factorsfrom various studies, several key conclusions couldbe agreed upon. First, dietary caloric reduction is indeedassociated with weight loss. Low-calorie diets (LCDs)consisting of 1000–1200 kcal/day can reduce total bodyweight by an average of 8% over 3–12 months. Verylow-calorie diets (VLCDs) with 400–500 kcal/day producegreater initial weight loss than LCDs, but thelong-term (.1 year) weight loss is not different fromthat of LCDs. Second, a change in diet composition by fatreduction is associated with weight loss. Lower-fat diets(LFDs), deriving 20%–30% of calories from fat, helppromote weight loss by producing a reduced caloricintake. LFDs coupled with total caloric reduction producegreater weight loss than LFDs alone. LFDs produceweight loss primarily by decreasing caloric intake. Basedon these findings, the panel recommends (1) LCDs forweight loss in overweight and obese patients and (2)reduction of fat as part of an LCD to reduce calories.Because there is little evidence that LFDs per se causeweight loss independent of caloric reduction, reductionin total caloric intake is the most important factor inweight loss.Carbohydrate restriction has been the basis for severalpopular diets in the past, and has recently been resurrected

as the “Atkins diet” and the “zone diet.”55,56 Thebasic premise of low-carbohydrate diets is that excessivecarbohydrates induce increased levels of insulin that promotetransport and storage of fat. Low-carbohydrate diets,consisting inevitably of high levels of protein and fat,promptly induce depletion of liver glycogen storage andsystemic ketosis resulting from oxidation of fat. However,short-term weight loss on such diets seems to becaused in large part by the loss of water and electrolytes.57 This is particularly true during the initial dietphase, in which heavily hydrated glycogen is catabolizedto meet energy requirements and maintain blood glucose.Low-carbohydrate, high-protein diets seem particularlyeffective in suppressing hunger, possibly becauseof branched-chain amino acid content.58 Thus, theweight loss may not necessarily be attributable solely tothe composition of the diet, but also to reduced totalcaloric intake. Additionally, a more recent study showedthat weight loss is not different between low- and highcarbohydratediets,59 indicating that total energy intake,not nutrient composition, determines weight loss. Becauselong-term studies are not yet available, the longtermsafety and efficacy of low-carbohydrate, high-protein/fat diets remain to be established.ExerciseIn our modern, technology-driven age, an everincreasingnumber of labor-saving conveniences and gadgetscontribute to the reduction in average daily energyexpenditure that favors the development of obesity.60Although physical activity and exercise are key factors insuccessful weight reduction programs, the contribution672 MUN ET AL. GASTROENTEROLOGY Vol. 120, No. 3of exercise to weight loss is modest at best. For example,approximately 40 miles of walking is required to metabolize1 kg of fat. The effect of exercise on weight loss isvariable, but most studies show only a small reduction(;2 kg),37,61,62 and some show no benefit at all.63,64Furthermore, most studies show that weight loss inducedby exercise alone is inferior to that achieved by dietalone.37,64,65 However, exercise is probably independentlyimportant to the well-being of overweight andobese individuals because physical activity increases maximaloxygen uptake and thus cardiorespiratory fitness.37,63,64 Moreover, maintenance of weight loss isfacilitated by regular exercise.66 Unfortunately, the unrealisticexpectations of weight loss from exercise regimensthat are promoted by many commercial enterprisesmay lead to disappointment and discontinuation of exercise.Development of a consistently achievable exerciseprogram for each overweight patient is essential. Although

it is difficult, even the most obese patients maybe able to participate in some form of appropriatelydesigned physical activity.Behavior TherapyConditioning probably plays a major role in manybehavioral disorders. Like Pavlov’s dog, who salivated atthe sound of a bell, overweight and obese individualsbecome conditioned to the repeated association of, forexample, pizza and beer with watching sporting eventson television. Thus, behavior therapy in obesity is toidentify cravings and weaken or disconnect the triggeringevents that lead to overeating. However, the effectivenessof behavior therapy alone against obesity ismodest compared with that reported in other conditionssuch as depression, anxiety, and bulimia,67 and it is bestcombined with other weight loss modalities. When behaviortherapy was combined with diet therapy in theform of an LCD or VLCD, maintenance of weight loss at1 year was better than with diet alone.68,69 Similarly,drug therapy with fenfluramine was shown to achieve abetter weight loss at 6 months, and better maintenanceat 1 year, when combined with behavior therapy.70 Longterm(1–5 years) follow-up of these patients, however,indicates that most of the subjects in the group regainthe lost weight in the absence of continued behavioralintervention.69,71 A recent NIH expert panel recommendsthat behavior therapy be an adjunct treatment forweight loss and weight maintenance.5PharmacotherapyAlthough somewhat effective, drug therapy, likeother nonsurgical modalities, achieves only a modest(;10%) weight reduction and requires continued use tomaintain this result. In addition, the dangerous adverseeffects associated with some drugs, including addictionwith amphetamines72 and valvular heart disease withfenfluramine plus phentermine (fen-phen),73 have givenpharmacotherapy for obesity a bad reputation. However,recent advances in understanding of molecular mechanismsof weight regulation may yet lead to the developmentof new classes of antiobesity drugs.Most antiobesity drugs can be classified into 2 majorgroups by their mechanisms of action: appetite suppressionand/or stimulation of thermogenesis and intestinalfat absorption. Fenfluramine and its d isomer dexfenfluramineincrease central serotonin release and induce anorexiaand weight loss. In one study, dexfenfluramineshowed a small but statistically significant advantage inweight loss when 35% of the subjects taking dexfenfluramine(compared with 17% of the placebo group) wereable to lose at least 10% of their initial weight.74 Phentermine

is a central adrenergic agonist that leads toappetite suppression and weight loss.72 Combinationtherapy with serotonergic (fenfluramine) and the catecholaminergic(phentermine) agents was demonstratedto be effective in weight reduction75,76 and becamewidely popular until an unusual form of valvular heartdisease in women taking this combination was reportedin 199773 and confirmed in other studies.77–79 Theseagents were subsequently withdrawn from the market in1998. Phentermine alone does not appear to increasevalvular heart disease.Sibutramine, a b-phenethylamine, is a selective inhibitorof the reuptake of both serotonin and norepinephrineand is used more widely since the discontinuation offenfluramine and dexfenfluramine. It induces both decreasedfood intake and increased thermogenesis.80–82Weight loss induced by sibutramine was shown to becomparable to that induced by dexfenfluramine.83 Inanother study, patients taking sibutramine maintained acontinued weight loss of 15% over a 1-year period afteran initial diet-induced weight loss, and those in theplacebo group regained the weight they initially lost.84Patients using sibutramine should be monitored for sympathomimeticside effects, including any tachycardia andhypertension.Orlistat, an inhibitor of pancreatic lipase, decreases fatabsorption in the intestine. Orlistat blocks digestion ofapproximately 30% of ingested dietary triglyceride85 andhas been shown to achieve a weight loss of ;10%(compared with ;5% in the control group).86,87 However,since the induction of fat malabsorption is its basisof action, it is rather common that steatorrhea developswith orlistat if enough fat is ingested during a meal.88Caffeine, ephedrine, and the combination of these 2drugs can reduce food intake and cause thermogenesis byincreasing oxygen consumption.89,90 The combination ofcaffeine and ephedrine has been shown to be more effectivein inducing weight loss than placebo, caffeine, orephedrine alone.91,92 The weight loss difference betweenthe combination regimen and placebo is relatively small,and neither agent alone produced significantly moreweight loss than placebo.91 Major adverse effects of theseagents include tachycardia and palpitations, and thecombination of caffeine and ephedrine is not currentlyapproved by the U.S. Food and Drug Administration.Recent insights into the molecular mechanisms ofsatiety and thermogenesis present a window of opportunityfor novel pharmacotherapies. Potential strategiesinclude targeting the central regulation of food intakeusing leptin (and leptin analogues), leptin receptor agonists,

melanocortin receptor (MC4R) agonists, NPY antagonists,and cocaine- and amphetamine-regulated transcriptreceptor agonists. Potential drugs that targetthermogenesis regulation are also promising, and theseinclude b3-adrenergic receptor agonists and agents thatactivate or increase uncoupling proteins. A major intestinalfatty acid transporter (FATP4) has recently beenidentified93; specific inhibitors of this transporter couldbe effective in blocking fat absorption, although thepotential for steatorrhea, as with orlistat, must be acknowledged.Currently, indications for the use of pharmacotherapyfor obesity are a BMI of 30 or a BMI of 27 withobesity-related comorbidities. However, no data showthat drug treatment of otherwise healthy obese subjectsprevents future complications or improves long-termoutcome. Moreover, long-term weight loss data are notavailable. Most patients regain weight once any drugtherapy is discontinued, and the success of pharmacotherapydemands life-style changes in diet, exercise, andbehavior to increase its effectiveness.Surgical TherapyBariatric procedures for weight reduction share 2major designs: intestinal malabsorption and gastric restriction.Malabsorptive procedures (Figure 3) involverearrangement of the small intestine to decrease thefunctional length or efficiency of the intestinal mucosafor nutrient absorption. Restrictive operations (Figure 4)involve creation of a small neogastric pouch and gastricFigure 3. Malabsorptive bariatricprocedures. (A) Jejunoilealbypass; (B) biliopancreatic diversion;(C) duodenal switch.Figure 4. Restrictive bariatricprocedures. (A) VBG; (B) adjustablegastric banding; (C)Roux-en-Y GBP.674 MUN ET AL. GASTROENTEROLOGY Vol. 120, No. 3outlet to decrease food intake. Various procedures haveevolved from combinations of these 2 principles. Patientselection criteria for surgical treatment of obesity weredeveloped at a 1991 NIH Consensus Development ConferencePanel and include patients with BMI . 40 orBMI . 35 with obesity-related medical comorbidities.94Additional criteria used by most bariatric surgeons asgeneral guidelines are listed in Table 3. The goals ofsurgery are to induce and maintain significant loss ofexcess weight through a safe operation and to improve orresolve many of the comorbid medical problems so thatquality of life is enhanced and prolonged. A successfuloutcome of bariatric surgery depends on several factors.First and foremost is a well-informed and well-educatedpatient with realistic expectations. It is simply unrealistic

to expect total or near-total loss of excess weight withunchanged dietary habits and this expectation can resultin patient dissatisfaction postoperatively. A multidisciplinaryteam capable of providing all aspects of preoperativeand postoperative care is thought to be crucial inaddressing multiple potential difficulties in the care ofthese complex patients. Optimally, the team should includededicated surgeons, internists, psychiatrists, dieticians,nutritionists, and nurses.Malabsorptive ProceduresJejunoileal BypassThe first bariatric operation was jejunoileal bypass,95 in which an anastomosis of proximal jejunum (14inches from the ligament of Treitz) to the terminal ileum(4 inches from the ileocecal valve) is created, leaving anextended loop excluded from the food stream (Figure3A). The jejunoileal bypass is exclusively a malabsorptiveprocedure because the stomach is not modified tolimit food intake. Although this operation requires nosignificant changes in eating habits to induce weightloss, it was plagued by an unacceptable level of seriouscomplications, including hepatic failure,96 cirrhosis,97oxalate kidney stones, bypass enteritis, arthritis, andmultiple metabolic deficiencies such as protein malnutrition,metabolic bone disease, hypocalcemia, and vitaminB12 and vitamin D deficiency.98 This procedure is nolonger performed, and the poor experience with jejunoilealbypass caused a stigma to be associated with bariatricsurgery and probably hindered more widespreadapplication of improved operations for obesity. Survivorsof this procedure should be evaluated for liver and renaldysfunction and for conversion to a more acceptableanatomic construction whenever possible.Biliopancreatic Diversion andDuodenal SwitchThis procedure uses malabsorption of nutrientsbecause its principal antiobesity mechanism is diversionof biliary and pancreatic secretions to the distal 50 cm ofthe ileum (Figure 3B).99 A small degree of gastric restrictionis added by performing a distal (80%) gastrectomy.The combination of gastroileostomy (rather thangastrojejunostomy), a very long biliopancreatic limb, anda very short common channel results in significant maldigestionand malabsorption of nutrients. This procedureis highly effective in inducing weight loss, particularly in“supermorbid” obese patients (BMI . 50). However,significant metabolic complications can occur, such asprotein calorie malnutrition, metabolic bone disease, anddeficiencies in fat-soluble vitamins, iron, calcium, andvitamin B12.99,100 Most bariatric surgeons are reluctant to

perform biliopancreatic diversion as a first-line antiobesityprocedure.35,100 The duodenal switch procedure (Figure3C) is a modified form of biliopancreatic diversionthat connects the jejunum (rather than ileum) to the proximalduodenum, thus taking advantage of dumping physiologyas with Roux-en-Y gastric bypass (GBP).101–103

Restrictive ProceduresGastroplastyGastroplasty involves pure restriction of the storagecapacity of the stomach to decrease consumption ofsolid foods. These procedures entail the use of surgicalstapling devices and are thus commonly referred to asgastric stapling operations. Initially, gastroplasty consistedof horizontal partitioning of the stomach into asmall proximal pouch and a large distal remnant,104,105which communicate through a narrow channel or stoma.Table 3. Eligibility Criteria for SurgeryEligibility criteriaBMI . 40 or BMI . 35 with obesity-related comorbiditiesAge 16–65 yrAcceptable medical/operative risksProof of failed attempts at nonsurgical weight reductionMotivated, psychologically stable patient with realisticexpectationsPatient capable of understanding the procedure and possiblecomplicationsCommitment to prolonged life-style changesSupportive family/social environmentCommitment to long-term follow-upIneligibility criteriaUnsolved history of alcohol or substance abuseHistory of schizophrenia, severe depression, particularly suicidalideationHostile uncooperative behaviorUnacceptable medical riskHostile uncooperative family environmentFebruary 2001 MEDICAL AND SURGICAL THERAPY FOR OBESITY 675Later this was modified by Mason to a vertically orientedgastroplasty with the staple line extending upward fromthe angle of His, with a mesh-band reinforcement at thestoma on the lesser curvature, and was termed verticalbanded gastroplasty (VBG) (Figure 4A).106 VBG wasdesigned to avoid 2 common causes of failure of horizontalpartitioning gastroplasty: pouch and stomal dilation.The vertical staple line was primarily to exclude thefundus of the stomach, which was thought to dilaterelatively easily, while polypropylene mesh encirclingthe stoma was used to prevent dilation.However, a high incidence of stomal stenosis or stapleline dehiscence has also been reported with VBG.107Although the small pouch and stoma effectively deteringestion of large boluses of food, many patients learnout of frustration to cheat with high-calorie liquids suchas milkshakes. Long-term weight maintenance afterVBG has been disappointing, despite the rapid weightloss seen in the first 1–2 years: Nightengale et al.108

found that only 38% of the patients were able to maintainat least 50% of the excess weight loss at 3 years, andHoward et al.109 reported a smaller series in which nopatient could maintain a 50% excess weight loss by 5years. Sugerman et al.110 also found that the mean loss ofexcess weight was only approximately 38% at 3 years. Inthese randomized prospective trials, weight loss afterVBG was inferior to that after Roux-en-Y GBP, leadingmany bariatric surgeons to abandon gastroplasty as aprimary antiobesity procedure.35,111Gastric BandingGastric banding, another pure gastric restrictiveprocedure in which a prosthetic band is encircledaround the proximal stomach to compartmentalize itinto a small pouch and a large remnant, was initiallydescribed by Bo and Modalsli.112 The absence of astaple line and of the associated risk of staple linedehiscence is a theoretical advantage of this procedure.An adjustable gastric band (Figure 4B) was later introducedin which a subcutaneous saline port connectedto the adjustable band allows changes in thestoma size.113 This device may be placed laparoscopically,114,115 making it an attractive new device. Theresults are widely variable but will probably provecomparable to those of VBG.116 –118 Westling et al.119reported a 56% loss of excess weight at 2 years in 90patients, but with a disappointing 35% conversion toRoux-en-Y gastric bypass. The adjustable gastric bandis not yet available in the United States pending theresults of a multicenter trial in progress.Gastric BypassBased on the observation that patients with asmall proximal gastric remnant after subtotal gastrectomyexperienced significant weight loss, GBP was firstused to treat obesity by Mason and Ito in 1969.120,121The original operation partitioned the stomach into asmall proximal cardia and a distal bypassed stomach,with a loop gastrojejunostomy to drain the proximalpouch. Various modifications have been introduced sincethen, such as Roux-en-Y gastrojejunostomy (Figure4C),122 in situ compartmentalization of stomach withoutdivision,123 and lengthening of the Roux limb.124 Thisprocedure is primarily a restrictive operation in thatingestion of a large meal is prevented by a small gastricpouch and a narrow stoma. In addition, the gastrojejunostomyconfiguration of this operation uses dumpingphysiology (characterized by lightheadedness, nausea,palpitations, diaphoresis and/or abdominal pain, and diarrhea)as a negative conditioning response when a highcarbohydrateliquid meal is ingested. Thus, after a purely

restrictive operation such as VBG, sweets eaters resistantto weight loss because of dietary indiscretions such ashigh-carbohydrate liquid meals may lose significantweight if their anatomy is reconfigured to Roux-en-YGBP.52 Dumping symptoms in response to oral glucoseoccur specifically in GBP but not in VBG patients, andthis phenomenon is closely associated with an elevatedserum enteroglucagon level.125,126Both procedures, VBG and GBP, are endorsed by theNIH Consensus Development Panel, but GBP has beenshown to be superior to VBG in weight reduction inseveral randomized, prospective comparisons109,110,127and has emerged as the gold standard operation.8 Longtermmaintenance of weight loss after GBP has beenexcellent. Pories et al.48 reported a series with 58%,55%, and 49% loss of excess weight at 5, 10, and 14years from surgery, respectively. More recently, Jonesreported a 62% loss of excess weight at 10 years.111Modifications that enhance the malabsorptive effects ofRoux-en-Y GBP include lengthening the Roux limb(thus shortening the distal common digestive channel).“Long-limb GBP,” in which the Roux limb is 150 cmlong, compared to the standard 50–75 cm,124 “distalGBP,” and “very very long Roux limb GBP,” in whichthe distal common digestive channel is shortened to50–100 cm,100 may achieve better weight loss in superobese(BMI $ 50) patients, although the potential fordebilitating malnutrition and vitamin deficiencies exists.With recent advances in minimally invasive surgicaltechniques, laparoscopic GBP has become feasible.128–131This technically intensive procedure carries a significant