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-stim- ulated 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 adeno- sine 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 lep- tin (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.