chapter 6 neurotrophic support, gh/igf-1 and hpa axes vis...

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Chapter 6

Neurotrophic support, GH/IGF-1 and HPA axes vis-à-vis decreased longevity

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6.1 Introduction

6.2 Materials and methods

6.3 Results

6.4 Discussion

6.5 Summary

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6.1 Introduction

Neurotrophic factors (NTFs) are well known for their roles in supporting and

sustaining development, differentiation, maintenance and plasticity of various brain

regions and their functions throughout life. Any loss in NTF regulation and function

increase the risk of the nervous system for cognitive breakdown, degeneration and

different neuronal malfunctions. There exists a delicate balance between the trophic

support of various neurotrophic factors, which are programmed temporally as well as

spatially. Of these, brain-derived neurotrophic factor (BDNF) is well researched for

its role in neuronal maintenance, survival and growth of new neurons and synapses

(Huang and Reichardt 2001). Pituitary gland has a critical role in controlling many of

the physiological processes like growth, body composition, reproduction, stress-

adaptive responses, balance of sodium and water, lactation, thyroid function and many

others (Veldhuis 2013). Particularly, the anterior pituitary secretes growth hormone

(GH), thyroid-stimulating hormone (TSH), adrenocorticotropic hormone (ACTH),

follicle-stimulating hormone (FSH), luteinizing hormone (LH) and prolactin, the

hormones involved in a plethora of monitoring and regulatory functions (Veldhuis, et

al. 2013, Hohl, Ronsoni et al. 2014, Otto, Franca et al. 2014, Nicolaides, Kyratzi et al.

2015).

Insulin-like growth factor 1 (IGF-1) has anabolic effects in adults and has a

molecular structure similar to insulin. It is produced mainly as an endocrine hormone

in liver and acts in a paracrine or autocrine way in the target tissues after being

stimulated by GH. IGF-1 production is decreased by undernutrition, lack of GH

receptors, GH insensitivity or problems in downstream signaling pathways post GH

receptor including SHP2 and STAT5B. GH secretion has been shown to decreased in

old rats (Sonntag, Steger et al. 1980) as compared to the young ones. Insulin/IGF-1

like receptor pathway contributes significantly to the biologic ageing process.

Insulin/IGF-1-like signaling is conserved from worms like C. elegans to humans.

Indeed, in vitro experiments have shown that mutations (Kenyon, Chang et al. 1993)

reducing insulin/IGF-1 signalling extend life (Bartke 2011) by decelerating the

degenerative, ageing process. GH/IGF-1 axis is in fact, known to play an important

role in modulating the ageing process across species (Junnila, List et al. 2013).

BDNF helps neurons in their survival, growth and differentiation of new

neurons and synapses (Acheson, Conover et al. 1995). It is found in high

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concentrations in hippocampus, cerebral cortex and basal forebrain and is reported to

be involved in learning and memory (Yamada and Nabeshima 2003, Bekinschtein, et

al. 2008). Its levels show significant changes with ageing (Greising, Ermilov et al.

2014) and lack of BDNF has been reported to underlie / to be associated with various

neurodegenerative outcomes and depression (Calabrese, Guidotti et al. 2013, Dalby,

Elfving et al. 2013, Dwivedi 2013). BDNF is also known to regulate brain energy

metabolism and cardiovascular health (Rothman, Griffioen et al. 2012, van Praag,

Fleshner et al. 2014). Hence a study of BDNF in CNS and periphery is very important

in evaluating its role, if any, in obesity and ageing.

Any disruption of the homeostasis through physical or psychological stressors

is defined as stress. The body tries to manage the situation through various

mechanisms and one of them is the neuroendocrine (NE) system, which plays a

critical role in responding to stress. The NE system includes hypothalamus, pituitary

and adrenal glands. On being activated by corticotropin-releasing hormone and

arginine-vasopressin, pituitary gland secretes ACTH that cascades in to the secretion

of cortisol / corticosterone and other glucocorticoids from adrenal glands. The

corticoids released as a stress response, work in close collaboration and terminate the

stress situation through a negative feedback loop to the hypothalamus. ACTH is an

important component of the Hypothalamo-Pituitary-Adrenal (HPA) axis and is often

produced in response to biological stress (Papadimitriou and Priftis 2009, Nicolaides,

Kyratzi et al. 2015). Hypothalamus also releases corticotropin-releasing hormone

simultaneously. ACTH regulates the levels of the steroid hormone cortisol /

corticosterone, which is released from the adrenal glands.

Melatonin is one of the universal / most fundamental hormone in the

evolution of cellular functions. It is identified primarily due to its antioxidant activity

(Hardeland 2005) and the protection it gives from the Sun’s radiation (Hardeland,

Pandi-Perumal et al. 2006, Tan, Zheng et al. 2014). It also acts to control sleep and its

deficiency has been implicated in sleep disorders (Chang, Wu et al. 2009). In small

animals, it is involved in energy metabolism and body weight control. Indeed studies

have shown that chronic melatonin supplementation reduces abdominal fat and body

weight (Wolden-Hanson, Mitton et al. 2000) and hence it is proposed as an approach

to treat obesity, basically due to its ability to regulate brown adipose tissue

metabolism (Tan, Manchester et al. 2011). There is also support for its anti-ageing

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effects (Brown, Young et al. 1979, Touitou 2001) and it has been proposed as a

biomarker for the intrinsic process of brain ageing (Sharma, Palacios-Bois et al.

1989). Melatonin also restores the basal concentrations of pituitary hormones and

pituitary responsiveness to the levels observed in young rats (Diaz, Pazo et al. 2000).

Hence quantification of ACTH, melatonin and corticosterone in the WNIN/Ob obese

rat provides a platform to study the basal stress response and systemic inflammation

and their probable role in the development of various pathologies including decreased

longevity.

6.2 Materials and methods

6.2.1 Maintenance of animals

The animal feeding and maintenance protocol used for the studies is same as

that reported in chapter 2 of the thesis. Therefore, only the deviations if any from it

are given herewith. Three groups of rats (n=5-7) were taken for the study i.e. parental

WNIN control (WN), WNIN/Ob obese (OO) and WNIN/Ob lean littermates (OL) of

3, 12 and 15 months of age.

6.2.2 Blood drawing

Blood was drawn from the ophthalmic venous plexus (orbital sinus) using a

thin walled heparinised capillary tube. The animal was held on to a platform, and the

skin on the head was stretched a little for proper positioning of the eye. The capillary

tube was positioned at the inner corner of the eye (beside the eye ball); by gentle and

firm push the fragile ophthalmic venous plexus was ruptured. By capillary action the

blood entered the capillary tube and the blood flow was started by slight rotation of

the capillary tube (Donovan and Brown 2006). Approximately 1.5 mL of blood

(Diehl, Hull et al. 2001) was collected in pre-coated EDTA vacuettes (Shanghai

International Holding Corp. GmbH (Europe), Hamburg, Germany). The capillary tube

was removed and the pressure was released from the animal.

6.2.3 Collection of plasma samples

The anticoagulant EDTA containing tubes were used for collecting blood for

plasma preparation. After collecting blood, the tubes were gently mixed for

homogeneity and kept on ice in an Esky box. The tubes were centrifuged (Eppendorf

Centrifuge 5810R; Swinging-bucket rotor A-4-62, Eppendorf AG, Hamburg,

Germany) at 2,230 rpm (or 1000x g) for 12 minutes at 4 0C. The supernatant plasma

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from the EDTA tubes was carefully collected without disturbing the lower layers and

stored as aliquots at -20 0C for 4 hours and then at -80 0C till they were further

analyzed.

6.2.4 Collection of cerebrospinal Fluid (CSF) samples

CSF concentrations of growth factors, various metabolites and many other

compounds are used as a surrogate measure of their CNS availability. CSF

concentration is logically, a good perceptive marker of biological availability to the

CNS for hydrophilic or large molecular weight compounds with poor to moderate

permeability (Shen, Artru et al. 2004). Hence, in order to check the bioavailability of

BDNF to CNS, we collected CSF from the cisterna magna by the procedure described

below. According to a report (Consiglio and Lucion 2000), cisterna magna can be

used to tap the largest volume (i.e. 50 – 120 µl) of CSF from the adult rat CNS.

6.2.4.1 Construction of collection syringe

The plastic part of the 24G needle (Hindustan Syringes & Medical Devices

Ltd., Faridabad, India) was cut and the stainless steel needle was taken. PE-50 tubing

(of internal diameter 0.023” and outer diameter 0.038”) (BPE-T50; Harvard

Apparatus Inc. (Holliston, USA) of 30 cm length was used to cover most part of the

stainless steel needle, excepting the first 5 mm portion so that the needle does not

penetrate deep during the procedure of CSF collection. 1 tuberculin mL syringe

(Hindustan Syringes & Medical Devices Ltd., Faridabad, India) was then attached to

the other end to develop the suction pressure.

6.2.4.2 Anesthetizing rats and collecting CSF

The rat was placed in an induction chamber and anesthetized using 5 %

halothane. Checking the eye reflex using a wet cotton bud did confirmation of the

complete anaesthetization of the animal. After complete loss of the consciousness, the

animal underwent shaving of fur on the back of neck region using electric shaver and

scrubbed with 70 % ethyl alcohol for disinfection. During the procedure, a mask was

kept near the nostrils of the rat and the halothane concentration was maintained at ≈ 2

– 3 %. The anesthetized rat was secured with the ear bars in the stereotaxic frame

(51600; Stoelting Co., Wood Dale, IL, USA) while keeping a 450 angle between head

and body axes. On the back of neck, a depressible surface was felt between occipital

protuberance and the atlas spine using the fingertip. After disinfecting with 70 % ethyl

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alcohol the needle of the collecting syringe was inserted horizontally and medially at

the cisterna magna up to the limit of cessation of resistance. Then the plunger of the

syringe was withdrawn to allow the flow of CSF in the PE-50 tubing under a

moderate to slow suction pressure. CSF sample was colorless and the blood

contamination was closely monitored. In case of blood contamination, the PE tubing

was cut little above the area of contamination and rest of the CSF was aspirated in to

the syringe. The collected CSF was rapidly transferred in to a polypropylene

microfuge tubes and stored at -80 0C until used for various assays.

6.2.5 Estimation of IGF-1, GH, ACTH, Melatonin, Corticosterone in plasma and

BDNF in plasma, CSF and various brain parts

IGF-1 was estimated in plasma using Milliplex MAP Rat / Mouse IGF-1 kit

(Item # RMIGF187K, Millipore Corporation, MA, USA), whereas GH, ACTH and

BDNF were estimated using Milliplex MAP Rat Pituitary kit (Item # RPT86K).

Melatonin and Corticosterone were determined using Milliplex MAP Rat Stress

Hormone kit (Item # RSH69K).

Different kits used for these estimations follow the same principle and

procedure and hence to avoid repetitions, details are given only for Rat Pituitary

Hormones analysis.

Principle:

BioPlex assays are bead based multiplex capture sandwich immunoassays in

which an antibody to the target protein is covalently bound to internally dyed beads

known as microspheres. The microspheres are internally colour-coded with two

fluorescent dyes. Through precise concentrations of these dyes, 100 distinctly

coloured beads are created, each of which is coated with a specific capture antibody.

The analyte in the sample binds to the specific capture antibody on the coated bead; a

biotinylated antibody detects the bound analyte. The above reaction mixture is then

incubated with Streptavidin-Phycoerythrin conjugate, the reporter molecule to

complete the reaction on the surface of each microsphere. These microspheres are

allowed to pass rapidly through a laser that excites the internal dyes marking the

microsphere set and a second laser excites the Phycoerythrin, the fluorescent dye on

the reporter molecule. High-speed digital-signal processors identify each individual

microsphere and quantify the result of its bioassay based on fluorescent reporter

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signals. Multiple conjugated beads (multiplex) are added to each sample to obtain

multiple results at a time.

Materials and reagents:

11. Rat Pituitary Standard (LRPT-8086) – reconstituted with 250 µl of deionized

water to get a concentration of 10,000 pg/mL (for BDNF, ACTH) and 50,000

pg/mL (for GH).

• Preparation of Working standard – 50 µl of reconstituted standard was

made up to 200 µl with assay buffer to prepare 2,000 pg/mL, etc.

concentration of working standard (Standard-5). Serial dilutions of the

working standard were made by taking 50 µl of previous standard and adding

it to 200 µl of assay buffer to get next dilution.

12. Rat Pituitary quality controls 1 and 2 (LRPT-6086) – reconstituted with 250 µl

of deionized water.

13. Assay Buffer (LE-ABGLP).

14. Serum matrix (LRPT-SM) – reconstituted with 2 mL of deionized water first

and then made up to 6 mL of final volume by adding 4 mL of assay buffer.

15. Rat Pituitary Detection Antibodies (LRPT-1086).

16. Streptavidin – Phycoerythrin (L-SAPE).

17. Bead Diluent.

18. 10x wash Buffer (L-WB) – diluted to 1x with deionized water.

19. Set of a 96-well filter plate with few sealers.

20. Mixing bottle.

21. Anti-BDNF beads, Anti-GH beads and Anti-ACTH Beads. Each individual

antibody-bead vial was sonicated for 25 seconds and vortexed for 1 minute.

150 µl of each antibody-bead was added to the mixing bottle and the final

volume was made up to 3 mL with bead diluent and vortexed again to prepare

the antibody-immobilized beads.

22. Luminex Sheath Fluid (#40-50000).

Procedure:

18. All the reagents were warmed to room temperature (20-25 0C) before use

in the assay.

19. 96-well filter plate was placed on a plate holder at all times to avoid the

bottom of the plate touching any surface.

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20. To prewet the filter plate, 200 µl of assay buffer was added to the filter

plate, sealed and mixed on a plate shaker for 10 minutes at room

temperature.

21. After the incubation, the assay buffer was removed by vacuum and the

excess assay buffer from the bottom of the plate was removed by blotting

on a tissue paper.

22. 25 µl of each of the standard, control and assay buffer (used as background

/ Standard 0) was added to the appropriate wells on the filter plate.

23. 25 µl of assay buffer was added to the sample wells.

24. 25 µl of serum matrix was added to the background, standards, and control

wells.

25. 25 µl of the sample (diluted 1:3 with serum matrix) was added to the

sample wells.

26. The mixing bottle containing the bead mix was vortexed and 25 µl of the

bead mix was added to each well, sealed with a plate sealer, covered with

the lid and incubated with agitation on a plate shaker overnight (16-18

hours) at 4 0C.

27. After overnight incubation, the fluid was gently removed by vacuum.

28. The filter plates were washed 3 times by adding 200 µl of wash buffer /

well. The wash buffer was removed by vacuum filtration between each

wash and the excess buffer from the bottom of the plate was removed by

blotting on a tissue paper.

29. 50 µl of detection antibody was added to each well, and then the filter

plate was sealed, covered with the lid and kept for incubation with

agitation on a plate shaker for 30 minutes at room temperature.

30. After incubation, 50 µl of Streptavidin – Phycoerythrin was added to each

well and then the filter plate was sealed, covered with the lid and kept for

incubation with agitation on a plate shaker for 30 minutes at room

temperature.

31. The contents were gently removed by vacuum.

32. The filter plates were washed 3 times by adding 200 µl of wash buffer /

well. The wash buffer was removed by vacuum filtration between each

wash and the excess buffer from the bottom of the plate was removed by

blotting on a tissue paper.

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33. 100 µl of Sheath fluid was added to each well and the beads were re-

suspended by agitation on a plate shaker for 5 minutes.

34. The filter plate with the re-suspended beads was run on a Luminex 200

system with BioPlex Manager, version 4.1, build 431, Bio-Rad

Laboratories, Inc., CA, USA.

Calculations:

The Median Fluorescent Intensity data using a weighted 5-parameter logistic

curve fitting method was used for calculating the analyte concentrations in samples.

One derivation of the 5PL equation is expressed below as an example:

where:

a = estimated response at zero concentration

b = slope factor

c = mid-range concentration (C50)

d = estimated response at infinite concentration

g = asymmetry factor

Statistical analyses

The data were analysed by Graphpad Prism 6.0f for Mac OS X (Graphpad

Software Inc., San Diego, CA, USA). Significance of difference among the 3 groups

of rats for each parameter studied at 3 age points was analysed by two-way analysis of

variance (ANOVA) followed by post hoc Tukey’s multiple comparison tests

appropriately. A p-value <0.05 was considered significant for the f ratio obtained by

two-way ANOVA and the t values obtained in the post hoc Tukey’s multiple

comparison tests between different groups.

y = d + [(a – d) / {1 + (x/c)b}g]

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6.3 Results

6.3.1 IGF-1 and GH axis

The plasma IGF-1 level (Table 6.1) in WNIN/Ob obese rats (OO) was

significantly higher than that in parental WNIN (WN) and WNIN/Ob lean littermates

(OL) at 3 and 15 months of age but not at 12 months. There were no such changes in

the normal rats across the ages. The pooled value representing the overall levels of an

animal group also was significantly higher (P<0.0001) in the OO than the two control

groups (WNIN and WNIN/Ob lean). On the other hand plasma levels of GH (Table

6.2) were significantly lower (P<0.0001) than that in both the controls groups. In line

with available literature the control rats were observed to show significant decreased

levels as an ageing effect.

Table 6.1 IGF-1 levels (ng/mL) in plasma of WNIN (WN), WNIN/Ob lean (OL)

and WNIN/Ob obese (OO) at 3, 12 and 15 months of age.

Age

Group 3 m 12 m 15 m Pooled Value

WN 266±9.4α 274±8.2 306±12.8α 282±12

OL 256±9.9aα 278±8.5ab 320±17.4bα 284±19

OO 360±16.6β 325±11.5 383±16.6β 356±17****

Across groups F(2, 54) = 32, P <0.0001

Across time points F(2, 54) = 11, P <0.0001

Interaction F(4, 54) = 1.5, P = 0.221

Data analysed using two-way ANOVA and represented as mean±S.E.M. The differences were

considered significant at minimum of P<0.05. The superscripts a, b and c represent the significant

differences (ageing changes) in an animal group across a row; α, β and γ represent the significant

differences among groups at a given age point, across a column. The pooled values (mean±S.E.M.)

represent the overall plasma level of IGF-1 in an animal group irrespective of age. Significant changes

among the groups for the pooled values are shown with asterisks (****P<0.0001).

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Table 6.2 GH levels (pg/mL) in plasma of WNIN (WN), WNIN/Ob lean (OL) and

WNIN/Ob obese (OO) at 3, 12 and 15 months of age.

Age


WN 6196±234aα 4357±284bα 3546±198bα 4700±784

OL 5882±453aα 3751±164bα 2679±195cα 4104±941

OO 832±165β 578±91β 368±79β 592±134****

Across groups F(2, 54) = 273, P <0.0001


Interaction F(4, 54) = 10, P <0.0001





represent the overall plasma level of GH in an animal group irrespective of age. Significant changes


6.3.2 BDNF as the neurotrophic support in central and peripheral systems

Plasma BDNF levels decreased (Table 6.3) with increasing age in normal rats.

Interestingly, plasma BDNF levels were significantly lower in young OO rats than

both the control groups and the lower BDNF levels continued at all age points

studied. However the decrease in CSF BDNF levels (Table 6.4) was only significant

at 15 months age in all the animal groups as compared to the levels at 3 months of

age. Also there were no changes in BDNF levels in CSF across the groups as such.

Still the pooled value for OO showed a significant decrease as compared to both of

the control groups.

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Table 6.3 BDNF levels (pg/mL) in plasma of different groups of animals, WNIN

(WN), WNIN/Ob lean (OL) and WNIN/Ob obese (OO) at the age of 3, 12 and 15

months.

Age


WN 2610±155aα 1984±219aα 1647±162bα 2080±282

OL 2478±132aα 1844±158abα 1596±167bα 1973±263

OO 495±75β 597±93β 556.6±42β 550±30****

Across groups F(2, 54) = 107, P <0.0001


Interaction F(4, 54) = 4.3, P = 0.005





represent the overall plasma level of BDNF in an animal group irrespective of age. Significant changes


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Table 6.4 BDNF levels (pg/mL) in CSF of different groups of animals, WNIN


months.

Age


WN 50.5±3.18a 40.7±3.51ab 32.5±1.92b 41.24±5.200

OL 48.8±2.85a 38.6±3.89ab 35.3±2.17b 40.91±4.063

OO 44.7±3.05a 33.5±2.39ab 24.5±1.93b 34.22±5.834***

Across groups F(2, 54) = 5.8, P = 0.005


Interaction F(4, 54) = 0.44, P = 0.78



differences (ageing changes) in an animal group across a row. The pooled values (mean±S.E.M.)

represent the overall CSF level of BDNF in an animal group irrespective of age. Significant changes

among the groups for the pooled values are shown with asterisks (***P<0.001).

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The BDNF levels in cerebral cortex (Table 6.5) were not different across

various groups but showed a trend of decreasing levels during ageing, albeit the

pooled BDNF levels in cerebral cortex of OO were significantly lower (P<0.001) than

those in the control groups. In hippocampus (Table 6.6) the BDNF levels were

significantly lower at the young age in OO than the controls and so were the pooled

values (P<0.0001). Similar to cerebral cortex, there was a slight decrease in BDNF

levels in the hippocampus during ageing in all the groups. In hypothalamus (Table

6.7), BDNF levels were very high as compared to other brain regions (e.g. cerebral

cortex and hippocampus). Here again, the levels were significantly lower in OO at the

young age compared that in age-matched controls and a similar change was seen in

the pooled BDNF levels (P<0.0001).

Table 6.5 BDNF levels (pg/mg total protein) in the cerebral cortex of different

groups of animals, WNIN (WN), WNIN/Ob lean (OL) and WNIN/Ob obese (OO)

at the age of 3, 12 and 15 months.

Age


WN 7.79±0.596a 7.72±0.430ab 5.93±0.287b 7.15±0.606

OL 7.99±0.506 8.00±0.617 6.09±0.476 7.36±0.636

OO 6.66±0.780a 5.57±0.491b 4.67±0.294b 5.63±0.574***

Across groups F(2, 54) = 9.9, P = 0.0002


Interaction F(4, 54) = 0.42, P = 0.792




represent the overall BDNF level in cerebral cortex in an animal group irrespective of age. Significant

changes among the groups for the pooled values are shown with asterisks (***P<0.001).

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Table 6.6 BDNF levels (pg/mg total protein) in the hippocampus of different



Age


WN 21.4±1.58aα 19±0.7ab 15.5±0.71b 18.7±1.70

OL 19.6±1.07α 19.2±1.02 15.1±0.48 18±1.43

OO 9.8±0.81aβ 17.9±1.90b 13.7±0.66ab 13.8±2.34****

Across groups F(2, 54) = 18, P <0.0001

Across time points F(2, 54) = 9.9, P = 0.0002

Interaction F(4, 54) = 8.3, P <0.0001





represent the overall BDNF level in hippocampus in an animal group irrespective of age. Significant

changes among the groups for the pooled values are shown with asterisks (****P<0.0001).

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Table 6.7 BDNF levels (pg/mg total protein) in the hypothalamus of different



Age


WN 39.4±4.69α 33.4±2.66 31.5±2.63α 34.8±2.38

OL 31.1±3.11α 27.1±2.37 26.1±2.07αβ 28.1±1.54

OO 14.8±1.11β 22.5±2.93 18.4±2.17β 18.6±2.23****

Across groups F(2, 54) = 26, P <0.0001

Across time points F(2, 54) = 1.0, P = 0.372

Interaction F(4, 54) = 2.0, P = 0.107


considered significant at minimum of P<0.05. The superscripts α, β and γ represent the significant


represent the overall BDNF level in hypothalamus in an animal group irrespective of age. Significant


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6.3.3 Hormones in stress response

Plasma ACTH levels (Table 6.8) of OO rats were significantly higher than

WN as well as OL control groups at all the time points studied. Also there was a

significant increase in plasma ACTH levels at the age of 15 months in control groups

indicating the effect of ageing. Interestingly, these levels were higher in 3 months old

OO than in 15 months old WN and 3OL group animals. The pooled ACTH levels also

showed a significant increase of comparable magnitude. (P<0.0001) in OO rats.

Table 6.8 ACTH levels (pg/mL) in plasma of different groups of animals, WNIN


months.

Age


WN 193.1±10.16aα 273.0±32.68aα 460.6±43.00bα 308.9±79.29

OL 237.8±13.80aα 345.4±27.64aα 592.7±44.65bα 392.0±105.1

OO 1480.7±73.91β 1441.73±60.95β 1646.3±106.61β 1523±62.72****

Across groups F(2, 54) = 468, P <0.0001


Interaction F(4, 54) = 0.90, P = 0.468





represent the overall plasma level of ACTH in an animal group irrespective of age. Significant changes

among the groups for the pooled values are shown with asterisks (****P<0.0001)

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Plasma melatonin levels (Table 6.9) were significantly lower in 3 months old

OO rats than the corresponding controls. Further, Plasma melatonin levels were

observed to decrease with age in all groups of rats. The pooled value of melatonin

levels also showed a significant decrease (P<0.0001) in OO rats compared to the

controls.

Table 6.9 Melatonin levels (pg/mL) in plasma of different groups of animals,

WNIN (WN), WNIN/Ob lean (OL) and WNIN/Ob obese (OO) at the age of 3, 12

and 15 months.

Age


WN 84.6±6.27aα 60.0±3.08b 48.6±3.76b 64.4±10.6

OL 83.1±6.95aα 67.8±3.84a 41.9±4.10b 64.3±12.0

OO 54.1±6.86β 40±3.5 32.8±2.59 42.3±6.24****

Across groups F(2, 54) = 21, P <0.0001


Interaction F(4, 54) = 1.7, P = 0.163





represent the overall plasma level of melatonin in an animal group irrespective of age. Significant


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Plasma corticosterone levels (Table 6.10) were significantly higher in OO rats

at 15 months of age but not earlier. Compared to the values at 3 months of age,

plasma corticosterone levels decreased at 12 months, but at 15 months the values

increased significantly in all three groups. However the pooled values were

comparable among the three groups.

Table 6.10 Corticosterone levels (pg/mL) in plasma of different groups of

animals, WNIN (WN), WNIN/Ob lean (OL) and WNIN/Ob obese (OO) at the

age of 3, 12 and 15 months.

Age


WN 54.6±4.46 47.5±2.45 72.3±12.79 58.1±7.39

OL 34.7±6.68a 25.8±5.60a 72.9±7.73b 44.5±14.5

OO 37.7±1.79a 33.0±5.57a 86.6±10.71b 52.4±17.1

Across groups F(2, 54) = 2.7, P = 0.08


Interaction F(4, 54) = 1.5, P = 0.21




represent the overall plasma level of corticosterone in an animal group irrespective of age.

6.4 Discussion

WNIN/Ob obese rat presents a unique animal model combining the features of

obesity and accelerated ageing. Considering their established role in / association with

ageing, we endeavoured to evaluate various neurotrophic factors, anterior pituitary

hormones, and stress hormones in the WNIN/Ob obese rats that are established rat

models of obesity and metabolic syndrome and show reduced longevity / accelerated

ageing. Accumulation of proinflammatory cytokines during ageing increases the risk

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of brain for cognitive decline and dementia (Cotman 2005) by generating a state of

neurotrophic resistance. Under these circumstances even if the levels of neurotrophic

factors are normal there is a greater probability of their dysfunction in the ageing

brain.

GH secreted from anterior pituitary, circulates all over body to exercise

important actions on growth and metabolism. It signals the secretion of IGF-1 that

mediates growth-promoting functions of GH. The role of GH/IGF-I axis is well

recognized in the longevity of organisms ranging from Caenorhabditis elegans to

mice (Bartke, Sun et al. 2013, Ding, Sackmann-Sala et al. 2013). GH/IGF-1 axis plays

an important role in modulating the ageing process across species from worms to

mammals (Holzenberger 2004, Junnila, List et al. 2013). Therefore, to start with we

checked the levels of GH and IGF-1 in the plasma of WNIN/Ob obese, WNIN/Ob

lean and parental WNIN control rats of different ages and observed that the levels of

GH to decrease with age (Sonntag, Steger et al. 1980) in the WN as well as OL

groups. In line with previous observations (Sonntag, Steger et al. 1980), GH levels

were significantly low (p<0.001) at 3 months of age in OO rats, and the levels

remained so throughout its ageing. On the other hand, levels of IGF-1 were

significantly increased in the WNIN/Ob obese rats compared to controls at 3 months

of age. This increase became less significant at 12 months of age, but a significant

increase was observed again at 15 months of age. IGF-1 levels are high in children as

it is required for their normal growth and development of various tissues. Although

IGF-1 level is known to decrease in adults and during normal ageing (Nessi, De Hoz

et al. 1995, Kuwahara, Kesuma Sari et al. 2004, Bartke 2005), a few studies have

reported that it increases during ageing where high risks of cancer are involved

(Bartke 2008, Bartke 2009). Considering that high levels of plasma IGF-1 have been

reported in cancer patients (Yu, Spitz et al. 1999, Otake, Takeda et al. 2010, Llanos,

Brasky et al. 2013, Guevara-Aguirre and Rosenbloom 2014), the high plasma IGF-1

levels in WNIN/Ob obese rats may explain / be associated with the greater incidence

of different types of tumours and increased DNA damage (Harishankar, et al. 2011,

Reddy, et al. 2014, Sinha, et al. 2014b). Deficiency in GH signaling is known to delay

ageing and remarkably extend longevity in laboratory mice (Brown-Borg, Borg et al.

1996, Flurkey, Papaconstantinou et al. 2001, Bartke, Sun et al. 2013) and similarly

humans with similar mutations are also reported to be benefitted (Guevara-Aguirre,

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Balasubramanian et al. 2011). Reduced levels of GH is consistent in elderly people

(Rudman 1985, Giustina and Veldhuis 1998, Muniyappa, Sorkin et al. 2007, Veldhuis

2008) and hence it has been proposed as a result or symptom or even as a ‘biomarker’

of ageing (Bartke, Sun et al. 2013). So the drastically low levels of GH may be an

important cause of the disturbed physiology of the WNIN/Ob obese rats.

BDNF is indispensable for neuronal maintenance, survival and growth of new

neurons and synapses (Lewin and Barde 1996, Huang and Reichardt 2001). Its

involvement is also reported in mood and eating disorders (Nakazato, Hashimoto et

al. 2006, Monteleone, Castaldo et al. 2008), mental health or psychiatric disorders

(Weickert, Hyde et al. 2003, Kim, Lee et al. 2007, Roth, Lubin et al. 2009, Castren

2014, Mitchelmore and Gede 2014), depression, cognition (Roth, Lubin et al. 2009),

neurodegeneration and ageing (Sohrabji and Lewis 2006, Komulainen, Pedersen et al.

2008, Erickson, Prakash et al. 2010, Driscoll, Martin et al. 2012). Considering that

BDNF is essential for survival and function of hippocampal, cortical, basal forebrain,

and entorhinal cortex neurons, our finding of extremely low BDNF levels in plasma,

hypothalamus and hippocampus at an early age of 3 months suggests strongly about

the lack of essential microenvironment for the normal maintenance of the neurons in

different parts of the brain. That the levels didn’t improve much even at later age

points probably corroborates the above inference that the deficiency of BDNF may be

correlated with / underlie the accumulation of different negative factors in the brain.

Also, our study is in line with some recent reports that through epigenetic

programming obesity reduced the expression of BDNF that had long-term deleterious

effects in the brain. Further this could contribute to the early onset of cognitive

decline during ageing (Greising, Ermilov et al. 2014, Wang, Freire et al. 2014).

Epigenetic studies in the WNIN/Ob obese rats would help to find the plausible causes

of the disturbed physiology.

The anterior pituitary (or adenohypophysis) regulates several biological

processes like stress, growth, reproduction, and lactation (Veldhuis 2013). It contains

five specialized hormone-secreting cell types that have many regulatory functions and

maintains homeostasis (Nicolaides, Kyratzi et al. 2015). They are: (a) Corticotropes

produce ACTH that acts on the adrenal gland, (b) Gonadotropes secreting FSH and

LH that regulate gonadal functions, (c) Lactotropes producing prolactin that acts on

the mammary glands, (d) Somatotropes secreting GH that targets the liver and bone,

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and (e) Thyrotropes secreting TSH that targets the thyroid gland (Watkins-Chow and

Camper 1998, Savage, Yaden et al. 2003, Gumbel, Patterson et al. 2012) In terms of

stress and its response, ACTH plays important role. After being activated by

corticotropin-releasing hormone and arginine-vasopressin, pituitary gland secretes

ACTH that signals adrenal glands to release cortisol / corticosterone and other

glucocorticoids and this cocktail of corticoids lays off the condition of biological

stress and hence ACTH secretion is considered important to check the initiation of

stress management (Papadimitriou and Priftis 2009, Nicolaides, Kyratzi et al. 2015).

As compared to the controls rats the levels of corticosterone (Sakamuri, et al. 2011)

and ACTH in WNIN/Ob obese rats were significantly higher at all the ages studied.

These findings clearly indicate increased oxidative stress in the animal including its

brain and hence increased plasma levels of ACTH and corticosterone in them in

response to the increased oxidative stress, probably to cope up with the stress

situation. In our studies, the ACTH levels were observed to increase slowly but not

significantly with age in control as well as WNIN/Ob obese rats. These findings

suggest the probable utility of plasma ACTH as a marker to assess the effectiveness /

efficacy of anti-oxidants in future studies, by determining whether or not the pituitary

gland is getting the signal of decreased biological stress and responding accordingly

by modulating the expression of ACTH and corticosterone. Although corticosterone

levels were higher in WNIN/Ob obese rats than WNIN controls at 15 months of age

around which the morbidity rate is high, but not at younger age. As reported in

chapter 2 regarding CRP levels that are known to put individuals at high risk of CVDs

and morbidity, corticosterone levels were also observed to follow similar trend. These

results suggest increased biological stress in the rats and hence in the same context we

evaluated the levels of melatonin as it is known to have strong anti-oxidant activity

(Hardeland 2005). In parental WNIN controls we observed a significant decrease in

the melatonin levels with age whereas the WNIN/Ob obese rats the levels were

significantly low right from the age of 3 months. The low levels of melatonin in

WNIN/Ob obese rats perhaps represent a compromised situation where the stress is

high and antioxidants are low, which can disturb sleep-wake cycle causing sleep

disorders (Chang, Wu et al. 2009) and may be associated with neurodegeneration and

ageing (Videnovic, Lazar et al. 2014). Considering the reports that rats supplemented

with melatonin had decreased abdominal fat and body weight (Wolden-Hanson,

Mitton et al. 2000), monitoring melatonin levels may be a useful approach to treat

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obesity as melatonin can regulate brown adipose tissue metabolism (Tan, Manchester

et al. 2011), is considered as having anti-ageing effects (Brown, Young et al. 1979,

Touitou 2001) and its deficiency is considered a biomarker for the intrinsic process of

the brain ageing (Sharma, Palacios-Bois et al. 1989). In view of the literature referred

above, our findings that are in line with these reports indicate that many of the

important biological processes were affected in WNIN/Ob obese rats at an early age

of 3-6 months. The neural circuits regulating food intake converge at the

paraventricular nucleus that also contains corticotrophin releasing hormone (CRH)

and urocortin containing neurons. Considering that hypothalamo-pituitary-adrenal or

HPA axis regulates biological stress responses as well as feeding behavior of an

animal (due to sharing same neuroanatomy), it becomes evident that both systems can

influence each other in stimulating a response (Maniam and Morris, 2012). Future

studies with supplementation of melatonin in WNIN/Ob obese rats appears to be a

possible way out for modulating obesity / early ageing in them as it has been shown

that melatonin reinstates pituitary responsiveness and base levels of pituitary

hormones in old rats to the levels observed in young ones (Diaz, Pazo et al. 2000).

6.5 Summary

• Significantly lower levels of GH and increased levels of IGF-1 in WNIN/Ob

rats points to an altered GH/IGF-1 axis, which is an important determinant of

longevity. This finding is suggestive of the increased susceptibility of

WNIN/Ob obese rats towards various types of tumors and cancers.

• BDNF levels were significantly low in plasma, hippocampus and

hypothalamus which shows poor neurotrophic support to brain regions,

thereby leading to neurodegeneration and accelerated ageing phenotype.

• Increased levels of ACTH and corticosterone, and concomitant decreased

levels of melatonin in plasma of young WNIN/Ob obese rats clearly reveals

increased biological stress and decreased antioxidant like activity, probably

accelerating the ageing process.

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Chapter 7

General discussion and conclusion

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7.1 General discussion

7.2 Conclusions

7.3 Limitations of the study

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7.1 General discussion

Lifespan is the duration of time an organism lives and longevity is the

expected average lifespan under ideal conditions. Lifespan is for an individual and

differs as per the prevailing conditions; but longevity covers mathematical terms like

average lifespan of a given population that is normally observed in absence of certain

disorders like cancer, stroke, etc. Ageing is a part of the lifespan that constitutes the

second half of the life story and is indeed very important (Sinha and Ghosh, 2010). As

the ageing population is on rise all over the globe, we need to be prepared with more

knowledge of the other co-existent disorders with different mechanisms playing their

roles to further deteriorate the normal ageing process. These include CVDs,

Alzheimer’s disease, Parkinson’s disease, diabetes, macular degeneration, various

cancers and obesity among others. These are well known to accelerate the ageing

process and cause reduced longevity of various organisms (Butterfield et al., 2001).

Numerous strategies have been tried in the past and continue to be tried almost

everywhere mainly to increase the longevity of human beings in general and a healthy

ageing in particular. It is very important to understand the basic mechanisms of

normal ageing and also the changes in different pathways in different disorders that

may be a reason or causally related to the ageing process. Accelerated ageing is even

more important to study because the mechanisms work out much faster and in an

unregulated way (Oliver et al., 1987). If the points where this disarrangement or

mismanagement occurs can be found out, that would be a great milestone in ageing

research. For such a kind of research, good animal models are required representing

real human situations or afflictions.

The factors that determine the longevity of an organism can be divided into

genetic and environmental (modifiable) ones. Obesity is one of the co-existing health

problems, which is known to cause an early death. Different metabolic pathways are

being researched upon to unravel the mystery but still the actual mechanism is

elusive. In such a scenario, it is of immense importance to develop appropriate animal

models that show the characteristics of both the co-morbid health situations. At NIN,

researchers have developed the WNIN/Ob obese rat strain using selective back

crossing, which shows morbid obesity (Giridharan, 1998) as well as significantly

reduced longevity. These are the first inbred mutant obese rat model which is also the

heaviest known till date (maximum ~1.47 Kg). Studies on these rats show the

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presence of various complications like retinal degeneration, cataract, compromised

immunity, hyperinsulinemia, hypercholesterolemia, hyperleptinemia, infertility,

polycystic ovaries and other kinds of tumors, etc. (Reddy et al., 2009, Harishankar et

al., 2011, Bandaru et al., 2013). Recently the mutation has been shown to be in 4.3cM

region on chromosome 5 upstream of the leptin receptor (Kalashikam et al., 2013) and

also altered leptin gene promoter methylation (Kalashikam et al., 2014). To the best of

our knowledge, no studies have been undertaken to establish them as an appropriate

model of reduced longevity / accelerated ageing and also the associated / underlying

mechanisms specially in their brain, considering the pivotal role the brain plays in

modulating a variety of physiological functions including ageing. Therefore, these

studies were conducted in the WNIN/Ob, the obese mutant rat with reduced longevity.

Evaluation of growth characteristics, survival and regional brain volume

changes

WNIN/Ob obese rat is the obese rat model with significantly decreased

longevity and hence could be a very useful model to study obesity associated

accelerated ageing. Although WNIN/Ob obese rats have been known to have

decreased lifespan, it has not been established / validated statistically nor reported

scientifically. In order to find out the probable mechanisms underlying the early death

in these rats, it is essential to establish them first, as models for accelerated ageing.

Therefore we attempted to scientifically establish and statistically validate the

accelerated ageing / decreased longevity of the WNIN/Ob obese rats. One of the

established experiments to examine the effects of genetic manipulations and various

chemical compounds on ageing is the measurement of lifespan. This has traditionally

been accomplished by survival analysis over the lifetime (Yang et al., 2011) because

one can derive interesting and useful information by appropriate statistical survival

analysis of the survival data (Valenzano et al., 2006, Harrison et al., 2009, Honjoh et

al., 2009, Yang et al., 2011). Using Kaplan – Meier estimation, we found a

significantly lower mean lifespan of 420 days in WNIN/Ob obese rats as compared to

the parental WNIN normal as well as WNIN/Ob lean littermates where it was 1067

and 1035 days respectively, which is around 60% reduction. Considering the

calculations of George Sacher using least squares regression of log lifespan (Sacher,

2008), there is relationship between brain weight and lifespan, and hence we checked

the brain wet weights of these rats. We found significantly decreased brain weights in

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WNIN/Ob obese rats as compared to both the control groups at all the time points

studied. It was of interest that the brain weights of WNIN/Ob obese rats at 3 months

of age were lower than that of WNIN control and WNIN/Ob lean littermates at the

age of 18 months, indicating very premature occurance of age related decrease in the

brain weight in the WNIN/Ob obese rats (at a very young age itself) probably

suggesting accelerated ageing in them. It is reported that with advancing age, there

was a reduction in the volume of prefrontal and cingulated cortices’ (Shamy et al.,

2011), thinning of prefrontal and superior temporal cortices (Alexander et al., 2008),

reduction in gray and white matter volume (Wisco et al., 2008), thinning of

somatosensory and motor cortices and thickening of superior temporal and cingulate

cortices (Koo et al., 2012). As the brain weights were significantly lower in

WNIN/Ob obese rats at 3 months of age itself, we looked for the possibility of

neuronal loss and / or shrinkage of various brain regions reported in brain during old

age. Therefore the regional brain volumetric analysis was performed using MRI.

Although there was decreasing trend observed in different brain volumes, there were

no significant changes. Nevertheless it is possible that even in the absence of any

volumetric change in the brain, there could be a compromised microenvironment in

the brain causing the molecular pathways to go awry (Miranda et al., 2012).

Therefore, we conducted cellular studies to examine different cellular subsets i.e. glial

and neuronal evaluations.

Cellular (neuronal and glial) changes in the brain vis-à-vis decreased longevity

During normal ageing, many structural and functional changes occur in the

brain and for evaluating the gross cytoarchitechture changes in brain, Nissl staining is

widely used (Huang et al., 2013). Therefore to evaluate cytostructural changes we

used Nissl staining and observed the paucity of neurons in the cerebral cortical layers

in the brain of WNIN/Ob obese rats compared to WNIN controls and WNIN/Ob lean

littermates , which is line with previous reports of similar nature (Brizzee et al., 1980).

To our surprise, increased neuronal populations were seen in the peri-ventricular

region where various hypothalamic nuclei are present. As WNIN/Ob obese rats are

hyperphagic and orexin-A (Ox-A) is known to increase food intake by delaying the

onset of normal satiety sequence (Rodgers et al., 2002), we quantitated by counting

the absolute numbers of Ox-A positive neurons and found it to be significantly higher

in lateral and dorsomedial areas of hypothalamus of WNIN/Ob obese rats as

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compared to the normal rats. These findings indicate that the hyperphagy seen in these

rats could be due to increased activity of the orexigenic neurons.

C-reactive protein (CRP) is known to increase the blood-brain-barrier

permeability and enter the brain at high levels and is implicated in obesity and

inflammation (Hsuchou et al., 2012). Therefore we checked the CRP levels in the

plasma of these rats and found it be increased in the WNIN/Ob obese rats pointing its

possible role in the complex mechanisms of obesity and the associated ageing. High

CRP levels are known to cause astrogliosis (Hsuchou et al., 2012) and hence we

checked its marker GFAP in the brain of these rats. In immunohistochemical analysis

of the brain sections, we found an increased expression of GFAP in the hippocampus

of 6 months of old WNIN/Ob obese rats but at the age of 12 months it was observed

to decrease as compared to the control rats. This may be due to the operation of some

age dependent compensatory mechanisms in the brains of WNIN/Ob obese rats. To

further confirm this finding we did quantitative analysis of GFAP using Western

blotting and found similarly increased GFAP protein expression in the WNIN/Ob

obese rat brains compared to the WNIN and WNIN/Ob lean littermates of comparable

age. Also there was an increased GFAP expression observed in the lateral

hypothalamus and arcuate nucleus in the obese rats suggesting the probable damage to

neuronal integrity of these hypothalamic nuclei that are involved in regulation of

feeding behavior and hormonal control. This also seems to suggest that altered

signaling pathways in the brain cause / underlie obesity and reduced longevity of

WNIN/Ob obese rats. In view of these findings, it was considered pertinent to assess

the alterations if any in the various neurochemicals and brain metabolites (Haga et al.,

2009, Rothman et al., 2011) in order to analyze the changes in the microenvironment

of the brains of these rats. Therefore, as the next step we evaluated the neurochemical

profile and estimated the brain metabolism of these rats using 13C-glucose and NMR

spectroscopy.

Evaluation of neurochemical profile and brain metabolism of WNIN/Ob obese

rats using Nuclear Magnetic Resonance Spectroscopy

Neurochemical profiling using NMR is a sensitive approach to simultaneuosly

measure numerous key neurochemicals that are present in minute amounts. During

ageing, concentration of most of the metabolites in the brain reflecting structural and

functional properties of the specific region are altered to a significant level

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(Boumezbeur et al., 2010, Duarte et al., 2012, Duarte et al., 2014). Therefore the

complex communication among the neurometabolites gets modified either due to

ageing or in the process of adapting to the changing microenvironment of the nervous

system. This makes them good canditates to be considered as biomarkers of different

patterns of health and disease (Duarte et al., 2014). It is well known that the level of

different neurometabolites such as N-acetylaspartate (NAA), total creatine (Cr),

phosphorylethanoloamine (PE), taurine (Tau), glutamate (Glu), glutamine (Gln),

gamma-aminobutyrate (GABA), alanine (Ala), get altered under disturbed

physiological conditions, and could be reliably measured using in vivo and in vitro

MRS (Dedeoglu et al., 2004, Duarte et al., 2014, Harris et al., 2014). As compared to

in vivo MRS, a wider range of neurochemicals can be quantitated using in vitro MRS

(Dedeoglu et al., 2004) and hence we did the in vitro study to find the changes in the

neurochemical profile in cerebral cortex of WNIN/Ob obese rats as compared to the

controls rats. The neurochemical profile and the brain metabolism (of 13C-glucose)

have been determined in the cerebral cortex considering its importance in the process

of ageing (Villa et al., 2012). Glu is the main excitatory neurotransmitter and NAA is

an indicator of neuronal integrity. Tau is well known for its neuroprotective and

antioxidant-like activities in the brain. A decrease in the levels of these

neurometabolites indicates neuronal dysfunction and compromised state of

neuroprotection (Duarte et al., 2012). In this study, the concentrations of Glu, NAA

and Tau were significantly lower in the cerebral cortex of WNIN/Ob obese rats (than

that of controls), which suggests the altered microenvironment in the cerebral neurons

posing a damaging situation where neuronal death and associated changes like

astrogliosis can occur. The neurochemicals considered as markers of astrogliosis viz,

Gln and Inositol (Ino; also shows osmotic stress dysfunction) have been reported to be

increased during ageing (Zahr et al., 2014). We observed increased concentrations of

Gln and Ino in the cerebral cortex of these obese rats of 3 months age (compared to

the corresponding controls) again confirming the negative balance of the

neurochemicals favouring neurodegeneration and ageing. As these observations were

done in the obese rats at 3 months of age, our findings indicate that at a young age

only the neurochemical profile of these animals is getting altered and resembles the

changes seen in the control rat brains at later age (e.g. 15 – 18 months)(Duarte et al.,

2014), corroborating the accelerated ageing of the WNIN/Ob obese rats.

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In terms of metabolism, the energy needs of brain are very high. According to

the neuronal activity (or energy needs) this demand is normally fulfilled with stringent

regulatory mechanisms to deliver sufficient energy substrates (Hyder et al., 2006).

Therefore different metabolite concentrations reveal the activity of various metabolic

processes. To evaluate this, we infused 13C-labeled glucose in these rats through vein

and measured amount of 13C being incorporated in the brain metabolites involved in

neurotransmitter cycling. Neurons release neurotransmitters Glu and GABA (based on

the type of neuron i.e. either glutamatergic or GABAergic) into the synaptic cleft.

From the cleft, astrocytes take up the neurotransmitters and convert into Gln and

release to the neurons. Inside neurons Gln is hydrolyzed to Glu or GABA, re-

packaged into vesicles and be ready for the next release at the arrival of action

potential. Considering that GluC4 and GABAC2 labeling occurs via glucose

metabolism through the TCA cycle and their determination provides an estimate of

TCA cycle flux associated with glutamatergic and GABAergic neurons respectively,

the GlnC4 labeling represents the total neurotransmitter cycling associated with

glutamatergic and GABAergic neurons. Hence we evaluated the concentrations of

GluC4, GABAC2 and GlnC4 from the 1H-[13C] NMR spectra of the cortical extract and

found these levels to be significantly decreased in the WNIN/Ob obese rats compared

to controls, indicating hypoactivity and decreased metabolism. This hypometabolism

concomitant to probable neuronal damage (as evident from the observed changes in

neuronal and glial profile) and increased lipid peroxidation (shown by decreased

NAA and Tau) probably indicates insufficient supply of the energy to the obese rat

brain even at a young age of three months. That these microenvironmental alterations

and metabolic changes normally seen in the ageing brain (Duarte et al., 2014) were

observed in the young age in the obese rats seems to further strengthen the inference

that changes associated with normal ageing appear to be occurring much earlier in the

brain of the WNIN/Ob obese rats and could therefore underlie their accelerated ageing

/ decreased longevity. Considering that despite no changes observed in the volume of

different brain regions in the WNIN/Ob obese rat, the neurochemical profile and

metabolism were altered in the WNIN/Ob obese rat brain suggestive of accelerated

ageing and since ageing in general is known to be associated with stress (e.g.

oxidative and corticosteroid), it was considered imperative to assess the level of

oxidative stress, antioxidant enzymes’ activities and the attendant damage of

macromolecules in the brain of these rats.

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Extent of DNA damage, oxidative stress status and activity of antioxidant

enzymes in the brain of WNIN/Ob obese rats

Ageing is well known to be associated with increased oxidative stress (Finkel

and Holbrook, 2000, Marosi et al., 2012), different types of macromolecular damage

and accumulation of DNA damage, especially in the brain (Freitas and de Magalhaes,

2011, Moskalev et al., 2013). When these events are set in to motion prematurely, it is

termed as accelerated ageing, causing an early build-up of deteriorating factors

normally seen at an advanced age. Factors contributing to accelerated ageing include

genetic conditions, chronic disorders and unbalanced lifestyle related disorders like

obesity, cardiovascular diseases and type 2 diabetes mellitus (Tzanetakou et al., 2012,

Zhu and van der Harst, 2014). In addition, obesity is well demonstrated to be

associated with ageing (Harrington and Lee-Chiong, 2009, Tzanetakou et al., 2012,

Alfadda et al., 2013) with the probable mechanism being through chronic

inflammation caused by altered adipokine signaling (Michaud et al., 2013) and

increased oxidative stress in the body. Obesity has also been correlated with increased

oxidative stress (Furukawa et al., 2004). Enhanced and uncontrolled production of

reactive oxygen species (ROS) and an inefficient machinery of antioxidant enzymes

in turn results in DNA damage (Barzilai and Yamamoto, 2004). The brain has been

observed to be more vulnerable to age-related DNA damage compared to other tissues

(Price et al., 1971, Mori and Goto, 1982). DNA damage in the form of single strand

breaks has been found to be maximum in neurons of cerebral cortex in aging rat brain

(Mandavilli and Rao, 1996). Thus, brain can be considered as the appropriate tissue to

decipher role of oxidative stress and DNA damage in ageing.

Early accumulation of deteriorating factors (especialy in terms of

macromolecular damage) in the brain (Barzilai and Yamamoto, 2004) can cause

accelerated ageing in organism. Therefore, we checked the oxidative stress markers:

levels of lipid peroxidation and protein carbonyls in the cerebral cortex and

hippocampus of these young (3 month age) obese rats and found it to be significantly

higher than age matched parental WNIN and lean littermates. Indeed these levels

were as high as what was seen at an later time point (15 months) in the control rats,

indicating an early increase of oxidative stress in the obese rat brain. Considering the

signifcant increase observed in lipid peroxidation and protein carbonyl levels in the

obese rat brain, it was of interest to check for the damage of DNA if any in these

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brains because the study of Rutten et al. suggests that neurons show age-related

increase in the accumulation of DNA damage in mouse brain (Rutten et al., 2007).

The extent of DNA damage in the brain of these rats was assessed in terms of single-

and double-stranded breaks (SSBs and DSBs respectively) using Comet assay (Singh

et al., 1988). To our surprise these results were also in the line of our previous

observations, i.e. significantly higher DNA damage both in terms of SSBs and DSBs,

in both regions of brain of the WNIN/Ob obese rats than controls. Considering the

fact that macromolecular damage is a part of normal wear-and-tear processes occuring

in a cell which is constantly rejevunated by the activity of antioxidant enzymes and

other factors which prevent / repair the damage caused by oxidative stress, we

checked if the activites of superoxide dismutase and catalase, the two important

antioxidant enzymes in the brain and found it to be significantly lower in the obese

rats than controls. These observations clearly suggest the early accumulation of DNA

damage due to increased oxidative stress in the brain of these rats could be due to the

compromised antioxidant capacity of the brain. In addition it could also be due to the

lack of sufficient neurotrophic support and neuroprotection (due to low levels of

taurine as observed in the neurochemical profiling studies) causing these rats to die

earlier. Therefore, we checked the availability of brain-derived neurotrophic factor

(BDNF) in the brain and also evaluated the axes involved in longevity pathways i.e.

GH/IGF-1 and HPA axes in the brains of WNIN/Ob obese rats and the controls.

Neurotrophic support, GH/IGF-1 & HPA axes vis-à-vis decreased longevity

Importance of neurotrophic factors (NTFs) in the development, differentiation,

maintenance and plasticity of various brain regions is well known. There is always a

spatial and temporal balance of trophic support required in the CNS for normal

functioning. BDNF is well known for its role in neuronal maintenance, survival and

growth of new neurons and synapses (Huang and Reichardt, 2001). BDNF support is

known to decrease as the age of an animal advances (Lommatzsch et al., 2005,

Ziegenhorn et al., 2007, Sen et al., 2008, Tapia-Arancibia et al., 2008, Greising et al.,

2014) and this forms one of the reasons for decreased probability of neuronal

viability. Considering this we checked BDNF levels at 3, 12 and 15 months of age, in

various brain regions mostly affected during ageing (i.e. cerebral cortex, hippocampus

and hypothalamus) as well as the periphery (i.e. plasma and cerebrospinal fluid

(CSF)) of the WNIN/Ob obese rats and compared it with those of age matched

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parental WNIN control and lean littermates. We found a significantly lower BDNF

concentration in plasma at all the age points of these rats. In CSF, there was a

significant decrease during normal ageing but we saw only a decreasing trend in

WNIN/Ob obese rats where the levels were not significantly lower. When we checked

BDNF concentration in brain regions, we found a decreasing trend in the cerebral

cortex, whereas these levels were significantly lower in hippocampus and

hypothalamus. At the age of 3 months the obese rats showed more depressed BDNF

levels both in hippocampus and hypothalamus, which indicates an insult at an early

age changing the microenvironment, which is required for normal development and

maintenance of neural tissues. At 12 and 15 months of age the BDNF levels were

significantly lower than control rats but among obese rats, it improved as compared to

that observed at 3 months of age. This probably shows a compensatory mechanism

trying to improve the microenvironment by helping in the maintenance of the neural

tissue.

GH/IGF-1 axis plays an important role in modulating longevity across species

(Holzenberger et al., 2003). IGF-1 also plays a major role in mediating the effects of

longevity genes on ageing and life span (Bartke et al., 2003). GH secretion has been

shown to decrease in old rats (Sonntag et al., 1980) as compared to the young ones.

Insulin/IGF-1 like signaling pathway contributes significantly to the biologic ageing

process. Insulin/IGF-1-like signaling is conserved from worms like C. elegans to

humans. Indeed, in vitro experiments have shown that mutations (Kenyon et al., 1993)

reducing insulin/IGF-1 signalling extend life (Bartke, 2011) by decelerating the

degenerative, ageing process. GH/IGF-1 axis is in fact, known to play an important

role in modulating the ageing process across species and hence it is very important to

measure the levels of GH and IGF-1 in animal models to check if these are also

involved in reducing longevity (Junnila et al., 2013) of WNIN/Ob obese rats. In line

with available literature, we observed that the levels of GH to decrease with age

(Sonntag et al., 1980) in the rats of control groups and it was significantly low at 3

months of age in obese rats, and the levels remained so throughout its ageing. On the

other hand, levels of IGF-1 were significantly increased in the WNIN/Ob obese rats

compared to controls at 3 months of age. This increase became less significant at 12

months of age, but a significant increase was observed again at 15 months of age.

IGF-1 levels are high in children as it is required for their normal growth and

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development of various tissues. Although IGF-1 level is known to decrease in adults

and during normal ageing (Nessi et al., 1995, Kuwahara et al., 2004, Bartke, 2005), a

few studies have reported that it increases during ageing where high risks of cancer

are involved (Bartke, 2008, 2009). Considering that high levels of plasma IGF-1 have

been reported in cancer patients (Yu et al., 1999, Otake et al., 2010, Llanos et al.,

2013, Guevara-Aguirre and Rosenbloom, 2014), the high plasma IGF-1 levels in

WNIN/Ob obese rats may explain / be associated with the greater incidence of

different types of tumours and increased DNA damage (Harishankar et al., 2011,

Reddy et al., 2014, Sinha et al., 2014b) observed / reported in them. Reduced levels of

GH is consistent in elderly people (Rudman, 1985, Giustina and Veldhuis, 1998,

Muniyappa et al., 2007, Veldhuis, 2008) and hence it has been proposed as a result or

symptom or even as a ‘biomarker’ of ageing (Bartke et al., 2013). So the drastically

low levels of GH may be an important cause of the disturbed physiology of the

WNIN/Ob obese rats. Future studies are required to check the downstream signalling

and see if there are any other alterations in this longevity determinant-signalling

pathway.

As we observed an increased oxidative stress in the brain of WNIN/Ob obese

rats at an early age (Sinha et al., 2014b), we wanted to see if signalling from brain is

affecting biological stress (if any) in the body. Hypothalamo-pituitary-adrenal (HPA)

axis forms an important direct influencing and feedback interaction neuroendocrine

system controlling reactions to stress and regulating body processes like digestion,

energy storage and expenditure, immunity, sexuality and other psychological status of

an organism. In case of any kind of stress in the body, it tries to manage the situation.

On being activated by corticotropin-releasing hormone (from hypothalamus) and

arginine-vasopressin, pituitary gland secretes ACTH that cascades in to the secretion

of corticosterone and other glucocorticoids from adrenal glands in rodents. The

corticoids released as a stress response, work in close collaboration and terminate the

stress situation through a negative feedback loop to the hypothalamus. ACTH is an

important element of the HPA axis and is often produced in response to biological

stress (Papadimitriou and Priftis, 2009, Nicolaides et al., 2015). As compared to the

controls rats the levels of corticosterone (Sakamuri et al., 2011) and ACTH in the

plasma of WNIN/Ob obese rats were significantly higher at all the ages studied

showing the activation of HPA axis in response to the stress in body. In our studies,

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the ACTH levels were observed to increase slowly but not significantly with age in

control as well as WNIN/Ob obese rats. These findings suggest the probable utility of

plasma ACTH as a marker to assess the effectiveness / efficacy of anti-oxidants in

future studies, by determining whether or not the pituitary gland is getting the signal

of decreased biological stress and responding accordingly by modulating the

expression of ACTH and corticosterone. Plasma corticosterone levels were indeed

higher in WNIN/Ob obese rats than WNIN controls at 15 months of age around which

the morbidity rate is high, but not at younger age.

Melatonin is one of the most fundamental hormones in the evolution of

cellular functions and is identified primarily due to its antioxidant activity (Hardeland,

2005) and the protection it gives from the Sun’s radiation (Hardeland et al., 2006, Tan

et al., 2014). It also acts to control sleep and its deficiency has been implicated in

sleep disorders (Chang et al., 2009). In small animals, it is involved in energy

metabolism and body weight control. It has been proposed as a biomarker for the

intrinsic process of brain ageing (Sharma et al., 1989). We observed a significant

decrease in the levels of melatonin in the plasma of WNIN/Ob obese rats as compared

to the lean littermates as well as parental WNIN control rats at an early age of 3

months and these decreased levels always remained low compared to the control rats

during ageing. In such a scenario where we see most of the damaging / deteriorating

factors to be present in a young age of WNIN/Ob obese rats concomitant with lack of

antioxidants and neuroprotective factors, it becomes very clear that the genetic output

or easily incitable epigenetic factors either get activated at 3 months of age or earlier.

Chronic melatonin supplementation is known to reduce abdominal fat and body

weight (Wolden-Hanson et al., 2000) and hence it is proposed as an approach to treat

obesity, basically due to its ability to regulate brown adipose tissue metabolism (Tan

et al., 2011). There is also support for its anti-ageing effects (Brown et al., 1979,

Touitou, 2001) and also that it restores the basal concentrations of pituitary hormones

and pituitary responsiveness to the levels observed in young rats (Diaz et al., 2000).

Therefore future supplementation studies may be done in these rats to check the

different pathways getting rectified.

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7.2 Conclusions

• WNIN/Ob obese rats can be useful model for decreased longevity and

accelerated ageing.

• Changes seen in normally ageing control WNIN rats (around 15 months of

age) are seen in the WNIN/Ob obese rats at a much younger age of 3-6 months

of age, confirming the accelerated ageing in them.

• That neuronal loss and astrogliosis, altered neurochemical profile, increased

oxidative stress, decreased neurotrophic support, the factors that underlie

normal ageing also were seen in WNIN/Ob obese rats albeit at a much

younger age suggest that they also underlie the accelerated ageing in

WNIN/Ob obese rats.

7.3 Limitations of the study

• Since this study is the first attempt to establish the WNIN/Ob obese rats as an

appropriate model to study accelerated ageing / reduced longevity, we had

started with the minimum age point as 3 months. As it is evident from this

study that most of the accumulation of ageing factors appeared by the age of 3

months, in our future studies we would start experiments from an earlier age

point like the weaning age or may be embryos, to see how early the deleterious

changes begin to appear in these rats.

• As WNIN/Ob obese rats are infertile, have reduced longevity and high

susceptibility to opportunistic infections, obtaining the available number of

male rats for various studies remained a big hurdle. Considering that these

studies were planned in male rats that are difficult to procure, in few of our

experiments the number of animals was less than six. As per the clues we have

obtained from the present studies, we will plan and perform future

experiments taking this factor into account.

• It is important to do rehabilitation studies (e.g. calorie restriction,

supplementation with various antioxidants and therapeutic substances like

resveratrol, etc.) in order to prove the causal relationship (proof-of-concept) of

various deleterious factors to accelerated ageing in WNIN/Ob obese rats. As

mentioned earlier, that these are the first exploratory studies on ageing in these

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rats, future studies will be executed taking care of all the factors including

present knowledge and evidences obtained.

Bibliography

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