Vol. 191. No. 4 I’nnted rn ii S.A.

Identification and Functional Studies of a Specific Peptide YY-Preferring Receptor in Dog Adipocytes ISABELLE CASTAN*, MARC LABURTHE,

PHILIPPE VALET, MAX LAFONTAN

THIERRY

VOISIN,

NATHALIE

QUIDEAU,

AND

Institut National de la Sante? et de la Recherche Mkdicale, INSERM U317, Institut L. Bugnard, CHU Rangueil, 31054 Toulouse Cedex, France; and Institut National de la Sank? et de la Recherche MGdicale, INSERM U239 (T. V., M.Lab.), Facultk de Mgdecine Xavier Bichat, 75018 Paris, France Lipolysis experiments performed with PYY, NPY, and NPY analogs confirm the relative order of potency found in competition experiments. The data agree with the definition of PYY-preferring receptor which resembles a Y2 receptor subtype since NPY-(13-36), a specific Y2 receptor agonist, inhibited binding and lipolysis in a similar way to PYY, whereas [Leu31-Pro34lNPY did not. No difference was observed in the antilipolytic response between I&,, values measured on omental, perirenal, and-subcutaneous fat deposits. Moreover, PYY and NPY (10m6 M) significantly attenuated forskolin-stimulated CAMP levels, involving inhibition of adenylyl cyclase as a transmembrane signaling mechanism. Cross-linking of bound [““I]PYY to membranes indicated that the mol wt of the receptor was 62K. The relative importance of such a receptor on fat cells alongside another powerful antilipolytic receptor-the &adrenoceptor-is discussed. (Endocrinology 131: 1970-1976,1992)

ABSTRACT Specific binding sites for peptide YY (PYY) and neuropeptide Y (NPY) as well as functional responses were identified in dog adipocytes. Studies were carried out using the radioligand [‘“‘I-Tyr’lmonoiodoPYY on crude adipocyte membranes. [‘““IIPYY bound to dog adipocyte membranes with a high affinity (156 + 24 PM) and binding capacity of 314 + 48 fmol/mg protein. Competition studies revealed a higher affinity of the binding sites for PYY than NPY (inhibition constants were 118 f 17 pM and 300 + 53 pM, respectively, P 5 0.001). NPY analogs displaced [“‘IIPYY specific binding with the following order of potency: NPY-(13-36) > NPY-(18-36) > NPY-(22-36) >> [Leu31Pro34lNPY. Neither adrenergic nor adenosine agents (activating or inhibiting other antilipolytic systems) interacted with [““I]PYY binding sites. So [‘““IIPYY binding was specific, saturable, and reversible.

S

EVERAL lines of evidence suggest a possible equivalence of activity of neuropeptide Y (NPY) and peptide YY (PYY) in a neuroendocrine tracts. NPY is a major regulatory peptide both in the central and peripheral nervous system and is costored with norepinephrine in autonomic neurones notably in the perivascular fibers of brown adipose tissue (1). PYY is a gut hormone produced from the endocrine cells of the lower intestine (2) and released into the blood in response to a meal (3) or after an intestinal perfusion of oleic acid (4). Previous data from our group showed that PYY and NPY promoted a dose-dependent inhibition of lipolysis in both human and dog isolated adipocytes through a pertussis toxin-sensitive G protein (5). The present study attempts to characterize more precisely the nature of the receptor mediating the inhibitory effects of NPY and PYY on dog adipocytes and the transmembrane signaling system involved. In various tissues such as small intestine (6), rat hepatocytes, and pancreatic islets (7), NPY and PYY have been described as sharing a common receptor site. Two main pathways for transmembrane signaling have been associated with NPY/PYY receptor sites: 1) inhibition of adenylyl cyclase activity in atria1 cells (8) and in the small intestine (6); and 2) elevation of intracellular calcium in human erythro-

leukemia cells (9). Mobilization of Ca++ can be attributed to an activation of Ca++ channels or to a dependent (or independent) inositol phosphate pathway as reported by Michel (10). In previous studies, signaling mechanisms were not linked to distinct receptor subtypes. The discrimination of NPY receptor subtypes (termed Yl and Y2) was first proposed by Wahlestedt et al. (11). The subtypes were distinguished by the relative affinity of the receptor site for the entire NPY or PYY molecule (Yl) or for the COOH-terminal fragments of NPY (Y2). PYY-preferring receptors, resembling the Y2 subtype of NPY receptors, have also been described

(2, 6, 12). Inhibition of adenylyl cyclase activity was found in cells possessing Yl or Y2 subtypes, but the association between mobilization of intracellular calcium and NPY/PYY receptor subtypes is not well defined yet. The aim of the present study was to characterize the dog adipocyte NPY/PYY receptivity. This study was performed on dog fat cells which, like human fat cells, possess a well defined adrenergic responsiveness controlled by cu2- and /3adrenoceptors and which are devoid of responsiveness to lipolytic peptides (glucagon and corticotropin), unlike the fat cells of various other species (hamster, rat, and rabbit). High affinity binding sites for PYY/NPY were characterized on dog adipocyte membranes, and a 62K specific PYY-preferring receptor protein able to inhibit adenylyl cyclase activity was identified. The receptor belongs to a Y2 subtype, since radioligand binding and lipolytic studies showed a good affinity for the NPY-(13-36) fragment and the lack of interaction with the [Leu31-Pro34lNPY analog.

Received March 23, 1992. Address all correspondence and requests for reprints to: Dr. Philippe Valet, lnstitut National de la Sante et de la Recherche Medicale, INSERM U317. Institut L. Bugnard, CHU Rangueil, Bit L3, 31054 Toulouse Cedex, France. *Recipient of a fellowship from the Centre de Recherches Pierre Fabre (Castanet, France).

1970

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A SPECIFIC Materials Preparation

of

PYY-PREFERRING

and Methods

dog adipocyte membranes

Adipose tissue was obtained from beagle dogs (weighing 15-20 kg; INSERM SC3 Breeding Center, Limeil-Brevannes, France) after an overnight fast; a biopsy was taken immediately after the induction of general anesthesia by 30 mg/kg pentobarbitone (iv). The isolated adipocytes, obtained as previously described (13), were washed four times in a hypotonic lysing medium to elicit total cell breakage and recover fat cell ghosts. The lysing medium was composed of 3.5 rnM MgC12, 1 mM KHCO,, 2 rnM Tris HCI, pH 7.5, and the following protease inhibitors: leupeptin (5 fig/ml), benzamidine (10 FM), phenylmethylsulfonyl fluoride (100 PM), and EGTA (3 mM). Homogenization was performed at 20-22 C to minimize trapping of plasma membranes in the coalescing fat layer. Crude adipocyte ghosts were pelleted by centrifugation (40,000 x g, 10 min) at 4 C, washed twice in the same buffer, and repelleted. At the end of the washing procedure, they were suspended in the lysing buffer at a final concentration of 2-2.5 mg protein/ml and immediately frozen. The membrane preparations were stored at -80 C and generally used within 1 week for binding analysis.

Radioligand

binding

studies

Radioiodinated peptide ([1251-Tyr’]monoiodo-PYY, SA, 2000 Ci/ mmol) was prepared as previously described (14). Binding of [izsI]PYY was studied using 1 mg/ml crude adipocyte membranes incubated with 25-30 PM [“?]PYY and various concentrations of unlabeled agent for 90 min at 37 C in a final vol of 200 ~1 buffer [60 rnM HEPES, 5 mM MgClz containing 2% (wt/vol) BSA and 0.1% (wt/vol) bacitracin, pH 7.41. At the end of the incubation 190.~1 portions of the incubation medium were transferred into microtubes containing 150 ~1 cold HEPES buffer. Bound and free peptide were separated by centrifugation at 20,000 X g for 10 min at 4 C and the pellet counted in a Packard yspectrometer (Packard Instruments, Meriden, CT). Specific binding was taken as the amount of radioactivity bound to the membranes and defined as the difference between the total binding and binding in the presence of 1 FM PYY. Specific binding ranged from 70-80% of the total binding for [“‘I]I’YY at a concentration of 30 PM. Protein was assayed by the method of Lowry et al. (15) using BSA as standard. It was verified that specific [“‘IJPYY binding is proportional to membrane concentrations up to at least 2 mg/ml.

1971

RECEPTOR

pH 7.4 with 1 N NaOH just before use, was used as previously described (20). After collagenase action (1 mg/ml buffer), isolated fat cells were washed three times, and the packed cells were brought to a suitable dilution in buffer. The cells were incubated in plastic vials (1 ml incubation medium) with gentle shaking in a water bath under an air phase at 37 C. After 90 min, the incubation tubes were placed in an ice bath, the adipocytes were separated from the buffer, and 200 ~1 of the infranatant removed for the enzymatic determination of glycerol according to the method of Wieland (21). The glycerol produced by the cells was taken as an index of lipolysis. Total lipid was evaluated gravimetrically after extraction by the method of Dole and Meinertz (22). Pharmacological agents were added just before the beginning of the incubation in 10 ~1 portions in vehicle to obtain a suitable final concentration. Adenosine deaminase (ADA) was included in the incubation medium to remove the inhibiting effect of adenosine released by the fat cells and, thus, to promote an increment of basal lipolysis; this procedure allows, as previously reported, a more accurate definition of the antilipolytic effects (20). The lipolytic activity of all isolated fat cell batches was checked using 1 I.‘M isoproterenol (a nonselective P-agonist).

Adenylyl

cyclase activity

Adenylyl cyclase activity was measured in the crude membrane fraction according to the method described by Alvarez and Daniels (23). Briefly, 30 pg membrane protein were incubated with 0.5-l @Zi [a-32P] ATP in 40 ~1 Tris-HCI buffer (40 mM, pH 7.5) containing 1.5 mM MgClz, 5 rnt.4 creatine phosphate, 0.2 mg/ml creatine kinase, 1 rnM CAMP, 0.5 rnM isobutylmethylxanthine, 100 PM EGTA, 1 PM GTP, 0.2 mM ATP, 0.2% BSA, and 1 pg/ml adenosine deaminase. After 15 min at 30 C the reaction was stopped by addition of 20 11 of a solution containing 2.2 N HCl and 12 nCi [3H]cAMP as an internal standard. CAMP was separated from ATP on a column packed with alumina and eluted with 4 ml 100 rnM ammonium acetate. Activities are expressed as picomoles of CAMP produced per mg protein/min or in percent of inhibition.

Data analysis All experiments were performed in duplicate. Values are given as means f SE. Student’s t test was used for comparisons; differences were considered significant when P was less than than 0.05.

Drugs and chemicals Cross-linking of bound f”“I]PYY to membranes dodecyl sulfate-polyacrylamide gel electrophoresis

and sodium

Adipocyte membranes containing bound [‘?]PYY were suspended in 1 ml 20 mM HEPES buffer (pH 7.5) and incubated for 15 min at 4 C with 1 mM disuccinimidyl-suberate (DSS) as previously described (16). At the end of the incubation, the reaction was quenched by addition of 50 ~1 1 mM Tris, pH 7.5. The cross-linked material obtained was centrifuged at 20,000 X g for 10 min at 4 C, and the pellet was washed with 1 ml 20 rnM HEPES buffer, pH 7.5, containing 1 rnM NaCI. It was then resuspended in 50 ~1 60 mM Tris HCI buffer, pH 6.8, containing 10% (vol/vol) glycerol, 3% (wt/vol) sodium dodecyl sulfate (SDS), and 0.001% (wt/vol) bromophenol blue. After heating for 3 min at 100 C, the suspension was centrifuged for 10 min at 20,000 x g and the resulting supernatant used for electrophoresis, SDS-polyacrylamide gel electrophoresis (PAGE) was run according to the procedure of Laemmli (17). The cross-linked extracts containing up to 100 pg protein were loaded onto a 10% polyacrylamide gel with a 5% stacking gel as described previously (18). The gels were calibrated with prestained SDSPAGE standards. They stained, dried, and exposed for 7-14 days at -80 C.

Lipolysis

measurements

The lipolytic activity was analyzed on isolated adipocytes from omental, perirenal, and subcutaneous fat deposits, prepared according to the technique of Rodbell (19) with minor modifications. Krebs-Ringer bicarbonate buffer containing BSA (3.5%) and glucose (6 mM), adjusted to

NPY, PYY, NPY-(13-36), NPY-(18-36), NPY-(22-36), and [Leu31Pro34]NPY were purchased from Neosystem Laboratories (Strasbourg, France). Collagenase, BSA, and enzymes for glycerol assays came from Boehringer Mannheim (Mannheim, Germany). All other chemicals and organic solvents were of reagent grade.

Results Radioligand

binding

studies

As suggested in a previous publication (5) and confirmed in this paper (see below) PYY presents a higher affinity in functional studies than NPY. So we decided to use labeled PYY rather than labeled NPY in the binding studies. Preliminary studies were performed with either [1251-Tyr36]l’YY or [‘251-Tyr1]PYY. No significant difference was observed in the maximal number of sites or in dissociation constant (K,) values (not shown), and [‘251-Tyr’]PYY was chosen for this study. Binding of [‘251]PYY to dog adipocyte membranes at 37 C was rapid, with a half-maximal specific binding (t%) of 10 min (Fig. 1). Binding remained stable for at least 3 h at 37 C; at 25 C, binding of [1251]PYY was slower, reaching the steady state within 120 min (data not shown). In subsequent exper-

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A SPECIFIC

1972

I g3000 fl i0 .i I L

PYY-PREFERRING

4000

Y 2000 B ii = 1000 0 .t =.

I

Of 0

.

60

120

160 TIME

.

100

s

iz

.--. 1

240

300

I

360

1-

“. 0

420

2-

1. Kinetics of association and dissociation of [““I]PYY specific binding to dog adipocyte membranes. Membranes were incubated at 37 C at the indicated times with 30 _DM .1”“IlPYY in the absence (association) or presence of 1 PM PYY (dissociation). The data presented are means of triplicate determinations in a typical experiment.

iments, an incubation time of 90 min was therefore chosen to represent steady state binding conditions at 37 C. The dissociation of bound [i2”I]PYY was determined by incubating adipocyte membranesto equilibrium and then adding 1 PM PYY at time zero and measuring the residual specific at subsequent time intervals (Fig. 1). Kinetic parameters have been determined by linearization of associationand dissociation curves and conducted to the evaluation of the equilibrium KU, which was between the range of 50-150 PM. KD was determined from the ratio of K2/Kl, where K2 was the first order dissociation rate constant (0.005 < K2 < 0.0085 min-I), and Kl was the secondorder associationrate constant (0.06 < Kl < 0.2 min-’ m-l). These values are within the range of those defined in saturation studies (60-280 PM). Specific binding was clearly saturable and of high affinity. Apparent saturation was observed at a ligand concentration of 1 nM with half-maximal binding occurring at about 150170 PM (Fig. 2, top). Scatchard analysis of these data (Fig. 2, bottom) yielded a straight line, indicating that the ligand binds to a single class of binding sites. This was confirmed by Hill coefficient determination (nH close to 0.9). The equilibrium dissociation constant was 156 & 24 PM, and the total number of binding siteswas 314 f 48 fmol/mg protein (n = 12). The properties of these sites were delineated by displacement of specific [‘251]PYY binding by various compounds acting on adipocyte receptors such as (~2-, P-adrenoceptors, and adenosine Al receptor. Neither cu2- or /I-adrenergic agonists (epinephrine and norepinephrine, not shown) nor an cY2-adrenergicspecific antagonist (RX 821002) or adenosine Al-receptor specific antagonist (diphenylcyclopropylxanthin) were able to displace binding of [12sI]PYY to dog adipocyte membranes(Fig. 3). The pharmacological subtype specificity of the PYY/NPY receptor was characterized using peptide YY, neuropeptide Y, selective fragments of neuropeptide Y, and the synthetic analog of NPY, [Leu31-Pro34lNPY. All the fragments competed with [‘2sI]PYY in the following order of potency: PYY > NPY-(18-36)

i

PYY

(min)

FIG.

> NPY > NPY-(13-36)

Endo. 1992 Vol 131. No 4

300* 5=E .*200 5=f!@ I

.

J 1 z

RECEPTOR

> NPY-(22-36)

>>

4-

5’

(nM)

0.6 1 6

W

E ;;

0.6 -

0.4.

f $

0.2 -

0-l

1

-0

50 BOUND

150 (lmol/mg

250 protdn)

350

FIG. 2. Top, Concentration dependence curve of specific binding of [““I]PYY to dog adipocyte membranes at equilibrium. Membranes were incubated for 90 min at 37 C with the indicated concentrations of [“‘I]PYY as described in Materials and Methods. Nonspecific binding was determined by 1 jtM PYY. Each point is the mean of triplicate determinations of one typical experiment. Bottom, Scatchard plot of the same data. The equilibrium Kn and the maximal number of binding sites were calculated by regression analysis (r = 0.99).

[Leu31-Pro34]NPY, indicating that dog adipocyte receptor is a PYY-preferring receptor resembling a Y2 subtype (Fig. 4). Mean inhibition constants (K,) are indicated in Table 1. Determination

of

the molecular

weight of the receptor

The apparent mol wt of the dog adipocyte PYY-preferring receptor was determined by cross-linking of bound [‘251]PYY to membranesby DSS followed by SDS-PAGE. SDS-PAGE analysis revealed, in three separate experiments, a single band of M, 66,000 whose labeling was prevented by 1 PM unlabeled PYY. A 62K apparent mol wt for the receptor protein was obtained when the peptide weight was subtracted from that determined on the band (Fig. 5). Thus the cross-linker DSS made it possible to covalently attach [125I] PYY to a macromolecular component in adipocyte membranes. Adenylyl

cyclase activity

In dog adipocyte membranes, forskolin (10e5 M) significantly increased (7.4-fold) CAMP accumulation (basal and stimulated values were 29 f 5 and 215 + 37 pmol/mg protein .min, respectively, n = 4). Inhibition assays were performed on forskolin-stimulated cyclase with increasing

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A SPECIFIC

PYY-PREFERRING

RECEPTOR

1973

o-“-Q* A 2 0

0

\ 0

\

0 \ \ OO-

o-13

-12

-11

-10

log [drug

-9

-8

-7

(WI

FIG. 3. Displacement curves of specific [““I]PYY binding to dog adipocyte membranes by adrenergic and adenosine compounds. Membranes were incubated with [““I]PYY and the indicated concentrations of RX 821002 (A) n2-antagonist diphenylcyclopropyl-xanthin (0) Al antagonist, or PYY (0). Binding assays were performed as described in Materials and Methods. Results are expressed as a percentage of radioligand specifically bound in the absence of drugs and are means of three separate experiments. For the sake of clarity SEs are not indicated, but in any case SEs are always lo-15% of the mean value. concentrations of PYY and NPY. Both peptides exerted a dose-dependent inhibitory effect (Fig. 6). NPY-(13-36) also promoted inhibition of adenylyl cyclase activity (16 + 3% of inhibition at 10m5M), whereas [Leu31-Pro34lNPY did not. An Lu2-agonist(UK 14304) was also usedto test the inhibitory effects on the same membrane preparations (20 + 4% inhibition). Dog adipocyte membraneswere alsoincubated under basal conditions with each drug alone, and no modification on the basal level of CAMP was noticed.

Lipolytic

-13

-11

-10

-9

-8

-7

log [drug (WI FIG. 4. Displacement curves of specific [““IIPYY binding to dog adipocyte membranes by various NPY fragments and analogs. Membranes were incubated with [““IIPYY and the indicated concentrations of NPY-(13-36) (O), NPY-(E-36) (O), NPY-(22-36) (A), [Leu31-Pro341 NPY (A), NPY (W) and PYY (0). Binding assays were performed as described in Materials and Methods. Results are expressed as a percentage of radioligand specifically bound in the absence of drugs and are means of five separate experiments. For the sake of clarity SEs are not indicated, but in any case SEs are always lo-15% of the mean value. Calculated K; values are given in Table 1.

TABLE adipocyte

1. Inhibition membranes

of specific [““I]PYY binding on dog by PYY, NPY, and NPY fragments

Competitors

K (PM)

PYY NPY NPY-(13-36) NPY-(18-36) NPY-(22-36) ILeu31-Pro341NPY

measurements

The lipolytic consequencesof receptor stimulation by NPY, PYY, and analogs were measured under various conditions. Neither agent used had any lipolytic effect on isolated dog fat cells. Thus, the investigations were focused on the exploration of the putative antilipolytic pathways requiring specific experimental conditions leading to the activation of the basal rate of fat cell lipolysis. As previously reported (24), the basal lipolytic rate of the isolated fat cell can be severely restrained by adenosine released endogenously or by cell breakage. Therefore, when the antilipolytic effects were explored, the lipolytic activity of dog fat cells was preactivated by removing adenosine by addition of 4 pg/ml adenosine deaminaseto the incubation buffer. The antilipolytic effects of the samecompounds as those usedin binding experiments were investigated in three different fat deposits. Figure 7 shows the dose-dependent inhibition of lipolysis by PYY, NPY, NPY-(13-36) and [Leu31-Pro34lNPY. Table 2 indicates the 50% inhibitory concentrations (ICsO)of all the compounds used. There is a good agreement between the order of

-12

118 300 1,105 1,431 4,340 50.923

f + f f f f

17 53 197 440 1,858 9.941

performed as described in Materials and using a computer curve-fitting program for one-site inhibition model. The K, values were calculated from the equation: K, = ICSo/(l + [radioligand/K,,]). The reported values are means + SE of five different determinations. Inhibition

studies

were

Methods. Data were analyzed

efficiency defined in the antilipolytic assaysand the order of potency described in the binding studies (PYY > NPY > NPY-(13-36) > NPY-(18-36) > NPY-(22-36) >> [Leu31Pro34lNPY). No significant difference was observed in the I& values between adipocytes from omental, perirenal, and subcutaneousfat deposits(Table 2). Discussion

The present work shows that dog adipocytes possessa 62K specific PYY-preferring receptor resembling a Y2-sub-

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1974

A SPECIFIC f25 I ]-PYY

+NPYlpM

+ PYY 1pM

PYY-PREFERRING

M r (kDa)

RECEPTOR

Endo. Vol131.

1.8-

.+-I .% .\ \ -i \Yi, t4 f \ \ 0 0\

ALONE -

67

-66 1.2-

0.9 -

-

18

-

12

FIG. 5. Affinity cross-linking of [iz51]PYY in dog adipocyte membranes. Membranes were incubated with [‘251]PYY alone, with 1 PM PYY, or with 1 pM NPY as specified. After washing, membranes were treated with 1 mM DSS and submitted to SDS-PAGE.

\\

0.6 -

0.3 ’

1992 No 4

* BAS

ADA

-10

-9

f 4\ %

-8

-7

log [drug (WI FIG. 7. Dose-response curve of inhibition of ADA-stimulated lipolysis (4 rg/ml) by NPY-(13-36) (O), [Leu31-Pro34lNPY (A), NPY (O), and PYY (0) in isolated dog adipocytes. Values are expressed as micromoles of glycerol released by 100 mg lipids during 90 min. Results are mean + SE of five separate experiments. TABLE 2. Inhibition of ADA-stimulated lipolysis by PYY, NPY, and NPY fragments on dog fat cells of subcutaneous, perirenal and omentum adipose tissue Fat deposit I&o (nM) Omentum

log I drugWI 1 FIG. 6. Inhibition of the forskolin-stimulated CAMP level (B) by PYY @I) and NPY (f@. UK 14304, an cu2-adrenergic agonist, can also inhibit 20 + 4% of the adenylyl cyclase activity (not shown). Levels of CAMP were: basal, 29 + 5; and forskolin-stimulated (FK 10m5M), 215 + 37 pmol/mg protein. min. Values are the mean z? SE of four experiments performed in duplicate.

type which is coupled negatively with adenylyl cyclase. This supports the fact that PYY has a greater potency than NPY to inhibit lipolysis in various fat deposits of the dog. Stimulation by PYY and NPY promotes the inhibition of adenylyl cyclase activity, which can easily explain the reduction of CAMP levels and inhibition of lipolysis. The inhibition effect is not strikingly different from that described after a2adrenergic receptor stimulation. The present work used forskolin in order to activate the catalytic subunit of adenylyl cyclase and to elevate CAMP levels without involving the associatedregulatory GTP-binding protein. Previous results (5) showed that inhibitory effects of PYY and NPY on lipolysis were completely abolished when cells were pretreated with pertussistoxin, suggestingthe involvement of a Gi protein in the transmembrane pathway. In dog adipocyte

PYY NPY NPY-(13-36) NPY-(18-36) NPY-(22-36) [Leu31-Pro34lNPY

0.77 5.05 25.5 43.9 183 352

k 0.29 + 0.63 + 3.9 + 9.6 + 79 zk 81

Perirenal

3.61 5.04 23.3 79.7 78.4 422

+ + f + + f

0.32 1.07 2.5 7.4 12.1 161

Subcutaneous 1.46 + 0.42 5.55 + 1.87 43.1 2 8.6 54.6 -+ 14.2 212 + 89 882 f 235

Lipolysis studies were performed as described in Materials and Methods. Isolated fat cells were incubated in Krebs-Rinber bicarbonate buffer containing 4 rg/ml adenosine deaminase. Values are means + SE of five separate experiments.

membranessharing a PYY-preferring receptor, at least one of the putative paths for signal transduction requires a pertussis-toxin-sensitive Gi protein involved in the negative coupling with adenylyl cyclase. [‘251-Tyr’]PYY binds specifically, saturably, and reversibly to high affinity PYY-preferring receptor sitesin dog adipocyte membranes and exhibits a rather low level of nonspecific binding on this crude membrane preparation (~20% at Ku value). [lz51]PYY binding (i.e. mean Kn of 156 f 24 PM) is consistentwith the values previously reported in rat intestinal epithelial plasma membranes(12) or described for [‘251]NPY binding in other tissuessuch asrat brain (25) and pig spleen (26, 27). The rank order of potency of NPY fragments or analogs to inhibit [‘251]PYYbinding matched with the definition of a Y2 receptor. Y2 receptorsare describedin jejunum mucosa (28), cardiac ventricular membranes (29), and hippocampal regions of the brain (30, 31), whereas the Yl

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A SPECIFIC

PYY-PREFERRING

subtype is defined in brain cortex (32), human neuroblastoma cells (33), and vascular smooth muscle in pancreas (34). Thus, the initial proposal localizing Y 1 receptors postsynaptically and Y2 presynaptically as suggested by Wahlested (11) probably needs reconsideration. More recently, a third subtype (Y3) has been described in chromaffin cells which specifically binds labeled NPY (35). Affinity labeling was used to extend the results given by the pharmacological delineation of NPY or PYY-preferring receptors. A single band of M, 66,000 was clearly identified in dog fat cell membranes. Although selective affinity labeling has demonstrated that Yl and Y2 subtypes are structurally distinct glycoproteins, some confusion still persits concerning the YZ-receptor glycoprotein which is identified by affinity-labeling techniques. In rat hippocampus and rabbit kidney membranes (36), the Y2 receptor subtype is represented by a labeled protein having an M, of 50,000; it is also the same kind of labeling which is obtained in the pig hippocampal area (37). The intestinal Y2 receptor, which has a slightly higher affinity for PYY than for NPY, as in fat cells, has an apparently lower mol wt (M, 44,000) than in fat cells (38). When referring to other more recent studies, a band of [‘251]PYY affinity-labeled protein at M, 62,000 was found in rat cerebral cortex (25) and at M, 65,000 in rat liver and pancreas (7). For the moment, the confusion probably stems from the fact that all the labeled proteins corresponding to PYY or NPY receptors have not been fully delineated. The use of tissues possessing various kinds of cells bearing NPY or PYY receptors does not facilitate subclassification. Some tissues probably contain a mixture of Yl and Y2 receptors, which could be located on nerves or on various kinds of cells (i.e. prejunctional or postjunctional receptors). Inside the Y2 receptor family it is not impossible to differentiate subtypes based on their anatomical localization or cell distribution. The glycosylation state of the receptor protein can also be at the origin of the large variations in mol wt values reported by various authors. Whatever the outcome of the large and still open debate, it is clear that the dog fat cell PYY-preferring receptor is a postsynaptic receptor of the pharmacologically defined Y2 subtype which was defined as having an M, of 62,000 in affinity labeling studies performed with [‘251]PYY. The isolated fat cell conveniently provides a system for the study of only one Y receptor type (Y2 type) having a higher affinity for PYY than NPY. This postsynaptic Y2 receptor is negatively coupled with adenylate cyclase, and it is very easy to carry out binding approaches and functional assays in parallel. Antilipolytic experiments were also done in an attempt to determine whether the PYY or NPY potency was similar in various fat deposits. In fact, previous data showed that in man, adrenergic stimulation leads to different responses between subcutaneous (inhibition of lipolysis) and omental (stimulation of lipolysis) adipose tissues (13). In dog, whatever the anatomical location of the fat cells studied, it is noticeable that PYY presents a higher efficacy than NPY in inhibiting lipolysis. Moreover, there is no significant difference between the I& values when comparing omental, perire~~al, and subcutaneous fat deposits. These results sug-

RECEPTOR

1975

gest the equipotency of the PYY/NPY antilipolytic system whatever the fat deposit. This cell model could be an easy and reliable system for testing different analogs and fragments of NPY or PYY in the search for receptor antagonists of this kind of postsynaptic Y2 receptor subtype. The clearly identified existence of an antilipolytic receptor in fat cell membranes alongside the a2-adrenergic receptor and leading to the same biological effect brings forward the question of the relative importance of these systems. It now seems necessary to study the possible regulation of both Y2 and a2-adipocyte receptors at the same time.

References 1. Cannon B, Nedergaard J, Lundberg J, Hokfelt T, Terenius L, Goldstein M 1986 Neuropeptide tyrosine (NPY) is costored with noradrenaline in vascular but not in parenchymal sympathetic nerves of brown adipose tissue. Exp Cell Res 164:546-550 2. Laburthe M 1990 Peptide YY and neuropeptide Y in the gut: availability, biological action and receptors. Trends Endocrinol Metab 1:168-174 3. Adrian T, Ferri G, Bacarese-Hamilton A, Fuessl H, Polak J, Bloom S 1985 Human distribution and release of a putative new gut hormone, peptide YY. Gastroenterology 89:1070-1077 4. Pappas T, Debas H, Goto Y, Taylor I 1985 Peptide YY inhibits meal-stimulated pancreatic and gastric secretion. Am J Physiol 248:G118-G123 5. Valet I’, Berlan M, Beauville M, Crampes F, Montastruc J, Lafontan M 1990 Neuropeptide Y and peptide YY inhibit lipolysis in human and dog fat cells through a pertussis toxin sensitive Gprotein. J Clin Invest 85:291-295 6. Servin A, Rouyer-Fessard C, Balasubramaniam A, Saint-Pierre S, Laburthe M 1989 Peptide-YY and neuropeptide-Y inhibit vasoactive intestinal peptide-stimulated adenosine 3’%A 5’-mono-phosphate production in rat small intestine: structural requirements of peptides for interacting with peptide-YY preferring receptors. Endocrinology 124:692-700 7. Nata K, Yonekura H, Yamamoto H, Okamoto H 1990 Identification of a novel 65 kDa cell surface receptor common to pancreatic polypeptide, neuropeptide Y and peptide YY. Biochem Biophys Res Commun 171:330-335 8. Kassis S, Olasmaa M, Terenjust L, Fishman H 1987 Neuropeptide Y inhibits cardiac adenylate cyclase through a pertussis toxin-sensitive G protein. J Biol Chem 262:3429-3431 9. Motulsky H, Michel M 1988 Neuropeptide Y mobilizes calcium and inhibits adenylate cyclase in human erythroleukemia cells. Am J Physiol 255:E880-E885 10. Michel C 1991 Receptors for neuropeptide Y: multiple subtypes and multiple second messengers. Trends Pharmacol Sci 12:389-394 11. Wahlestedt C, Yanaihara N, Hakanson R 1986 Evidence for different pre- and post-junctional receptors for neuropeptide Y and related peptides. Regul Peptides 13:307-318 12. Laburthe M, Chenut 8, Rouyer-Fessard C, Tatemoto K, Couvineau A, Servin A, Amiranoff B 1986 Interaction of peptide YY with rat intestinal epithelial plasma membranes: binding of the radioiodinated peptide. Endocrinology 118:1910-1917 13. Mauriege P, Galitzky J, Berlan M, Lafontan M 1987 Heterogenous distribution of beta- and alpha2-adrenoceptor binding sites in human fat cells from various deposits. Functional consequences. Eur J Clin Invest 17:156-165 14. Voisin T, Royer-Fessard C, Laburthe M 1990 PYY/NPY receptors in small intestine: characterization, signal transduction and expression during cell differentiation. Ann NY Acad Sci 611:343-346 15. Lowry 0, Rosenbrough N, Farr A, Randal R 1951 Protein measurement with the folin phenol reagent. J Biol Chem 193:265-275 16. Laburthe M, Brtiant B, Rouyer-Fessard C 1984 Molecular identification of receptors for vasoactive intestinal peptide in rat intestinal epithelium by covalent cross-linking. Eur J Biochem 139:181-186

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 23 November 2015. at 18:53 For personal use only. No other uses without permission. . All rights reserved.

A SPECIFIC

1976 17.

Laemmli UK 1970 Cleavage

of structural proteins during the assemT4. Nature 227:680-685 Couvineau A, Amiranofi B, iaburthe M 1986 Solubilization of the liver vasoactive intestinal peptide receptor. Hydrodynamic characterization and evidence for an association with a functional GTP regulatory protein. J Biol Chem 261:14482-14489 Rodbell M 1964 Effects of hormones on glucose metabolism and lipolysis. J Biol Chem 239:375-380 Berlan M, Lafontan M 1985 Evidence that epinephrine acts preferentially as an antilipolytic agent in abdominal human subcutaneous fat cells: assessment by analysis of beta- and alpha2-adrenoceptor properties. Eur J Clin Invest 15:341-348 Wieland 0 1957 Eine enzymatiche methode zur bestimmung von glycerin. Biochem Z 239:313-319 Dole V, Meinertz H 1960 Microdetermination of long chain fatty acids in plasma and tissues. J Biol Chem 235:2595-2599 Alvarez R, Daniels D 1990 A single column method for the assay of adenylate cyclase. Anal Biochem 187:93-103 Kather H, Wieland E, Fisher B, Schlierf G 1985 Antilipolytic effects of N6-phenylisopropyladenosine and prostaglandin E2 in fat cells of obese volunteers before and during energy restriction. Biochem J 231:531-535 Mannon P, Mervin S, Taylor I 1991 Solubilization of the neuropeptide Y receptor from rat brain membranes. J Neurochem 56:1804-1809 blv of the head

18.

19. 20.

21. 22. 23. 24.

25.

26.

PYY-PREFERRING

tics, reduction of cyclic AMP formation and calcium antagonist inhibition of vasoconstriction. Eur J Pharmacol 145:21-29 27. Price J, Brown M 1990 ?Neuropeptide Y binding activity of pig spleen cell membranes: effect of solubilization. Life Sci 47:22992306 28. Cox H, Krstenansky J 1991 The effects of selective amino acid

Endo. 1992 Vol 131. No 4

substitution upon neuropeptide iunum. Peptides 12:323-327

of bacteriouhaae

Lundberg J, Hemsen A, Larsson 0, Rudehill A, Saria A, Fredholm B 1988 Neuropeptide Y receptor in pig spleen: binding characteris-

RECEPTOR Y antisecretory

potency

in rat je-

A, Sheriff S 1990 Neuropeptide Y (18-36) is a antagonist of neuropeptide Y in rat cardiac ventricular J Biol Chem 265:14724-14727 30. Sheikh S, Hakanson R, Schwartz T 1989 Yl and Y2 receptors for neuropeptide Y. FEBS Lett 245:209-214 31. Hedlund P, Bjelke 8, Aguirre J, Fuxe K 1991 Preferential increases of ‘251-NPY l-36 binding in the hippocampal formation produced by the NPY Y2-receptor agonist NPY13-36. Acta Physiol Stand 141:279-280 32. Dumont Y, Fournier A, Pierre SS, Schwartz T, Quirion R 1990 Differential distribution of neuropeptide Yl and Y2 receptors in the rat brain. Eur J Pharmacol 191:501-503 33. Fuhlendorff J, Gether U, Aakerlund L, Langeland-Johansen N, 29.

balasubrahaniam competitive membranes.

Thogersen H, Melberg S, Bang-Olsen U, Thastrup 0, Schwartz T 1990 [Leu31-Pro341 neuropeptide Y: a specific Yl receptor agonist. Proc Natl

Acad Sci USA

87:182-186

J, Williams J 1991 Localization of for NPY and PYY on vascular smooth muscle cells in Am J Physiol 260:G250-G257 35. Wahlestedt C, Reeunathan S, Reis D 1992 Identification of cultured cells sel&tivzy expressing Yl-, Y2-, or Y3-type receptors for neuropeptide Y/peptide YY. Life Sci 50:7-12 36. Sheikh SP, Williams JA 1990 Structural characterization of Yl and Y2 receptors for neuropeptide Y and peptide YY by affinity crosslinking. J Biol Chem 265:8304-8310 37. Inui A, Okita M, Inoue T, Sakatani N, Oya N, Morioka H, Shihi K, Yokono K, Mizuno H, Baba S 1989 Characterization of PYY receptors in the brain. Endocrinology 124:402-409 38. Voisin T, Couvineau A, Rouyer-Fessard C, Laburthe M 1991 Solubilization and hydrodynamic properties of active peptide YY receptor from rat jejunal crypts. Characterization as a M, 44,000 glycoprotein. J Biol Chem 266:10762-10767 34.

Sheikh S, Roach E, Fuhlendorff Yl-receptors rat pancreas.

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