Christian Carpene, Michel Berlan, and Max Lafontan

Transcription

1 Influene of development and redution of fat stores on the antilipolyti a,-adrenoeptor in hamster adipoytes: omparison with adenosine and,&adrenergi lipolyti responses' Christian Carpene, Mihel Berlan, and Max Lafontan Institut de Physiologie, Universiti: Paul Sabatier,' ERA 412 CNRS, rue F. Magendie, 314 Toulouse, Frane, and Laboratoire de Pharmaologie MC.diale, Fault6 de Mkdiine,' all6es Jules Guesde, 31 1 Toulouse, Frane Abstrat The response of the hamster adipoyte to various lipolyti (@-adrenergi) and antilipolyti (an-adrenergi and adenosine-dependent) stimuli was studied during the development and after old-indued regression of fat stores. Alphanadrenergi binding (['Hllonidine binding sites) was also investigated. Adipoytes ame from young animals (4-5 weeks), adults (2-25 weeks), and adults submitted to a 6-week old exposure (6OC) that promoted a large derease in fat stores and in fat ell size. The lipolyti response indued by isoproterenol (@-agonist) was equivalent in the different groups. Adenosine and a2-adrenergi antilipolyti effets were estimated through the inhibition of theophylline-indued lipolysis by phenylisopropyladenosine and lonidine, respetively. The adenosine effet was unhanged in all the groups. In ontrast, the an-adrenergi effet, whih was not present in young hamsters, inreased simultaneously with fat ell size, was fully effetive in adult hamsters, and had ompletely disappeared in small adipoytes from old-exposed hamsters. In fat ell ghosts, an-adrenoeptors (['H]lonidine binding sites), followed similar modifiations: they inreased with fat ell enlargement and disappeared after ell size redution following old expsure.l These results suggest that: I) the inreased a2-adrenergi antilipolyti response whih is onomitant with fat ell enlargement ould partly explain the growth-related derease in the previously reported lipolyti effet of epinephrine; 2) the u2-reeptivity of the adipoyte seems to be stritly fat ell size-dependent while the -adrenergi and adenosine responses are unaffeted; and 3) the regulation in the adipoytes of the adenosine, yz- seems to be unrelated.-carpene, C., M. Berlan, and M. Lafontan. Influene of development and redution of fat stores on the antilipolyti a'-adrenoeptor in hamster adipoytes: omparison with adenosine lipolyti responses. J. Lipid Res : branes of various speies (1-6). The stimulation of lipolysis is mediated by the -adrenoeptor while the a*-adrenoeptor subtype is involved in the inhibition of lipolysis. So, it is now well aepted that the lipolyti differenes in the responsiveness of the adipose tissue between various speies are mainly dependent on the balane between a- and 8-adrenergi reeptors (6). Most of the studies on the effet of aging on hormone responsiveness and sensitivity of adipose tissue have been arried out in the rat. Conerning the adrenergi agents, the most striking result is that aging is assoiated with a derease in the lipolyti response to ateholamines (7-15) and that this effet is related to a derease in P-adrenoeptor number (1 4) and adenylate ylase ativity (16, 17). Although the rat has been the most ommonly used speies for investigations into the agedependent effet of ateholamines on adipose tissue, its adipoytes lak the a*-adrenergi reeptors ontrolling lipolyti proesses (1 8, 19). Little is known onerning hanges of the a*-antilipolyti adrenoeptor during aging. We have shown that in rabbit (2) and in dog adipoytes (21), the absene (22) or the loss (23) of the lipolyti response to epinephrine in the aging animal is linked to the involvement of an inreased a-adrenergi response. Moreover, Pequery and Giudielli (24) reported that the a-adrenergi responsiveness inreases with age in hamster adipoytes onomitantly with [3H]dihydroergoryptine Downloaded from by guest, on September 8, 218 Supplementary key words fat ell size ap-reeptor sites adenosine reeptor sites ['H]lonidine isoproterenol phenylisopropyladenosine lonidine A great number of studies have shown that both a- are present in adipoyte mem- Abbreviations: KRBA, Krebs-Ringer biarbonate buffer ontaining albumin; PIA, phenylisopropyladenosine. ' A preliminary report of a portion of these data has been published in abstrat form at the International Conferene on the Adipoyte and Obesity: Cellular and Moleular Mehanisms, held in Toronto, June ' C. Carpene and M. Lafontan. '' M. Berlan. 766 Journal of Lipid Researh Volume 24, 1983

2 binding site number. In the hamster, as in the dog and the rabbit, aging is assoiated with an extension of the adipose tissue as demonstrated by the development of large fat pads with bigger adipoytes. It is of interest to distinguish the relative importane of aging and obesity in the appearane and the inrement of the a-adrenergi antilipolyti effet in fat ells. In the present report, a desription of the antilipolyti a-adrenergi response in adipoytes of young and mature hamsters is assoiated with a study of a2-adrenergi reeptor binding sites. Moreover, we also present evidene suggesting that the main fator involved in the genesis of a2-adrenergi responsiveness is the inrease of the fat ell size rather than aging as previously supposed. In this study we also ompare the antilipolyti effet initiated by y2-adrenoeptor stimulation to that of phenylisopropyladenosine, a well known antilipolyti agent ating on inhibitory adenosine reeptors of the fat ell membrane (25). Animals MATERIALS AND METHODS All the studies were performed within a period ranging from May to July. The male golden hamsters used were reared in our laboratory. Hamsters that were 4-5 weeks old were onsidered as young, immature animals in our experiment. They were weaned 1 week before use. At this time, the mean body weight was 4-5 g. The diameters of the perirenal adipoytes ranged from 3 to 4 pm. We also used adult hamsters, 2-25 weeks of age, housed in a temperature-ontrolled room (22 C) and exposed to light-dark intervals of 12 hr. Another group of adult hamsters of the same age was housed in a old temperature-ontrolled room (SOC), TABLE 1. 6 weeks before use, under the same light-dark yle. These animals had redued physial ativity and spent most of their time in a otton bed inluded in eah age. They were normothermi over the entire ourse of the experiment. The experimental onditions were suffiient to produe signifiant weight loss and inreased food intake (1-2%). All the animals were fed ad libitum with a standard diet (UAR, Paris) omposed of protein (22%), fat (5%), arbohydrate (5 1 %), ellulose (4%), water (1 2%), minerals, and vitamins. Body weight and other harateristis of the groups of hamsters are given in Table 1. Preparation of isolated adipoytes The fat ells were prepared as follows. The hamsters were fasted overnight. They were killed by deapitation and the perirenal and epididymal adipose tissue was immediately removed. Adipose tissue from 3-5 adults or 25-3 young or old-treated hamsters was pooled to obtain suffiient yield of fat ells for lipolysis and binding studies. The tissue was ut into small piees and inubated for 2-3 min at 37 C in 1 ml of Krebs-Ringer biarbonate buffer ontaining 35 mg/ml of defatted bovine serum albumin (KRBA) and 6 mg of rude ollagenase. The isolated fat ells were then prepared as previously desribed (3, 21, 22). Lipolysis measurements The isolated adipoytes (2-3 mg of ell lipid) were dispersed in plasti vials in 2 ml of KRBA and 6 pmol/ ml gluose. Theophylline and other agents were added in portions of 2 p1 just before starting the inubation proedure. After 9 min, an aliquot of the inubation medium was taken to determine glyerol release by Wieland's method (26). The metaboli ativity is expressed as pmol of glyerol produed per 1 mg total Charateristis of various hamster groups: weight, fat ell size, basal lipolysis, and theophylline-indued lipolysis Downloaded from by guest, on September 8, 218 Weight Theophylline- After Cold Fat Cell Size indued Ratio Stimulated/ n Before Exposure Diameter Basal Lipolysis Lipolysis Basal Lipolysis Adult hamsters (2-25 weeks) f 3 Young hamsters (4-5 weeks) 7 6f f.25" 2.76 f f.53' 7.47 k f f f f Adult hamsters plaed in f f f f temperature ontrolled.33 f f room (6'C, 6 weeks) Results are means f SEM. Weight and fat ell diameters were estimated from the whole population of hamsters (n) used for all the experiments. In eah group basal and theophylline-stimulated lipolysis are estimated from five experiments and expressed as: " rmol glyero1/1 mg lipid per 9 min, or ' pmol giyeroi/l6 ells per 9 min. Carpene, Berlan, and Lafontan Cell size and up-adrenergi response in hamster adipoytes 767

3 lipid determined gravimetrially after extration aording to Dole and Meinertz's method (27) or as pmol of glyerol produed by lo6 ells. The fat ells of two vials were stained with Giemsa stain and the diameters of 4 ells were determined by optial sizing. The mean fat ell diameter and number were determined as previously desribed (22). Binding studies Pooled perirenal and epididymal adipoytes were washed three times in a lysing medium (MgCI2, 2 mm; KHC3, 1 mm: ATP, 1 mm; Tris-HCI, 2 mm, ph 7.6). Crude adipoyte membranes were pelleted by entrifugation (1, X g, 1 min, 4 C). They were washed twie in a buffer (Tris, 5 mm ph 7.6; 1 mm EDTA) and finally suspended (2-3 mg protein/ml) in an inubation medium (1 mm MgCI,; 5 mm Tris-HCI, ph 7.5). They were used immediately in binding assays or stored, at most for 1 week, at -8 C. Protein was determined using the method of Lowry et al. (28) with bovine serum albumin as standard. Binding assays were arried out as follows. One hundred 111 of membrane suspension (2-3 pg of protein) was inubated for 15-2 min at 25 C with 1 rl aqueous [3H]lonidine solution in total volume of Tris-HC1 5 mm, ph 7.5, ontaining 1 mm MgCI2. Nonspeifi binding was determined by addition of 1 pl of phentolamine M). At the end of the inubation, dupliate 15-pl aliquots were diluted with 4 ml of ie-old buffer and filtered immediately under vauum, using Whatman GF/C glass fiber filters. The filters were washed rapidly with two 1-ml portions of inubation buffer at 4OC, dried at 5 C for 3-4 min, and ounted in minivials in 4 ml of ACS sintillation mixture (Amersham-Searle) with an effiieny of 4-45%. All values refer to speifi binding whih represented 754% (adult hamsters) of the radioativity retained on the filters. Reeptor binding assay results were analyzed aording to the general priniples found in Williams and Lefkowitz's monograph (29). Chemials Chemials were obtained from the following soures: [3H]lonidine (sp at 22.2 Ci/mmol) from New England Nulear; lonidine hlorhydrate from Boehringer Ingelheim; phentolamine base from Giba-Geigy (Bade); and theophylline from Bruneau Labs (Frane). Bovine serum albumin (fration V) and yohimbine-hci were obtained from Sigma and rude ollagenase was from the Worthington Biohemial Corporation (Freehold, NJ). Enzymes and phenylisopropyladenosine ame from Boehringer-Mannheim Corporation. All other hemials were of analytial grade. RESULTS In order to measure the biologial effets of a2-adrenoeptor stimulation on fat ell lipolysis, the ation of lonidine, an a2-adrenergi agonist, was studied during theophylline-stimulated lipolysis. The same experimental design, based on fat ell lipolysis stimulated by theophylline, was also used to test the inhibitory effet of phenylisopropyladenosine (PIA). This sort of experiment is an aurate model for the exploration of the inhibitory effet linked to as-adrenoeptor or adenosine-reeptor stimulation (3, 6). Similar results were obtained when adenosine-deaminase was used instead of theophylline to promote stimulation of lipolyti ativity (data not shown). When expressed as pmol glyerol/ 1 mg lipid, the basal lipolyti ativity was very similar in young and adult hamster adipoytes (Table 1). However, when the lipolyti ativity was related to lo6 fat ells, the glyerol release was 6- to 7-fold higher in adults than in young or old-exposed hamster adipoytes. Sine non-stimulated fat ells showed a low basal lipolyti rate that did not permit lipolysis inhibition to be evaluated, the inhibiting effets were studied after theophylline stimulation of the ells. Theophylline (.33 mm) promoted an inrement of lipolyti ativity of the fat ells that was of the same order of magnitude in the three groups, whatever the expression of the lipolyti rate as indiated by the values of the ratio of stimu- 1ated:basal lipolysis (Table 1). Comparison of lonidine and PIA effets on theophylline-indued lipolysis in fat ells from young and adult hamsters The effet of the a2-adrenoeptor agonist, lonidine, on theophylline-indued lipolysis in fat ells from young and adult hamsters is shown in Fig. 1. Clonidine aused dose-dependent inhibition of the lipolyti response indued by theophylline (.33 mm) in adult hamster adipoytes. The maximal inhibitory effet (65% inhibition) was reahed with 5. M lonidine (lipolysis fell from 2.8 pmol glyerol/ 1 mg lipid in theophylline-stimulated state to.98 pmo1/1 mg lipid with M lonidine). A signifiant antilipolyti effet was observed with lo-' M lonidine. In ontrast, in adipoytes from young hamsters, lonidine had no inhibitory effet until 1-6 M; a weak inhibitory response appeared with large amounts of lonidine (5.1OP6 and 5.1OP5 M); the maximal effet did not exeed 1%. The results obtained with the antilipolyti agent PIA are shown on the right of the Fig. 1. In both ases, PIA provoked an antilipolyti effet. Inexpliably, adipoytes from young animals were more sensitive to this agent sine a signifiant antilipolyti effet was observed Downloaded from by guest, on September 8, Journal of Lipid Researh Volume 24, 1983

4 PIA (MI HiM log 1Q' 1' lo z S - 5 ae 75 * * ***,*** p 5 ap 75 1 ontrols 2-25 weeks 1 o young hamsters 4-5 weeks Fig. 1. Effets of inreasing onentrations of lonidine (a-agonist) and phenylisopropyladenosine (PIA) on theophylline-indued lipolysis in fat ells of young and adult hamsters. The effets of the drugs are expressed as perent inhibition of theophylline-stimulated lipolysis. Values shown are means k SEM of (n) different experiments indiated by the numbers. Statistial signifiane of the inhibition of lipolysis promoted by the agents was tested aording to Student's paired f test. (*, P <.5; **e, P <.1). at M PIA, while in adipoytes from adults, the antilipolyti effet was only seen at M PIA. Effet of old exposure on ar-adrenergi response in adipoytes from adult hamsters As shown in Table 1, the major onsequene of a 6- week old exposure was a signifiant redution of hamster body weight. It is of interest to mention that, during the experimental period (from May to July), old exposure did not promote hypothermia in the hamsters although a redution of physial ativity was observed in old-exposed animals. In spite of over-feeding (food intake was inreased 1-2%), this hange in weight was brought about by a notieable derease in epididymal and perirenal rat pads and fat ell size. Thus, adult hamsters, of the same age as the ontrols, with a redued fat ell size were obtained; mean fat ell size was similar to that found in younger hamsters. The ability of inreasing onentrations of lonidine to inhibit lipolysis in adipoytes from ontrol and old-exposed hamsters is presented in Fig. 2. The lak of inhibitory effet of lonidine an be seen in old-exposed hamster adipoytes even at high onentrations of the y2-adrenergi agonist. On the other hand the antilipolyti effet of PIA was observed in ontrol and old-exposed hamster fat ells. The dose-response urves of PIA are superimposable, suggesting that the adenosine-inhibiting system is not altered in the adipoytes of old-exposed animals. Effet of isoproterenol on lipolysis of fat ells from young, adult, and old-exposed adult hamsters Sine both y- and p-adrenoeptors exist in hamster fat ells, it was of interest to observe the effet of p- adrenergi stimulation on the adipoytes from the three groups of hamsters. The study of the effet of inreasing onentrations of isoproterenol (a 6-adrenergi agonist) on the lipolyti ativity of isolated fat ells was arried out in eah group (Table 2). When the lipolyti poteny of isoproterenol is expressed by the ratio of stimulated 1ipolysis:basal lipolysis, we obtain information on p-reeptor effiieny. The dose-response studies showed that in the three groups, the maximal P-mediated response was obtained with.5 PM isoproterenol in eah group. However the lipolyti ativity of the adipoyte in the different groups is expressed (on a per lo6 ells or per 1 mg total lipid basis) (Table 2), the ratio is not sig- Downloaded from by guest, on September 8, 218 Carpene, Berlan, and Lafontan Cell size and ap-adrenergi response in hamster adipoytes 769

5 Clonidine (MI 19 IO-* 17 lo6 15 PIA (MI n ae 75 - Adult ontrols o Adult plaed 1. in temperature ontrolled room 6'-6 weeks Fig. 2. Effet of inreasing onentrations of lonidine and PIA on theophylline-indued lipolysis in fat ells of adult ontrols and adult hamsters with a redued adipose tissue mass after old exposure. The effets of drugs are expressed as perent inhibition of theophylline-stimulated lipolysis. Values are means k SEM of (n) different experiments indiated by the numbers. Statistial signifiane of the inhibition of lipolysis promoted by the agents was tested aording to Student's paired t test. (*a, P <.1; **e, P <.1). nifiantly different at any onentration. Thus, the p- adrenergi effet is not notieably modified in adipoytes from young animals and from old-exposed emaiated animals. [3H]Clonidine binding on fat ell membranes from young, adult, and old-exposed adult hamsters In order to assess the results obtained onerning the a2-adrenergi response, we attempted to haraterize the a2-adrenoeptors using ['Hllonidine on fat ell membranes from the three groups of animals. The binding of ['Hllonidine to hamster fat ell membranes was rapid. Half-maximal binding was reahed in 2 min at 25 C. The assoiation of the radioligand to its binding sites reahed equilibrium within 1-15 min and was stable for at least 6 min. Fig. 3 shows a typial saturation urve of the speifi binding of ['Hllonidine as a funtion of ['Hllonidine onen- Downloaded from by guest, on September 8, 218 TABLE 2. Comparison of isoproterenol-stimulated lipolysis in isolated fat ells of young, adult ontrol, and adult hamsters plaed in a temperature-ontrolled room (6OC, 6 weeks) Isoproterenol ( p ~ ) n' Basal Lipolysis Adult hamsters (2-25 weeks) 5.89 f k f f f k.53' 2.91 f f k k 1.9 Young hamsters (4-5 weeks) f f f k f f.4.42 f.9.92 f f f.11 Adult hamsters plaed in f.22 temperature ontrolled.33 f.3 room (SOC, 6 weeks) 2.78 f f f.95.7 f f f.42 a Results are means f SEM of (n) different experiments. Lipolysis is expressed as pmol glyero1/1 mg lipid per 9 min. Lipolysis is expressed as pmol glyerol/16 ells per 9 min. However the lipolyti ativity is expressed, the ratio of stimulated lipolysis/basal lipolysis is not signifiantly different. Moreover, there are no signifiant differenes in the ratio (at orresponding onentrations) between the experimental groups. 77 Journal of Lipid Researh Volume 24, 1983

6 KD Bmax Adult F '2 - E 25 C 2 15 n 9) C.- e C - I Y 5 I A A A p G li (3H) Clonidine A (nm) B Young Cold-exposed Clonidlne bound fmolhg prot. Undetetable Undetetable Fig. 3. An example of speifi binding of ['Hllonidine to fat ell membranes from adult hamsters (). young hamsters () and adult hamsters with redued adipose tissue after old exposure (A). Panel A, as funtion of ['Hllonidine onentration. Fat ell membranes were inubated with inreasing onentrations of ['Hllonidine for 15 min at 25 C and speifi binding was determined as desribed in Materials and Methods. Panel B, Sathard plot of speifi binding of ['Hllonidine derived from the saturation urves (Panel A). The ratio (bound/free) of bound ligand to free ligand (nm) is plotted against bound ligand (fmol/mg protein). The slope of the plot (-l/k,,) was determined by linear regression analysis. The number of binding sites (B,,,,,) is alulated from the x interept of the regression line. Inset table, ['Hllonidine binding harateristis in fat ell membranes of adult hamsters (n = 6), young hamsters, and adult hamsters with redued adipose tissue after old exposure (6OC, 6 weeks) (old-exposed). Parameters of binding annot be alulated in either young or old-exposed hamster fat ell membranes sine the ratio bound/free is onstant whatever the onentration of ['HH]lonidine used. The term "undetetable" indiates that binding parameters ould not be determined. The experiments were repeated three times for young and old-exposed hamsters (fat ell membranes pooled from 25-3 hamsters). tration. Nonspeifi binding represented 2-25% of the total binding in adult ontrol fat ell membranes and 8% in the young and old-exposed adult groups at 6 nm ['H]lonidine. The binding exhibited saturation and Sathard analyses indiated that there was only one lass of binding site (n Hill = 1). The relative order of poteny of antagonists in ompetition with the [3H]lonidine binding sites was that expeted for an a2-adrenoeptor (not shown, see Ref. 19). The alulated B,,,, and KD values obtained from Sathard plots of various saturation urves are given in the inset in Fig. 3. It is notieable that in young hamsters or adult hamsters submitted to old exposure typial a2-adrenoeptor binding was not detetable. Nonspeifi binding values were higher than in the ontrols. Bound/Free values of Sathard plots were low and there was no possibility of onduting linear regression analysis of the data (Fig. 3). Thus, under our experimental onditions, ['H]lonidine binding in young or old-treated hamsters annot be aurately quantified and is very low. The large number of animals needed for a determination, 25-3, did not allow the binding experiments to be repeated more than three times. Nevertheless, it is notieable that the binding results are in good agreement with lipolysis studies. DISCUSSION The present studies on isolated fat ells of hamsters were arried out to extend our earlier results on a-adrenergi responses in fat ells (22, 23) and to investigate the relationship between aging, fat ell enlargement, and ellular responses to ateholamines. In the rabbit and the dog, the inreased &*-adrenergi responses, whih are onomitant to fat ell enlargement, ould explain, in part, the age-related derease in the lipolyti effet of physiologial amines (epinephrine and norepinephrine) previously reported (22, 23). The present results demonstrate that fat ell enlargement as a result of their maturation, ourring during Downloaded from by guest, on September 8, 218 Carpene, Berlan, and Lafontan Cell size and a2-adrenergi response in hamster adipoytes 771

7 the growth of the hamsters (from 4-5 to 2-25 weeks), is assoiated with an inrement of a,-adrenergi response. The latter part of the life span will not be onsidered here. The ourrene and extent of this effet is not assoiated with a parallel variation (inrement or deline) in P-adrenergi response (Table 2) as desribed previously in dog or rabbit adipoytes (22, 23). So the physiologial amines, ating on the two sites, stimulate more a,-adrenoeptors in the larger fat ells of week-old hamsters than in younger animals. In parallel to a,- and -adrenoeptor-mediated responses, we also investigated the adenosine-dependent inhibition of lipolysis to ompare it with the a,-mediated inhibiting effet. Sine adenosine reeptors and a2-adrenoeptors are oupled to plasma membrane adenylate ylase, at through an inhibition of the enzyme (3-33), and share a ommon pathway for their antilipolyti ations (34), it was of interest to hek, by indiret means, if the enzyme was able to be inhibited by these two mehanisms under the various experimental onditions, espeially when a,-adrenergi responses were laking. It is lear (Fig. 1) that the adenosine-dependent inhibition system is always funtional and not subjeted to notieable variations during the development of hamster adipose tissue. Suh a result supports the idea that the lak of a,-adrenergi responses is not linked to the fat that adenylate ylase annot be inhibited and suggests investigation of the binding site variation. Studies on a,-adrenoeptor sites (['H]lonidine binding sites) revealed that these reeptors are not detetable in small fat ells of young animals (Fig. 3). Thus, it appears that during fat ell development, a,-adrenergi sites mature and a,-adrenergi responses takes plae onomitantly. A determination of -adrenoeptor sites and adenosine sites was not arried out sine their biologial effets are not strikingly modified in the various groups. Thus, it would appear that during adipoyte growth and maturation, the fat ell plasma membrane undergoes strutural hanges mainly involving a,-adrenergi sites and their probable oupling to adenylate ylase. The -adrenergi responses and adenosine-dependent effets indiate that adenylate ylase an be either stimulated or inhibited, respetively, by the two agents. Suh a result is quite provoative, sine deline of various hormone reeptors generally ours during fat ell aging or maturation (35-38), and probably explains the derease of gluagon (36) or ACTH-dependent adenylate ylase ativation (1 7). Sine the ourrene of a,-adrenergi responsiveness an be ausally related to aging or fat ell enlargement, we attempted to separate the influene of age from that of fat ell size and to establish the major determinant in the maturation of a,-adrenergi responsiveness. Experiments were arried out to ompare two groups of hamsters of similar ages with lear-ut differenes in their adipose tissue mass. Instead of submitting animals to alori restrition, as previously done in the rabbit (39), the redution of adipose mass was promoted by exposure of hamsters to a old environment (6OC) for 6 weeks. Under suh onditions, a signifiant weight loss assoiated with a notieable fat ell size redution was observed; the mean fat ell diameter of old-exposed adult hamsters equalled that of 4-5-week-old hamsters (Table 1). Fat ell size redution was assoiated with a omplete disappearane of a,-binding sites (Fig. 3) and a2-adrenergi responsiveness (Fig. 2). No notieable modifiations in the sensitivity of the adipoytes to the P-agonist (Table 2) or PIA (Fig. 2) were found. The inrement of a2-adrenergi responsiveness observed during fat ell maturation and enlargement was ompletely reversed after weight loss and fat ell size redution. Thus, the appearane and disappearane of the a,-adrenoeptors of the adipose tissue seems to be orrelated with the extent of perirenal and epididymal fat stores. The striking differenes, observed under our experimental onditions, in a,-, P-, and adenosine-reeptor modifiations suggest that the regulation of these reeptors in the adipose tissue is partly unrelated. Diret measurements of the senstivity of isoproterenol-stimulated and PIA-inhibited adenylate ylase ativities assoiated to the determination of P- and adenosine-reeptor sites will probably improve our data on adenosine- and P-adrenoeptor-mediated effets. Among the mehanisms that an be advaned to explain the derease in the a,-adrenergi response in the adipoytes of old-exposed hamsters, the hormonal hanges ourring during old adaptation ould play a ritial role in the ontrol of adrenoeptor-mediated effets. It is well known that ateholamine exretion (mainly norepinephrine) is inreased by old exposure; this adaptative mehanism enhanes old resistane in nonhibernating animals (4-42). The hroni exposure of various ells to ateholamines is well known to indue hyporesponsiveness of the ells to the amines, a phenomenon ommonly known as desensitization (43). Is a,-adrenoeptor depletion due to a desensitization promoted by higher ateholamine levels resulting from old-exposure? The absene of apparent desensitization of P-adrenergi reeptors makes this hypothesis rather diffiult to uphold. P-Adrenoeptor desensitization has been shown to exist in fat ells (43-45), while a2-adrenoeptor desensitization is still questionable (45). This aspet requires further investigations and ateholamine determinations; moreover, the effet of a shorter old exposure should also be investigated. Another hormonal fator able to play a notieable role in the ontrol of a,-adrenergi responsiveness in adipoytes of oldexposed hamsters is thyroxine. Radioimmunoassay teh- Downloaded from by guest, on September 8, Journal of Lipid Researh Volume 24, 1983

8 niques have revealed high plasma triiodothyronine (T,) levels in old-alimated rats without any symptom of hyperthyroidism (41, 42). If we refer to Garia-Sainz et al. (46), a2-adrenergi sensitivity of hamster adipoytes is unaltered by thyroid status while a onfliting result showed that hypothyroidism inreases the a2-antilipolyti response (47). This aspet is still unsolved. Our data do not provide answers with respet to the auses of the inrease and derease in fat ell a2-adrenoeptors and a2-adrenergi responses, but they provide a useful model for further investigations into the interplay between the various families of reeptors involved in the ontrol of lipolyti ativity of the fat ell. More researh is needed to shed light on these problems. Nevertheless, in order to find the ause of a2- adrenoeptor variations and to gain further insight into y2-adrenoeptor regulation, adipose tissue has the unique apaity, among the other body tissues, to alter its mass under various onditions depending on age, endorine status, and energy ba1ane.l The authors are grateful to Mihele Dauzats for her exellent tehnial assistane and preparation of the figures and to Elise Bluthe and Odette Azimon for animal breeding. This work was supported by CNRS (ERA 4 12 and ATP Pharmaologie des rkepteurs des neuromkdiateurs) and by the Institut National de la Santk et de la Reherhe Mkdiale (C.R.L ). Manusript rereived 27 July 1982 and in rmisedform 31 January REFERENCES 1. Ostman, J., and S. Efendi Cateholamines and metabolism of human adipose tissue. Effet of isopropylnoradrenaline and adrenergi bloking agents on lypolysis in human omental adipose tissue in vitro. Ata Med. Sand. 187: Burns, T. W., P. E. Langley, and G. A. 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