Role of Adenosine in Insulin-Stimulated Release Of

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Role of Adenosine in Insulin-Stimulated Release of Leptin From Isolated White Adipocytes of Wistar Rats Ju e i - T ang Cheng, I-Min Liu, Tzong-Cherng Chi, Kazumasa Shinozuka, Feng-Hwa Lu, Ta-Jen Wu, and Chih Jen Chang

Leptin, the o b gene product that can decrease caloric intake and increase energy expenditure, is function- ally released by insulin from adipose tissue. Adenosine he ob gene, which encodes a 167-amino acid pep- is thought to be an important regulator of the action of tide named leptin in white adipocytes (1), has insulin in adipose tissue. The present study investi- received increasing attention for its role in the gated the role of adenosine in the release of leptin by regulation of food intake and whole-body energy insulin in isolated rat white adipocytes. Release of lep- T balance in rodents and humans (2,3). It has been demon- tin, measured by radioimmunoassay, from insulin-stimulated samples was seen after 30 min. Adenosine deam- strated that circulatory leptin levels in rats were modulated inase, at concentrations sufficient to metabolize by exogenous insulin (4) and ob gene expression was endogenous adenosine, decreased insulin-stimulated induced by corticosteroids (5). Insulin also stimulated the leptin release. Also, the insulin-stimulated leptin mRNA levels of the ob gene in rat adipocytes (6). Thus, release was completely blocked by the adenosine A1 insulin appears to be one of the important regulators of ob receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine gene expression and leptin secretion in adipose tissue. (DPCPX). Mediation of endogenous adenosine in this Adenosine is another endogenous regulator in adipose action of insulin was further supported by the assay of tissue. Under physiological concentrations, adenosine adenosine released into the medium from adipocytes increased the sensitivity of glucose transport (7,8) and glucose stimulated with insulin. In addition, activation of 6 oxidation and/or metabolism (9,10) to the stimulation with adenosine A1 receptors by N - c y c l o p e n t y l a d e n o s i n e ( C PA) induced an increase in leptin release in a con- insulin in adipocytes. Also, the action of adenosine in adipose centration-dependent manner that could be blocked by tissue appears to be mainly through activation of adenosine antagonists, either DPCPX or 8-(p- s u l f o p h e n y l ) t h e o- A1 receptors (10,11). The release of adenosine from adipose phylline (8-SPT). In the presence of U73312, a specific tissue has also been demonstrated (12,13). However, the role inhibitor of phospholipase C (PLC), CPA - s t i m u l a t e d of adenosine in mediating the effect of insulin on leptin leptin secretion from adipocytes was reduced in a con- release is still unknown. Thus, in the present study, we exam- centration-dependent manner, but it was not affected by ined the release of adenosine in response to insulin at con- U73343, the negative control for U73312. Moreover, centrations sufficient to stimulate release of leptin from rat chelerythrine and GF 109203X diminished the CPA - white adipocytes, and we studied the role of adenosine A1 stimulated leptin secretion at concentrations suff i c i e n t receptors using specific agonists and antagonists. to inhibit protein kinase C (PKC). These results suggest that, in isolated white adipocytes, the released adenosine acts as a helper and/or a positive regulator for RESEARCH DESIGN AND METHODS insulin in the release of leptin via an activation of Animal models. Male Wistar rats at the age of 8–10 weeks were obtained from the animal center of National Cheng Kung University Medical College. All animal adenosine A1 receptors that involves the PLC-PKC procedures were performed according to the Guide for the Care and Use of Lab - p a t h w a y. Diabetes 4 9 :2 0–24, 2000 oratory Animals of the National Institutes of Health, as well as the guidelines of the Animal Welfare Act. Rats were housed four per cage with wood chips for bed- ding, and food and water were available ad libitum. Animal cages were kept in a room where the temperature (23 ± 1°C) and light/dark cycle (12-h light:12-h dark) were closely controlled. From the Departments of Pharmacology (J.-T.C., I.-M.L., T.-C.C.), Family Adipocyte isolation and incubation. White adipocytes were prepared from the Medicine (F.-H.L., C.J.C.), and Internal Medicine (T. - J . W.), College of Med- epididymal fat pads of rats, as previously described (14). Briefly, the fat pads were icine, National Cheng Kung University, Tainan City, Taiwan, Republic of immersed in Krebs-Ringer bicarbonate buffer (KRBB) (37°C, pH 7.4) containing China; and the Department of Pharmacology (K.S.), School of Pharmacy, 1 mmol/l glucose and 1% fatty acid–free bovine serum albumin and were equili- Mukogawa Wo m e n ’s University, Koshiyen, Nishinomiya City, Japan. brated with 95% O –5% CO . Cells were minced and subjected to collagenase Address correspondence and reprint requests to Professor Juei-Ta n g 2 2 (Sigma, St. Louis, MO) (2 mg/g fat) for 1 h of digestion at 37°C with constant shak- Cheng, PhD, FCP, Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan City, Taiwan 70101, Republic of ing at 40 cycles/min. The cell suspension was filtered though a 500-µm nylon mesh China. E-mail: [email protected]. and was washed three times in KRBB. Cells were adjusted to a 20% concentration Received for publication 24 May 1999 and accepted in revised form 6 with KRBB and equilibrated at 37°C for 30 min with constant shaking. At the end October 1999. of this period, the conditioning medium was changed, and the cells were incubated ADA, adenosine deaminase; C PA, N6-cyclopentyladenosine; DPCPX, in the absence or presence of pharmacological inhibitors, either 8-cyclopentyl-1,3- 8-cyclopentyl-1,3-dipropylxanthine; KRBB, Krebs-Ringer bicarbonate dipropylxanthine (DPCPX) (Research Biochemical, Natick, MA), 8-(p- s u l f o- buffer; PKC, protein kinase C; PLC, phospholipase C; 8-SPT, 8-(p- s u l f o- phenyl)theophylline (8-SPT) (Research Biochemical), adenosine deaminase (ADA) p h e n y l ) t h e o p h y l l i n e . (Boehringer Mannheim Biochemical, Mannheim, Germany), U73122 (Research

20 DIABETES, VOL. 49, JANUARY 2000 J.-T. CHENG AND ASSOCIATES

TA B L E 1 Effects of ADA and DPCPX on the secretion of leptin stimulated by insulin from the isolated white adipocytes of Wistar rats

Leptin (ng/ml)

B a s a l 0.69 ± 0.05 DPCPX (1 µmol/l) 0.65 ± 0.04 ADA (2 µg/ml) 0.59 ± 0.06 Insulin (35 n m o l / l ) 1.63 ± 0.17* Ve h i c l e 0.60 ± 0.15 DPCPX (µmol/l) 0 . 0 1 1.29 ± 0.14† 0 . 1 1.08 ± 0.09‡ 1 0.93 ± 0.08‡ Vehicle ADA (µg/ml) 0 . 5 1.43 ± 0.09† 1 1.11 ± 0.07‡ Insulin (nmol/l) 2 0.88 ± 0.06‡ FIG. 1. Effect of insulin on the amount of adenosine released into medium containing isolated white adipocytes. Each column indicates Data are means ± SE from seven animals. The vehicle used to dis- the mean values from eight animals with SE bar. *P < 0.05 and **P < solve DPCPX or ADA was given in the same volume. The basal 0.01 relative to vehicle-treated control animals. level of leptin was obtained from adipocytes incubated with KRBB only. *P < 0.001 compared with basal level; †P < 0.05, ‡P < 0.01 relative to insulin-treated samples. a nonsignificant reduction in the spontaneous secretion of leptin (P > 0.05). Biochemical), U73343 (Research Biochemical), chelerythrine (Research Bio- Mo r e o v e r , as shown in Tab l e 1, preincubating adipocytes chemical), or GF 109203X (BIOMOL, Plymouth Meeting, PA) at the indicated con- with an antagonist of adenosine A1 receptors, DPCPX, for 30 centrations for 30 min at 37°C under continuous shaking (40 cycles/min). Then, min also caused a concentration-related inhibition of insulin- the cells were incubated with bovine insulin (Novo Nordisk, Bagsvaerd, Denmark) or N6-cyclopentyladenosine (CPA) (Research Biochemical) at the desired con- stimulated leptin secretion. Similarly, the basal secretion of centrations for another 30 min to stimulate the release. The medium from each leptin was not significantly modified by DPCPX. incubation was then collected and frozen at –70°C until the assay for leptin or Ef fect of insulin on adenosine release from isolated adenosine was performed. white adipocytes. Adenosine was determined in the Leptin analysis. Leptin secreted into the incubation medium was determined by medium containing adipocytes incubated with insulin at radioimmunoassay using a commercially available kit (Linco Research, St. Charles, MO), as described previously (15). The sensitivity of the assay was desired concentrations for 30 min. As shown in Fig. 1, insulin 0. 2 ng/ml. Samples from an individual were analyzed in triplicate at the same time. enhanced adenosine release from isolated adipocytes in a con- The obtained values were indicated as nanograms of leptin-like immunoreactiv- centration-dependent manner (n = 8). ity per milliliter of plasma. Insulin or other test compounds used in the present Ef fect of CPA on leptin secretion from isolated white study did not affect the binding of leptin with antibodies. Adenosine analysis. The amount of adenosine in the medium was measured by adipocytes. To examine the role of adenosine A1 re c e p t o r s high-performance liquid chromatography with fluorescence detection, as previ- in leptin secretion from isolated adipocytes, an agonist of ously described (16). The wavelengths for excitation and emission were set at 305 adenosine A1 receptors, CPA, was used. After a 30-min incu- and 420 nm, respectively. The mobile phase, 5 mmol/l citrate buffer with 1.5% ace- bation of isolated adipocytes with CPA, a concentration- tonitrile, was adjusted to pH 4.5 with 2-diethylaminoethanol and was run at a flow dependent increase of leptin release was seen in samples rate of 1.0 ml/min at ambient temperature (22–24°C). The concentration of adenosine was calibrated from the standards, as described previously (16). treated with CPA at concentrations from 0.1 to 10 µmol/l Statistics. Results are expressed as means ± SE from each group, and the (F i g . 2). To further examine whether these effects of the number (n) of rats is indicated. Data for concentrations of adenosine or leptin adenosine analog were mediated through adenosine recep- were analyzed using one-way analysis of variance. P values £0.05 were regarded tors, the selective adenosine antagonists DPCPX and 8-SPT as significa n t . were added into the incubation medium. The basal secretion of leptin was not significantly modified by DPCPX and 8-SP T , RE S U LT S but a concentration-dependent blockade of CPA- s t i m u l a t e d Ef fect of insulin on leptin release from the isolated leptin release was observed in adipocytes receiving a 30-min white adipocytes. The amount of leptin accumulated in the preincubation with DPCPX at concentrations from 0.01 to medium from adipocytes stimulated with insulin was deter- 1 µmol/l (Tab l e 2). Similar antagonism by 8-SPT to the action mined. In the preliminary study, the effective concentration of CPA was also obtained (Tab l e 2). Moreover, the action of of insulin (35 nmol/l) for induction of leptin secretion was C PA was enhanced by insulin. Coincubation of insulin obtained and used in subsequent studies. As shown in (7 nmol/l) and CPA (0.1 µmol/l) induced the release of le p t i n Tab l e 1, incubation of freshly isolated rat adipocytes with to 1.48 ± 0.14 ng/ml (n = 6), which was significantly different insulin produced a marked release of leptin into the incuba- (P < 0.05) from that of the insulin (7 nmol/l) alone–st i m u l a t e d tion medium. Under stimulation with insulin, the maximal group (1.16 ± 0.10 ng/ml; n = 6) or the CPA (0.1 µm o l / l ) - release of leptin was evident after 30 min. A 30-min preincu- induced release (1.01 ± 0.09 ng/ml; n = 6). bation of adipocytes with ADA, which can decrease endoge- E ffects of inhibitors of phospholipase C or protein nous adenosine by converting it to inosine, caused a con- kinase C on leptin secretion from isolated white centration-related inhibition of insulin-stimulated leptin ad i p o c y t e s . In the present study, U73122 was used to inhibit release (Tab l e 1). However, incubation with ADA produced phospholipase C (PLC) activity and U73343 was used as the

DIABETES, VOL. 49, JANUARY 2000 21 ADENOSINE IN LEPTIN RELEASE BYINSULIN

TA B L E 3 Effects of U73122 and U73343 on the secretion of adenosine stimulated by CPA from the isolated white adipocytes of Wistar rats

Leptin (ng/ml)

B a s a l 0.70 ± 0.06 U73122 (3.3 µmol/l) 0.67 ± 0.06 U73343 (3.3 µmol/l) 0.68 ± 0.07 C PA (10 µmol/l) 1.43 ± 0.09* Ve h i c l e 1.41 ± 0.08 U73122 (µmol/l) 0 . 2 5 1.07 ± 0.03† 1 0.91 ± 0.06‡ 3 . 3 0.69 ± 0.04‡ U73343 (µmol/l) 3 . 3 1.35 ± 0.08

FIG. 2. Effect of CPA on the secretion of leptin into medium contain- Data are means ± SE from six animals. *P < 0.001 compared with ing isolated white adipocytes. Each column indicates the mean values basal level; †P < 0.05, ‡P < 0.01 vs. CPA-treated samples. from eight animals with SE bar. *P < 0.05, **P < 0.01, and ***P < 0.01 relative to vehicle-treated control animals. tration-dependent manner (n = 6). Similarly, the basal secretion of leptin was not significantly (P > 0.05) modified by negative control. After a 30-min preincubation with U73122 either of these inhibitors. or U73343, adipocytes were treated with 10 µmol/l CPA for another 30 min. As shown in Tab l e 3, CPA-stimulated leptin D I S C U S S I O N secretion was decreased by U73122 in a concentration- In the present study, we found that adenosine appears to be dependent manner (n = 6). Addition of the inactive congener involved in the insulin-stimulated release of leptin from iso- U73343 failed to modify the action of CPA (P > 0.05), even at lated rat adipocytes. In addition to the regulation of leptin 3.3 µmol/l, which was the maximal concentration of U73122 gene expression by insulin (1), it has also been shown previ- used to abolish the action of CPA totally (Tab l e 3). However, ously that insulin produces a rapid and marked release of lep- neither U73122 nor U73343 significantly modified the basal tin from freshly isolated rat adipocytes (15). A similar release secretion of leptin from adipocytes. of leptin by insulin was observed in the present study. How- Mo r e o v e r , chelerythrine and GF 109203X were used to ev e r , mediation of this insulin action by endogenous adeno- inhibit the action of protein kinase C (PKC). After preincu- sine has not been reported before. bation with chelerythrine or GF 109203X at the indicated Adenosine has an ability to mimic insulin action in tissues, concentrations for 30 min, adipocytes were incubated with especially the adipose tissue, where the effects of insulin are CP A at 10 µmol/l for another 30 min. As shown in Tab l e 4, both m o d i fied by changes in adenosine levels, including the chelerythrine and GF 109203X decreased the CPA- s t i m u l a t e d effects on glucose transport (7,8), glucose oxidation (9,10), leptin release from isolated white adipocytes in a concen- lipolysis (10,11), and the activity of cAMP phosphodiesterase

TA B L E 2 TA B L E 4 Effects of DPCPX and 8-SPT on the secretion of leptin stimulated Effects of chelerythrine and GF 109203X on the secretion of by CPA from the isolated white adipocytes of Wistar rats adenosine stimulated by CPA from the isolated white adipocytes of Wistar rats Leptin (ng/ml) Leptin (ng/ml) B a s a l 0.69 ± 0.04 DPCPX (1 µmol/l) 0.66 ± 0.07 B a s a l 0.67 ± 0.07 8-SPT (1 µmol/l) 0.68 ± 0.06 Chelerythrine (10 µmol/l) 0.65 ± 0.06 C PA (10 µmol/l) 1.42 ± 0.09* GF 109203X (70 µmol/l) 0.63 ± 0.06 Ve h i c l e 1.38 ± 0.14 C PA (10 µmol/l) 1.45 ± 0.10* DPCPX (µmol/l) Ve h i c l e 1.42 ± 0.09 0 . 0 1 1.02 ± 0.07† Chelerythrine (µmol/l) 0 . 1 0.92 ± 0.06‡ 0 . 1 1.34 ± 0.09 1 0.64 ± 0.06‡ 1 1.21 ± 0.08† 8-SPT (µmol/l) 1 0 1.09 ± 0.07‡ 0 . 0 1 1.19 ± 0.06† GF 109203X (µmol/l) 0 . 1 1.02 ± 0.08‡ 2 0 1.38 ± 0.05 1 0.70 ± 0.05‡ 5 0 1.28 ± 0.04† 7 0 1.19 ± 0.05‡ Data are means ± SE from seven animals. *P < 0.001 compared with basal level; †P < 0.05, ‡P < 0.01 relative to CPA - t r e a t e d Data are means ± SE from six animals. *P < 0.001 compared with samples. basal level; †P < 0.05, ‡P < 0.01 vs. CPA-treated samples.

22 DIABETES, VOL. 49, JANUARY 2000 J.-T. CHENG AND ASSOCIATES

(17) or pyruvate dehydrogenase (18). Therefore, changes in we did not determine the activity of PKC directly, chelery- adenosine levels and/or stimulation of adenosine receptors thrine or GF 109203X inhibited the action of CPA at concen- may influence the function of insulin in both responsiveness trations that would be sufficient to inhibit PKC activity and sensitivity. The present study supports this view from the (27,28). Mediation of PKC in the action of CPA can thus be findings that ADA at concentrations sufficient to convert considered. Taken together, the involvement of the PLC-PKC endogenous adenosine to inosine (10,19) reduced the stimu- pathway in the stimulation of leptin secretion by an activation lation of insulin on leptin secretion from freshly isolated rat of adenosine A1 receptors can thus be considered. Currently, adipocytes (Tab l e 1). Actually, insulin caused a concentration- phosphatidylinositol 3-kinase and Map/Erk kinase are dependent release of adenosine from rat white adipocytes reported to have a key role in insulin-stimulated leptin syn- (F i g . 1). Mediation of adenosine in insulin-stimulated leptin thesis and secretion in rat adipocytes (29). The relationship release from adipocytes can thus be considered. However, the of this signal to the PLC-PKC pathway should be clarified amount of adenosine in the present study was not at the using molecular tools or other methods in the future. absolute level that needs to be estimated in the future. In conclusion, the present study shows that insulin may An important role of adenosine A1 receptors in rat increase the secretion of adenosine from adipocytes. An acti- adipocytes has been documented (20). The antagonists spe- vation of adenosine A1 receptors through the PLC-PKC path- ci fi c for the adenosine A1 re c e p t o r , either DPCPX (21) or way may increase the insulin-stimulated release of leptin in 8-SPT (22), were thus used to study the role of adenosine A1 isolated white adipocytes. receptors in insulin-stimulated leptin release. The stimulatory effect of insulin on leptin secretion was blocked by DPCPX A C K N O W L E D G M E N T S in a concentration-dependent manner (Tab l e 1). The mecha- The present study is supported in part by a grant from the nism would therefore appear to involve activation of adeno- National Science Council of the Republic of China (NSC88- sine A1 receptors. Moreover, pharmacological manipulation 2314-B006-043). with CPA to activate adenosine A1 receptors also enhanced We appreciate the help of Professor Brian Tomlinson and leptin secretion into the medium in a concentration-related Professor Y.C. Tong in editing the manuscript. Thanks also to manner (Fig. 2). The stimulatory effect of CPA on leptin S.M. Tai for technical assistance. release was reversed by the preincubation of adipocytes with DPCPX or 8-SPT at concentrations sufficient to block adeno- R E F E R E N C E S sine A receptors. Moreover, the action of CPA was facilitated 1 1. Leroy P, Dessolin S, Villageois P, Moon BC, Friedman JM, Ailhaud G, Dani C: by coincubation with insulin. This further indicates that the Expression of ob gene in adipose cells: regulation by insulin. J Biol Chem amount of adenosine released by insulin is sufficient to 271:2365–2368, 1996 mimic the CPA-stimulated leptin secretion by activation of 2. Trayhurn P, Rayner DV: Hormones and the ob gene product (leptin) in the con- adenosine A receptors in isolated adipocytes. However, the trol of energy balance. Biochem Soc Tra n s 24:565–570, 1996 1 3. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM: Posi- release of leptin by insulin was reduced but not abolished by tional cloning of the mouse obese gene and its human homologue. Na t u r e ADA or DPCPX (Tab l e 1). Also, the basal secretion of leptin 372:425–432, 1994 was not significantly affected by blockade of adenosine A1 4. Hardie LJ, Rayner DV, Holmes S, Trayhurn P: Circulating leptin levels are mod- receptors or ADA (Ta b l e 1). Therefore, the mediation of ulated by fasting, cold exposure, and insulin administration in lean but not insulin-stimulated leptin secretion by adenosine seems to be Zucker (fa / f a ) rats as measured by ELISA. Biochem Biophys Res Commun 223:660–665, 1996 only partial and not complete. A similar response in physio- 5. De Vos P, Saladin R, Auwerx J, Staels B: Induction of ob gene expression by logical function can thus be considered. corticosteroids is accompanied by body weight loss and reduced food intake. The accumulation of diacylglycerol and Ca2+ as a result of J Biol Chem 270:15958–15961, 1995 activation of PLC causes the activation of PKC (23). Activa- 6. Saladin R, De Vos P, Guerre-Millo M, Leturque A, Girard J, Stales B, Auwerx J: Transient increase in obese gene expression after food intake or insulin tion of adenosine A1 receptors may link to this subcellular sig- administration. Na t u r e 377:527–529, 1995 naling (24).We found that PLC-PKC may be a major signaling 7. Smith U, Kuroda M, Simpson IA: Counterregulation of insulin-stimulated glu- pathway of adenosine A1 receptors in CPA-stimulated leptin cose transport by catecholamines in the isolated rat adipose cell. J Biol Chem release using inhibitors specific for PLC or PKC. Recently, 259:8758–8763, 1984 U73122 has been widely used as a specific inhibitor of PLC 8. Lonnroth P, Davies JI, Lonnroth I, Smith U: The interaction between the adenylate cyclase system and insulin-stimulated glucose transport: evidence (25), and U73122 has been used as the negative control for for the importance of both cyclic-AMP-dependent and -independent mecha- U73343 (26). Preincubation with U73122 abolished the CPA- nisms. Biochem J 243:789–795, 1987 stimulated leptin secretion from isolated white adipocytes, 9. Schwabe U, Schonhofer PS, Ebert R: Facilitation by adenosine of the action indicating the participation of PLC. Also, U73343, the nega- of insulin on the accumulation of adenosine 3 :5 -monophosphate, lipolysis, tive control of U73122, failed to modify this stimulatory and glucose oxidation in isolated fat cells. Eur J Biochem 46:537–545, 1974 10 . Vannucci SJ, Klim CM, Martin LF, LaNoue KF: A1-adenosine receptor–induced action of CPA (Tab l e 3). This indicates that PLC mediates in adipocyte adenylate cyclase and lipolysis in Zucker rats. Am J Physiol 25 7 : the stimulation of leptin secretion by adenosine A1 re c e p t o r s E871–E878, 1989 in isolated white adipocytes. Direct determination of the PLC 11 . Mersmann HJ, Carey GB, Smith BO: Influence of nutritional weaning on porcine adipocyte -adrenergic and adenosine A receptors. J Anim Sci activity in adipocytes treated with CPA will provide helpful 1 75:2368–2377, 1997 evidence of this view. 12 . Schwabe U, Ebert R, Erbler HC: Adenosine release from fat cells: effect on cyclic The role of PKC was investigated using chelerythrine (27) AMP levels and hormone actions. Adv Cyclic Nucleotide Res 5:569–584, 1975 and GF 109203X (28), both of which can inhibit PKC in a com- 13 . Capogrossi MC, Di Girolamo M, Zimring H, Crandall D, Francendese A, Israili petitive manner with respect to the phosphate acceptor. In the ZH: Adenosine release from isolated rat adipocytes: influence of fat cell concentration and cell size. Proc Soc Exp Biol Med 182:15–22, 1986 presence of either of these PKC inhibitors, the release of lep- 14 . Chang CJ, Kao JT, Lee TL, Lai CW, Cheng JT: Comparison of isoproterenol with tin induced by CPA from isolated adipocytes was attenuated 14 BRL37344 in activation of 3-adrenoceptors to inhibit the uptake of [ C] - d e o x y - in a concentration-dependent manner (Tab l e 4). Although D-glucose and translocation of glucose transporter (GLUT4) to membrane frac-

DIABETES, VOL. 49, JANUARY 2000 23 ADENOSINE IN LEPTIN RELEASE BYINSULIN

tion in rat adipocytes. J Auton Nerv Syst 61:191–194, 1996 Proc Natl Acad Sci U S A 77:5547–5551, 1980

15 . Gettys TW, Harkness PJ, Watson PM: The 3-adrenergic receptor inhibits 23 . Gerwins P, Fredholm BB: Activation of adenosine A1 and bradykinin recep- insulin-stimulated leptin secretion from isolated rat adipocytes. En d o c r i n o l - tors increase protein kinase C and phospholipase D activity in smooth mus- ogy 137:4054–4057, 1996 cle cells. Naunyn Schmiedebergs Arch Pharmacol 351:186–193, 1995

16 . Mohri K, Takeuchi K, Shinozuka K, Bjur RA, Westfall DP: Simultaneous deter- 24 . Linden J: Structure and function of A1 adenosine receptors. FASEB J 5: 2 6 6 8 – mination of nerve-induced adenine nucleotides and nucleosides released 2676, 1991 from rabbit pulmonary artery. Anal Biochem 210:262–267, 1993 25 . Smallridge RC, Kiang JG, Gist ID, Fein HG, Galloway RJ: U-73122, an amino- 17 . Wong EH, Ooi SO, Loten EG, Sneyd JG: The action of adenosine in relation steroid phospholipase C antagonist, noncompetitively inhibits thyrotropin-

to that of insulin on the low-Km cyclic AMP phosphodiesterase in rat adipo- releasing hormone effects in GH3 rat pituitary cell. Endocrinology 13 1 : 1 8 8 3 – cytes. Biochem J 227:815–851, 1985 1888, 1992 18 . Wong EH, Smith JA, Jarett L: Effect of adenosine on insulin activation of rat 26 . Muto Y, Nagao T, Urushidani T: The putative phospholipase C inhibitor adipocyte pyruvate dehydrogenase. FEBS Lett 175:68–71, 1984 U73122 and its negative control, U73343, elicit unexpected effects on the 19 . Ciaraldi TP: The role of adenosine in insulin action coupling in rat adipocytes. rabbit parietal cell. J Pharmacol Exp Ther 282:1379–1388, 1997 Mol Cell Endocrinol 60:31–41, 1988 27 . Herbert JM, Augereau JM, Gleye J, Maffrand JP: Chelerythrine is a potent and

20 . Saggerson ED, Jamal Z: Differences in the properties of A1-type adenosine sp e c i ﬁ c inhibitor of protein kinase C. Biochem Biophys Res Commun 17 2 : 9 9 3 – receptors in rat white and brown adipocytes. Biochem J 269:157–161, 1990 999, 1990 21 . Lohse MJ, Klotz KN, Lindenborn-Fotinos J, Reddington M, Schwabe U, Olsson 28 . Toullec D, Pianetti P, Coste H, Bellevergue P, Grand-Perret T, Ajakane M, RA: 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX): a selective high-affinity Baudent V, Boissin P, Boursier E, Loriolle F, Duhamel L, Charon D, Kirilovsky

antagonist radioligand for A1 adenosine receptors. Naunyn Schmiedebergs J: The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of Arch Pharmacol 336:204–210, 1987 protein kinase C. J Biol Chem 266:15771–15781, 1991 22 . Bruns RF, Daly JW, Snyder SH: Adenosine receptors in brain membrane: 29 . Bradley RL, Cheatham B: Regulation of ob gene expression and leptin secre- binding of N6-cyclohexyl [3H]adenosine and 1,3-diethyl-8[3H] p h e n y l x a n t h i n e . tion by insulin and dexamethasone in rat adipocytes. Di a b e t e s 48:272–278, 1999

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