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Neuropeptide Y and Peptide YY Inhibit Lipolysis in Humanand DogFat Cells through a Pertussis Toxin-sensitive G ProteinPhilippe Valet,* Michel Berlan,* Michel Beauville,§ Franmois Crampes,* Jean L. Montastruc,t and Max Lafontan**Institut de Physiologie, Institut National de la Sante et de la Recherche Medicale (INSERM) U-31 7, Universite Paul Sabatier, 31400Toulouse, France; tLaboratoire de Pharmacologie Medicale and Clinique, INSERMU-317, 31073 Toulouse Cedex, France; and§Laboratoire de Physiologie, Hopital Purpan, 31052 Toulouse, France

Abstract

Neuropeptide Y (NPY) and peptide YY (PYY) are regulatorypeptides that have considerable sequence homology with pan-creatic polypeptide. Because (a) NPYhas been shown to becolocalized with noradrenaline in peripheral as well as centralcatecholaminergic neurons, and (b) alpha2-adrenergic recep-tors of adipocytes play a major role in the regulation of lipoly-sis, we investigated the effect of NPYand PYY on isolatedfat cells.

In human fat cells NPYand PYYpromoted a dose-depen-dent inhibition of lipolysis elicited by 2 jg/ml adenosine deam-inase (removal of adenosine) whatever the lipolytic index used(glycerol or nonesterified fatty acids). In dog fat cells NPYandPYY inhibited adenosine deaminase-, isoproterenol- and for-skolin-induced lipolysis. In humans and dogs the effects ofNPYor PYYwere abolished by treatment of cells with Borde-tella pertussis toxin, clearly indicating the involvement of a Giprotein in the antilipolytic effects.

This study indicates that, in addition to alpha2-adrenergicagonists, NPYand PYYare also involved in the regulation oflipolysis in human and dog adipose tissue as powerful antilipo-lytic agents. Further studies are needed to characterize thepharmacological nature of the receptor mediating the inhibi-tory effect of NPYand PYYin fat cells. (J. Clin. Invest. 1990.85:291-295.) adipocyte * Gprotein - lipolysis - neuropeptide Y* peptide YY

Introduction

Neuropeptide Y (NPY)' is a 36-amino acid tyrosine-rich pep-tide that has considerable sequence homology with regulatorypeptides from the gut such as peptide YY (PYY) and pancre-

Address correspondence to Dr. Philippe Valet, Institut de Physiologie,INSERM U-317, rue F. Magendie, Universite Paul Sabatier, 31400Toulouse, France.

Receivedfor publication 2 August 1989 and in revisedform 9 Oc-tober 1989.

1. Abbreviations used in this paper: ADA, adenosine deaminase;NEFA, nonesterified fatty acids; NPY, neuropeptide Y; PYY, peptideYY.

atic polypeptide (1). In contrast to PYYand pancreatic poly-peptide, NPYis strictly a neuropeptide. NPYhas been shownto be colocalized with NEin peripheral noradrenergic as wellas central catecholaminergic neurons. Apart from the vascularsmooth muscle sympathetic innervation where colocalizationof NE and NPY have clearly been revealed, NE and NPY-containing fibers also innervate the smooth muscle of theurino-genital tract, including the vas deferens and uterus. Veryrecently, it has also been shown by immunocytochemistry thatNPYis costored with NEin the perivascular fibers, whereas itis absent in the parenchyma of brown adipose tissue. It wassuggested that blood vessels and adipocytes in brown fat haveseparate innervation by subpopulations of autonomic neurons(2). As far as we know, no data are available in white fat, whichhas a more limited sympathetic innervation, with 95-98% ofthe fat cells not directly innervated.

Although not all the physiological functions of NPYhavebeen completely elucidated, the peptide produces several re-sponses when it is injected centrally or systemically. The re-sponses include inhibition of the evoked release of NE fromperipheral noradrenergic neurons, vasoconstriction after invivo administration, and potentiation of the contractile re-sponse promoted by epinephrine after in vitro administration.Various other physiological effects have also been describedafter central administration of NPYor stimulation of sympa-thetic nerves (3).

The contractile effect of NPY is attributed to reduced ac-cumulation of cAMPin cerebral blood vessel cells. Moreover,NPYand PYY have been found to inhibit vasoactive, intes-tinal peptide-stimulated cAMPproduction in epithelial cellsisolated from rat small intestine (4). In rat cerebral cortex NPYwas also found to inhibit NE-stimulated adenylate cyclase ac-tivity. Furthermore, NPY inhibited isoproterenol-stimulatedadenylate cyclase activity in cultured atrial cells as well as inatrial membranes (5).

The observed colocalization of NPYand NEin many cen-tral and peripheral neurons has certain physiological rele-vance. NPYand NE are released together upon sympatheticactivation during physiological exercise in man (6). Neverthe-less, in the rat a different pattern of NPYand NE responsedepending on the stressful conditions used may also suggestdifferences in the process of release of the two neurotransmit-ters or in the kinetics of their spillover into the blood flow (7).

Since there still has not been any demonstration of NPYeffects on tissues involved in the regulation of metabolismsuch as liver and white adipose tissue, which are highly sensi-tive to activation by NE, the present study attempts to charac-terize a possible action of NPYon the lipolytic function in fat

Neuropeptide Yand Peptide YYInhibit Lipolysis in Fat Cells 291

J. Clin. Invest.© The American Society for Clinical Investigation, Inc.0021-9738/90/01/0291/05 $2.00Volume 85, January 1990, 291-295

cells. PYYwas also used and a comparison with NPYeffectswas made because there is a strong structural homology be-tween both peptides. Moreover, NPY and PYY have beendescribed as sharing a common receptor site on small intes-tinal epithelial cells (4). Both agents have similar effects on thegut: PYYand NPYcause hydroelectric absorption and inhibitvasoactive intestinal peptide and PGE2-induced secretion inthe small intestine both in vivo and in vitro.

The present experiments were performed on human anddog fat cells, two cell models having functional similaritiessince they both possess a well-defined adrenergic responsive-ness controlled by alpha2- and beta adenoceptors and are de-void of responsiveness to lipolytic peptides (glucagon, cortico-tropin), unlike the fat cells of various other species (hamster,rat, rabbit). Moreover, the dog used for studies on the regula-tion of lipomobilization in the laboratory offers easier stan-dardization and homogeneity than the human for further stud-ies on regulating events in the area of NPY and PYY. Wedemonstrate that NPY and PYY are powerful antilipolyticagents. The antilipolytic effect is suppressed by treatment ofthe fat cells with pertussis toxin.

Methods

HumanstudiesSeven sedentary male volunteers (mean age, 28±4 yr; range, 24-35 yr)were included in this study. None of these subjects had any metabolicdisorder, received any pharmacological treatment, or performed anybout of exercise or training. All subjects were Caucasians and gaveinformed consent.

Biopsy procedure. Using a 2.3-mm-diam needle mounted on asyringe containing 2 ml of 9% NaCl, after anesthesia with 0.5% lido-caine solution without epinephrine, 200 mgof subcutaneous adiposetissue was drawn 5 cm from the navel in the right half of the abdomen.On all subjects this biopsy was performed between 10 a.m. andmidday.

Adipocyte isolation. Adipocytes were isolated using the Rodbellmethod (8) in KRB (pH 7.4) containing 0.5 mg/ml collagenase, 4%human albumin, and 90 mg/ 100 ml glucose. After 50 min of digestionat 37°C and filtration isolated adipocytes were obtained and suspendedin a volume so that the final concentration was the same in all subjects.

Measurements of adipocyte lipolysis. As an indicator of adipocytelipolysis, the quantity of glycerol and nonesterified fatty acids (NEFA)liberated in the medium was measured. 50-ml aliquots of the cellsuspension were placed in 1.5-ml conical tubes. 10 of these tubes wereused for cell counting and sizing. 10 others provided an evaluation ofthe initial quantity of glycerol and NEFAcontained in the medium.Agents for lipolysis stimulation or inhibition (10 ;d) in solution atvarious concentrations were added to the remaining tubes. For eachconcentration, three identical tubes were prepared.

After 3 h at 370C all the tubes were placed in a water bath at 95°Cfor 7 min to stop the incubation. Afterward, the Kather technique (9),based on the transformation of glycerol into 3-phosphoglycerol, al-lowed the reduction of NADto NADH. The latter was measured bybioluminescence with a luciferase solution using a Wallac luminom-eter (model 1251; LKB Instruments, Inc., Gaithersburg, MD). NEFAlevels were also determined with a luminometric technique (10). Theamounts of glycerol and NEFAwere taken as the average of the quan-tities obtained from the three tubes of incubation.

Estimating the quantity of lipids. To find out the quantity of lipidsincubated in each tube, the total volume of incubated cells was multi-plied by the average density (0.915, density of triolein). The total cellvolume incubated was obtained by multiplying the average number ofadipocytes contained in the tube by the average cell volume. Theaverage number of adipocytes was determined by counting. To do this,

the 50 Ml contained in each counted microtube was diluted with 450 MIKRB and albumin. A IO-Ml portion of this suspension was placeddropwise on a slide of plastic material and the adipocytes were countedwith a microscope. The average number of adipocytes per 10 Ml repre-sents the number of adipocytes per microliter of the initial suspension.

The average cell volume was calculated after measurement of adi-pocytes. This was done with a micrometer scale placed in the eyepiece.Each division corresponded to 13 Am, so the adipocytes were dividedinto classes by multiples of 13 Am. Cells with diameters < 39 Amwerenot counted. For each category of cells the total volume was obtainedby multiplying the average volume of the class (calculated from anaverage diameter, assimilating the adipocyte to a sphere) by the num-ber of cells in that class. By adding the total cellular volume of eachclass and dividing by the number of cells measured, the average adipo-cyte volume of the subject (in nanoliters) was obtained.

Animal studiesPreparation of dog adipocyte membranes. Adipose tissue was obtainedfrom six beagle dogs, weighing 15-20 kg. Omental adipose tissue wasobtained after an overnight fast; a biopsy was taken immediately afterthe induction of general anesthesia by 30 mg. kg-l i.v. pentobarbitone.The isolated adipocytes were obtained as previously described (1 1).

Lipolysis measurements. The lipolytic activity was analyzed onisolated adipocytes prepared according to the technique of Rodbell (8)with minor modifications. KRBcontaining BSA(3.5%) and glucose (6mM), adjusted to pH 7.4 with 1 NNaOHjust before use, was used aspreviously described (12). After collagenase action (1 mg/ml KRB),isolated fat cells were washed three times and the packed cells werebrought to a suitable dilution in KRB. The cells were incubated inplastic vials (1 ml incubation medium) with gentle shaking in a waterbath under an air phase at 370C. After 90 min the incubation tubeswere placed in an ice bath, the adipocytes were separated from thebuffer, and 200 Ml of the infranatant were removed for the enzymaticdetermination of glycerol which was taken as an index of the lipolyticrate. Total lipid was evaluated gravimetrically after extraction with themethod of Dole and Meinertz (13). Pharmacological agents wereadded just before the beginning of the incubation in IO-Ml portions invehicle to obtain a suitable final concentration. The lipolytic activity ofall isolated fat cell batches was controlled using 1 AMisoproterenol (anonselective beta agonist).

Treatment of human and dog fat cells with pertussis toxin wasperformed as described by Murayama and Ui (14).

Data analysis. All experiments were performed in duplicate.Values are means±SEM. Wilcoxon's test was used for comparisonsbetween matched pairs, differences being considered significant whenP < 0.05.

Drugs and chemicals. The following reagents were used: humanalbumin ORHA20/21 (Behring); isoproterenol and Bordetella per-tussis toxin (Sigma Chemical Co., St. Louis, MO); and tetradecyl alde-hyde (Aldrich Chemical Co., Milwaukee, WI). Collagenase, BSA, lucif-erase from Photobacterium fischeri, and enzymes for glycerol assayscame from Boehringer Mannheim GmbH(Mannheim, FRG). Allother chemicals and organic solvents were of reagent grade. NPYandPYYwere of human origin and purchased from Peninsula Laborato-ries, Inc. (Belmont, CA).

Results

This study was designed to assess whether NPYand PYYhavea functional impact on human and dog fat cell responsiveness.The lipolytic activity was measured under various conditions.Since there is no direct correlation between changes in cAMPlevels and lipolysis within certain concentration ranges of li-polytic agents, it was felt that lipolysis measurements were abetter index for exploration of a putative physiological impactof such peptides than cAMPor adenylate cyclase measure-ments.

292 P. Valet, M. Berlan, M. Beauville, F. Crampes, J. L. Montastruc, and M. Lafontan

NPYandPYYeffects on humanfat cells. Experiments werecarried out on isolated human fat cells incubated in the pres-ence of increasing concentrations of NPYand PYY. Neitheragent had any lipolytic effect on isolated fat cells. Thus, theinvestigations were focused on the exploration of the putativeantilipolytic potencies of these peptides requiring specific ex-perimental conditions leading to the activation of the rate offat cell lipolysis. However, the increment of this rate must becontrolled since activation of lipolysis, promoted higher thannecessary by strong lipolytic agents, cannot be counteracted byinhibitory agents (15). As previously reported, the basal lipo-lytic rate of the isolated fat cell can be severely restrained byendogenous adenosine released or resulting from cell breakage(9). Therefore, the lipolytic activity of human fat cells wasactivated by removing adenosine by addition of 2 ,gg/ml aden-osine deaminase (ADA) in the incubation medium. Underthese particular conditions spontaneous lipolysis was 0.84±0.2gmol/ 100 mgof lipid after 3 h of incubation, while spontane-ous lipolysis (in the absence of ADA) was only 0.41±0.07smol/100 mg lipid.

NPY promoted a dose-dependent inhibition of lipolysisregardless of which lipolytic index was used (glycerol orNEFA) (Fig. 1). PYYwas more potent than NPYin human fatcells.

The inhibiting concentrations of NPYappear somewhathigher under our assay conditions than those of PYY. How-ever, similar half-effective concentrations (ECm) have alreadybeen described in rat intestinal epithelial cells (4) and rat atrialcells (5). Nevertheless, local concentrations of NPYcould existin the adipocyte vascular bed and may achieve such highlevels. The maximal inhibition obtained at higher doses ofpeptides was slightly weaker than that defined with 1 OMepi-nephrine associated with 10 jM propranolol (beta-adrenergicantagonist) or 1 gM UK 14,304 (full alpha2-adrenergic ago-nist) tested under similar conditions.

Effects of PYYand NPYin dog fat cells. To extend thestudy of such an observation to an animal model the dog wasselected, since, as previously defined by our group, the fat cellof the dog possesses an adrenergic responsiveness that is very

120]

12 -11 -10 -9 -8 -7log [peptide (M)l

LI, I

Figure 1. Dose-response6 curve of inhibition of

ADA-stimulated lipolysis(2 jug/ml) by NPY(ni) andPYY(o) in human adipo-cytes. The values of lipoly-sis are given by both glyc-erol and NEFAreleased inthe medium and are givenin the upper and lowerpanels, respectively. Resultsare mean±SE of seven sep-arate experiments per-formed in triplicate.

similar to that described in the human fat cell (betal-, beta2-and alpha2 adrenoceptors coexist in human and dog fat cellmembranes; 16, 17). Moreover, as opposed to various com-monly used animal models such as rat, hamster, and rabbit,the fat cells of man and dog do not possess receptors for lipo-lytic peptides that are positively coupled with adenylate cy-clase (1 8).

In this cell model, the experimental approach for the evalu-ation of NPYeffects was conducted on fat cells whose lipolysiswas prestimulated by various lipolytic agents acting throughdifferent mechanisms. ADA (removal of adenosine), isopro-terenol (beta-adrenergic agonist), and forskolin (direct stimula-tor of the catalytic unit of adenylate cyclase) were used. Theinhibitory effects of l0-' MNPYwere tested in three differentsituations reported in Fig. 2. NPY clearly counteracted thelipolytic effect initiated by ADA (4 jig/ml), isoproterenol (1jiM), and forskolin (10 ,uM). These data suggest that NPYdoesnot interfere directly with stimulatory receptors (beta adreno-ceptors) in decreasing isoproterenol-induced lipolysis, butrather controls the cAMPproduction system itself as shown bythe counteraction of forskolin and ADAeffects. In other ex-periments we demonstrated that these effects are dose depen-dent (not shown). Inhibition of the lipolytic activity observedin the presence of ADAwas explored along the dose-responsecurve for NPYand PYY (Fig. 3).

PYYand NPYreduced lipolysis by - 95% at the highestdoses used. However, NPYwas apparently less efficient thanPYY in dog fat cells (ECm 0.72±0.22 and 8.95±2.71 jimolglycerol/100 mg lipids per 90 min for PYYand NPY, respec-tively) as already described in human fat cells.

Abolition of the effects of NPYand PYYby pertussis toxin.As regulation of lipolysis involves regulation of plasma mem-brane adenylate cyclase activity and changes in cAMPlevels, apossible action of NPYand PYYat the adenylate cyclase stepcould be proposed. Since an inhibition of lipolysis was ob-served, an involvement of a Gi protein could be considered.Such a hypothesis could be verified by treatment of fat cellswith pertussis toxin, which specifically ADP-ribosylates the Giproteins involved in the inhibition of adenylate cyclase block-ing the inhibiting process mediated. Thus, NPY and PYYresponsiveness was studied after treatment of fat cells withpertussis toxin and a comparison was performed with alpha2-adrenergic agonists in human fat cells. In control human fatcells, epinephrine alone or UK14,304 counteracted the lipoly-sis observed in the presence of ADA (2 jig/ml) as previouslyseen (15); the effect was slightly stronger than that observedwith NPYand PYY(Fig. 4). After pertussis toxin treatment of

ag 3 '-NY(. M

%a 21o I I O

NONE ISO(0.1 jM) ADA(4 ag/1) FK(10pM)

Figure 2. Effect of 10-'MNPY(open bars) onisoproterenol (ISO)-,ADA-, or forskolin(FK)-stimulated lipoly-sis in dog fat cells.Values of lipolysis areexpressed as gmol glyc-erol/100 mg lipid per90 min. Spontaneous li-polysis was 0.87±0.35

jmol glycerol/100 mg lipid per 90 min. Results are mean±SE of sixseparate experiments performed in duplicate. *P < 0.05; Wilcoxon'stest.

Neuropeptide Yand Peptide YYInhibit Lipolysis in Fat Cells 293

W 100

o so02.

a 60-

wi40-

-1

< 120-

< 100:

o 80

i 60,

-12 -11 -10 -9 -8 -7 -6log [peptide (M)]

uWU,

§80 Figure 3. Dose-responsecurve of inhibition ofADA-stimulated lipoly-

50 1\sis (4 ,ug/ml) by NPY

(U) and PYY (El) in dog40 ~~~~~~~~~adipocytes. Values are

expressed as percentage20 NPY of maximal inhibition.50;I° PYYI ^b Results are mean±SEof

0 , , , six separate experi-a -12 -11 -10 -9 -8 -7 -6 ments performed in du-

log [peptide (M)] plicate.

the cells, a significant enhancement of basal lipolysis was ob-served and no extra effect was noticed with ADA. All theinhibitory effects observed with epinephrine, UK 14,304,NPY, and PYYwere completely suppressed. This action wasalso confirmed in dog fat cells. Pertussis toxin treatment en-hanced basal lipolysis and suppressed the antilipolytic effectsof alpha2-adrenergic agonists, adenosine, PGE1, NPY, andPYY in dog fat cells (not shown).

To conclude, the inhibitory effect of NPYand PYY onlipolysis was abolished in human and dog fat cells, suggestingthe involvement of a Gi protein.

Discussion

The original finding of this work was that NPYand its relatedanalogue PYY can exert potent antilipolytic effects in humanand dog fat cells.

NPY, a major neuropeptide, is costored with NEin sympa-thetic nerve terminals, while PYY is located in the endocrinecells of the intestinal mucosa from various species (19). In theintestine, which appears to be the most fully studied, PYYandNPYhave been shown to have multiple interactions with dif-ferent second messenger systems for reducing intestinal secre-tion. In the fat cell, the putative receptors for these peptidesappear to be negatively coupled with adenylate cyclase sincepertussis toxin, which is known to specifically ADP-ribosylatethe two ai subunits of the GTP-binding regulatory proteins inhuman fat cells (20), uncouples the receptors from the ai sub-units and blocks the inhibitory effects of NPYand PYY (Fig.4) and also those of alpha2-adrenergic agonists. However, fur-ther studies which are considered to be out of the scope of the

Figure 4. Effect of alpha2-xo).8o) |-I ASAl adrenergic agonists (epi-I~ADA 2Pg/,dI

- I PMTi. nephrine [EPI], UK 14,0.60'i0 lK pM 304 [UK]), NPY, and PYY- iw|S on ADA-stimulated lipoly-

0.40 sis before and after treat-E|l t s ment of isolated fat cells

o.20 rr) ^ tlwithpertussis toxin.:IIH t-< * Humanadipocytes were in-

cubated for 90 min with(O-CONNTIR RKA I L 1) (treated) or without (con-

trol) 10 ,g/ml of pertussistoxin. Values of lipolysis are expressed as Mmol glycerol/ 100 mg lipidper 90 min. Results are mean±SE of six experiments. *P < 0.05;Wilcoxon's test.

present paper are required to clearly delineate the mecha-nism(s).

Based on our results, we propose that NPYand PYY in-hibit lipolysis through specific receptors coupled negativelywith adenylate cyclase via a pertussis toxin-sensitive protein.In this regard, like alpha2 adrenoceptors, NPYsites are cou-pled to the inhibition of adenylate cyclase and lipolysis; theireffect is also uncoupled by pertussis toxin treatment. Demon-stration of the physiological relevance of NPYand PYYin theregulation of adipocyte function needs further study. How-ever, several lines of evidence suggest its potential role on thefat cell. PYY-immunoreactive cells are numerous in the termi-nal ileum, colon, and rectum, whereas a limited number ofthese cells are found in the duodenum and jejunum. PYY isreleased into the blood in response to a meal or after intestinalperfusion with oleic acid (21, 22). The antilipolytic action de-scribed here fits reasonably with such physiological conditions.In contrast to PYY, NPY, which is strictly a neuropeptide, isfound throughout all regions of the intestinal tract and brownfat. A modulating action (counteraction of beta-adrenergic ef-fects) on fat cell lipolysis could be reasonably considered dur-ing the activation of the sympathetic nervous system since NEand NPYare colocalized in peripheral neurons, and the pep-tide and catecholamines may act as cotransmitters affectingthe same target fat cell through similar mechanisms. It is no-ticeable that the receptors of the fat cell could be stimulated orcostimulated by both endocrine (PYY) and neural (NPY)pathways. The detailed processes involved in the control ofboth transmitters as well as the mechanisms maintaining orcontrolling their spillover and degradation will probably re-quire further extended studies to delineate the interest of suchmechanisms in the control of fat cell function in physiologicaland pathological situations.

In conclusion, this work provides the first evidence for theexistence of NPY- and PYY-dependent antilipolytic effects infat cells. The action is probably mediated by adenylate cyclaseinhibition. It is now necessary to define, with improved bind-ing studies, the pharmacological nature of the receptor me-diating the inhibitory effects of NPYand PYYin fat cells.

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Neuropeptide Y and Peptide YYInhibit Lipolysis in Fat Cells 295