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Endocrinology Vol. 141, No. 4 1442-1445
Copyright © 2000 by The Endocrine Society

ARTICLES

A1 Adenosine Receptor Activation Increases Adipocyte Leptin Secretion1

Alan M. Rice, John N. Fain and Scott A. Rivkees

Department of Pediatrics, Yale University (A.M.R., S.A.R.), New Haven, Connecticut 06520; and Department of Biochemistry, University of Tennessee (J.N.F.), Memphis, Tennessee 38163

Address all correspondence and requests for reprints to: Scott A. Rivkees, M.D., Section of Pediatric Endocrinology, Department of Pediatrics, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520. E-mail: scott.rivkees{at}yale.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A1 adenosine receptors (A1ARs) are heavily expressed in adipocytes and influence fat cell metabolism. Because increasing evidence suggests a role for leptin in mediating appetite and fat cell metabolism, we tested whether A1ARs regulate leptin production. Rats were treated with the A1AR agonist N6-cyclopentyladenosine (CPA), and changes in circulating levels of leptin and leptin gene expression were examined. Serum leptin levels rose 2- to 10-fold, with peak increases seen 8–16 h after injection of CPA (P < 0.05). In contrast, CPA did not alter steady state levels of adipose tissue leptin mRNA. To assess the influence of endogenous adenosine on circulating leptin levels, rats were also injected with dipyridamole (DPY), an adenosine reuptake blocker. DPY induced 80% increases in serum levels at 8 h after injections (P < 0.05). Supporting the idea that stimulation of leptin production is A1AR mediated, pretreatment with the A1AR antagonist 8-cyclopentyl-1,3-dipropylxanthine completely blocked increases in leptin levels after DPY treatment. To complement in vivo studies, the effect of A1AR activation on leptin secretion was also studied in epididymal fat pad cultures. In cultures, CPA treatment increased leptin secretion by 37% (P < 0.05). Collectively, these data show that the adenosinergic system can increase leptin secretion by directly activating A1ARs in fat tissue.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE HORMONE LEPTIN is a 167-amino acid hydrophilic protein that is produced by white adipocytes (1). Considerable evidence suggests that leptin plays a key role in regulating body weight (2, 3, 4, 5, 6, 7, 8, 9). Mice and humans deficient in leptin have excessive appetite and obesity (2, 3, 4). Similarly, rodents and humans with defects in leptin receptors are obese (5, 6, 7, 8, 9, 10). It has also been shown that treatment of leptin-deficient mice and humans with leptin normalizes appetite and reduces body fat content (2, 3, 4, 11).

Currently, studies are in progress to assess the utility of exogenous leptin administration for the treatment of obesity in individuals without known leptin deficiency or defects in leptin receptors (12, 13). However, administration of leptin to humans is problematic due to poor gastrointestinal bioavailability, and local reactions to sc injections occur (13). Alternative strategies for increasing leptin levels, therefore, warrant consideration.

Various factors are known to influence circulating leptin levels. Glucocorticoids, tumor necrosis factor-{alpha}, and insulin increase circulating leptin levels (14, 15, 16, 17, 18). Of these, only glucocorticoids increase leptin gene expression (14, 15, 16, 17, 18). Leptin production in adipocytes is also influenced by changes in cellular cAMP levels (19, 20, 21, 22, 23, 24). ß-Adrenergic receptor activation, which increases intracellular cAMP levels, decreases both adipocyte leptin gene expression and serum leptin levels (19, 20, 21, 22, 23). It has also been shown that leptin production is directly influenced by alterations in cellular cAMP levels, with increases in cAMP decreasing leptin release (19, 24).

In addition to the recognized effects of the adrenergic system on fat cell metabolism, evidence gathered over the past 2 decades suggests that the adenosinergic system influences adipocyte physiology (25, 26, 27, 28). Adenosine is a nucleoside released by all cells that acts as an extracellular humoral chemical signal (29). Adenosine acts via specific receptors that contain seven transmembrane domains and couple to guanine nucleotide-binding proteins (30, 31). Activation of the Gi-coupled A1 and A3 receptors decreases cytosolic cAMP levels; activation of the Gs-coupled A2a and A2b receptors increases cAMP levels (30, 31).

In fat tissue, A1ARs are the most heavily expressed adenosine receptor subtype and mediate most of the effects of adenosine on adipocyte physiology (28). Because A1ARs act to lower cellular cAMP stores, we hypothesized that adenosine may act via A1ARs to stimulate leptin secretion (26, 27). Thus, we examined the effects of the A1AR activation on leptin secretion in vivo and in vitro. We now report that A1AR activation increases leptin secretion by acting directly on fat tissue.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Three hundred- to 700-g male Sprague Dawley rats (retired breeders) were obtained from Charles River Laboratories, Inc. (Raleigh, NC). Rats were individually housed, with free access to feed and water under 12-h light, 12-h dark lighting cycles. On the day of the study, food was removed from the cages at 0800 h, and rats were injected ip with study drugs, including N6-cyclopentyladenosine (CPA), dipyridamole (DPY), and 1,3-dipropyl-8-cyclopentylxanthine (CPX). The rats were then killed under anesthesia by CO2, and blood was obtained by cardiac puncture. Immediately after clotting, blood was centrifuged at 2000 x g for 20 min at 4 C. Individual samples of serum were then removed and stored at -80 C. All studies were approved by the Yale animal care and use committee.

Fat tissue cultures
Fat tissue was cultured as previously described (15). Briefly, epididymal fat tissue was removed and cut into 5- to 20-mg pieces and incubated in 50-ml polypropylene tubes in a gyratory water bath shaker at 100 rpm and 37 C. The incubation buffer was DMEM/Ham’s F-12 (1:1; no. 2906, Sigma, St. Louis, MO) containing 17.5 mM glucose, 121 mM NaCl, 4 mM KCl, 1 mM CaCl2, 25 mM HEPES, 2.4 mM sodium bicarbonate, 40 mg/ml BSA (Bovuminar, L59410, Intergen, Purchase, NY), 5 µg/ml ethanolamine, 0.1 ng/ml sodium selenite, 90 µg/ml penicillin G, 150 µg/ml streptomycin sulfate, 50 µg/ml gentamicin, 55 µM ascorbic acid, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 0.2 U/ml adenosine deaminase. The pH of the buffer was then adjusted to 7.4 and filtered through a 0.2-µm pore size filter. Vehicle or 100 nM CPA was added at the start of incubation. From the fat pads of each rat, one vehicle-treated and one CPA-treated primary culture were established. After 24 h of incubation, medium was removed and frozen at -80 C until leptin levels could be determined. The fat tissue was also frozen and stored at -80 C.

Serum leptin levels
Serum leptin levels were measured by rat leptin RIA (RL-83K Rat Leptin RIA Kit, Linco Research, Inc., St. Charles, MO) (32). This assay has a sensitivity of 0.5 ng/ml, an intraassay coefficient of variability of 2.4% at 1.6 ng/ml and 4.6% at 11.6 ng/ml, and an interassay coefficient of variability of 4.8% at 1.6 ng/ml and 5.7% at 11.6 ng/ml.

Northern slot blotting
Adipose tissue was obtained from epididymal fat pads as previously described (33). Briefly, total RNA was extracted immediately from the fat tissue in Trizol (Life Technologies, Inc., Gaithersburg, MD) in accordance with the manufacturer’s instructions (1 ml/100 mg tissue). Each sample was obtained and prepared from an individual rat or tissue culture plate. Until analyzed, RNA was stored at -80 C. Northern slot blotting was performed as described using a positively charged nylon membrane (Roche Molecular Biochemicals, Mannheim, Germany) and a Schleicher & Schuell, Inc. Minifold I Microsample Filtration Manifold (Keene, NH) (34). Blots were hybridized to 32P-labeled rat leptin or ß-actin cDNA probes that had been validated by Northern blotting of rat epididymal fat pad mRNA as previously described (34). Primer sequences for leptin probe synthesis were obtained from the PrimerSelect (Dnastar) program after analysis of a published sequence for leptin mRNA and consisted of nucleotides 117–138 (5'-GAC ACC AAA ACC CTC ATC AAG A-3') for the forward primer and 508–530 (5'-GCA TTC AGG GCT AAG GTC CAA CT-3') for the reverse primer (35, 36). Published primer sequences were used for ß-actin probe synthesis (37). After washing the blots, individual slots were cut and individually counted using a Wallac, Inc., 1450 Microbeta Tri Lux Liquid Scintillation and Luminescence Counter (Turku, Finland). To adjust for variation in the amount of extracted total RNA from each animal, leptin mRNA expression was normalized to ß-actin mRNA expression. For each animal, the ß-emission signal intensity (counts per min) of the leptin probe hybridized dot was divided by the ß-emission signal intensity (counts per minute) of the corresponding ß-actin probe hybridized dot.

Drugs
CPA, DPY, and CPX were obtained from Research Biochemicals International (Natick, MA). All drug solutions were prepared on the day of the study. CPA and CPX were dissolved in dimethylsulfoxide, and DPY was dissolved in 50% ethyl alcohol. Vehicle preparations contained the same concentrations of dimethylsulfoxide and ethyl alcohol as solutions containing drugs.

Statistical analysis
ANOVA with Bonferroni multiple comparison test analyses and paired t tests were used to compare in vivo and in vitro treatment groups, respectively (PRISM version 2, GraphPad Software, Inc., San Diego, CA). Values are expressed as the mean ± SEM. A significant difference was defined as P < 0.05. T bars on figures represent 1 SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Exp 1: influence of A1AR agonists on serum leptin levels
To determine whether A1AR activation alters circulating leptin levels, serum leptin levels were compared in rats treated with the A1AR-specific agonist CPA or vehicle. Time-course studies (Fig. 1Go) demonstrated peak stimulation of leptin production 8–16 h after treatment with 10 mg/kg CPA, whereas leptin levels in vehicle-treated animals remained unchanged (P < 0.05).



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  		Figure 1. Changes in circulating leptin levels at different times after injection of vehicle or 10 mg/kg CPA. Data for each time point and treatment condition are mean values from two separate studies involving three or more rats per treatment. *, P < 0.05 vs. control.

 
For dose-response studies, serum leptin levels were assessed 8 h after rats were injected with CPA or vehicle. CPA doses shown to influence other physiological systems were used in these studies (38, 39, 40, 41). Treatment of rats with CPA led to increased serum leptin levels in a dose-dependent manner (Fig. 2Go), with highest increases seen after the 10 mg/kg dose (P < 0.01).



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  		Figure 2. Changes in circulating leptin levels 8 h after injection of different doses of CPA. Data for each dose are mean values from two separate studies involving three rats per treatment. *, P < 0.01 vs. control.

 
Exp 2: influence of endogenous adenosine on circulating leptin levels
Next, we tested whether activation of A1ARs influences leptin secretion under basal conditions or when local adenosine levels are increased. To examine the role of basal A1AR activation, we examined responses to treatment with the A1AR antagonist CPX. For CPX treatments, two doses of CPX (10 mg/kg) were given 4 h apart to ensure effective blockade of the A1ARs. To examine responses to increased local adenosine levels, we treated rats with the adenosine reuptake blocker dipyridamole (DPY; 50 mg/kg) using a dose known to increase extracellular adenosine levels (42). To test the specificity of adenosine action, some animals were also treated with CPX and DPY, with the first CPX dose given 30 min before the DPY dose.

Results revealed that treating rats with CPX did not alter leptin levels after injections (P > 0.05, not significant; Fig. 3Go). In contrast, treatment of rats with DPY increased circulating leptin levels by 80% (P < 0.05). Showing that DPY action is A1AR mediated, CPX treatment completely blocked DPY-mediated increases in serum leptin levels (P < 0.05).



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  		Figure 3. Changes in circulating leptin levels at 8 h after injection with vehicle, CPX (two doses of 10 mg/kg given 4 h apart), the adenosine reuptake blocker DPY (50 mg/kg), or DPY and CPX (first dose 30 min before DPY). Data for each treatment condition are the mean values from two separate studies involving at least three rats per treatment. *, P < 0.05 vs. control, CPX, and CPX plus DPY.

 
Exp 3: influence of in vivo A1AR activation on leptin gene expression
In conjunction with the above studies, we also tested whether in vivo A1AR activation increases leptin gene expression in fat tissue. To determine whether changes in circulating leptin levels correlate with changes in mRNA levels, epididymal fat leptin mRNA levels were assessed in the rats treated with CPA (10 mg/kg) or vehicle in the time-course studies. We did not find any differences in leptin gene expression among the treatment groups at any time point (P > 0.05, not significant; n >= 6 for each time point and treatment condition).

Exp 4: influence of in vitro A1AR activation on fat cell leptin secretion and gene expression
Next, to determine whether the effects of CPA on serum leptin levels were due to direct effects on adipocytes, we tested whether A1AR activation altered leptin secretion in isolated fat tissue cultures. For these studies epididymal fat was incubated in 100 nM CPA or vehicle for 24 h, a time we found to be optimal in preliminary studies.

We found that treatment with CPA increased leptin production by 37% from 54.5 ± 13.9 to 74.6 ± 17.6 ng/g fat (P < 0.05; n = 16). In contrast to changes in leptin levels, steady state leptin mRNA levels were not altered by CPA treatment (P > 0.05, not significant; n = 3).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our observations identify the adenosinergic system as a regulator of leptin secretion. We found that A1AR activation leads to increased circulating leptin levels, and leptin secretion rises when endogenous adenosine levels increase. We also found that A1AR activation increases leptin secretion in isolated adipose tissue, showing that A1ARs act directly on fat tissue to stimulate leptin secretion.

The results from in vivo studies revealed that adenosine acts through A1ARs to alter leptin levels and demonstrated that pharmacological regulation of A1AR activation can be used to alter leptin levels. The finding that CPX inhibited DPY-mediated stimulation of leptin release supports the idea that adenosine acts through A1ARs to alter leptin secretion. However, our finding that leptin levels did not fall after CPX treatment suggests that A1ARs do not play a role in maintaining basal leptin secretion.

Because adenosine receptors are present at many sites, it is possible that in vivo results may be influenced by A1AR activation in nonfat tissue. However, our finding that CPA increased leptin secretion in cultured fat tissue demonstrates the direct action of A1ARs on fat cells. Interestingly, the magnitude of CPA-induced increases in leptin secretion in vitro was less than that in the in vivo studies. A1AR activation in other tissues may lead to effects such as altered blood flow, triglyceride, or glucose levels that may influence adipocyte leptin production (43, 44, 45, 46). Thus, it may not be possible to completely simulate in vivo conditions in tissue culture studies.

Because activation of Gs-coupled ß-adrenergic receptors decreases leptin gene expression and leptin secretion (19, 20, 21, 22, 23), we expected that activation of Gi-coupled A1ARs would increase leptin secretion and leptin gene expression. As expected, we found that A1AR activation increased leptin release. However, steady state leptin mRNA levels did not increase. Interestingly, insulin also increases leptin levels, but not steady state leptin mRNA levels (14, 15, 47). The effects of insulin on leptin secretion are believed to be mediated by stimulation of preformed leptin secretion in short term incubations and by increased synthesis of leptin during long term incubations (14, 15). Thus, the effects of A1AR may be mediated at the posttranscriptional level. It important to note, however, that as we only examined steady state mRNA levels, it is possible that transcriptional regulation occurs, but was not detected. Furthermore, our recent studies using transcription inhibitors suggest that A1AR-mediated stimulation of leptin production is transcriptionally regulated (work in progress).

Currently, the effects of long term treatment with A1AR agonists on fat metabolism are unknown. In adipocytes, A1AR activation inhibits lipolysis (26, 48), which is expected to increase body fat and weight. Yet, weight loss is seen in mice treated with adenosine deaminase inhibitors that increase adenosine levels (49). It is therefore possible that A1AR-mediated increases in circulating leptin levels may overcome the antilipolytic effects of adenosine and cause weight loss.

Overall, we now identify adenosine as a regulator of leptin production. Additional studies are indicated to determine whether adenosinergic modulation of leptin levels is of potential utility in the treatment of obesity.


   Acknowledgments
 
The authors thank Aida Groszmann, Andrea Belous, and Lisa Pouncey for technical assistance.


   Footnotes
 
1 This work was supported by a National Research Service Award F32-DK-09895 (to A.M.R.) and NIH Grant RO1-HL-58442 (to S.A.R.). Back

Received September 2, 1999.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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