Adenosine A1 receptors regulate lipolysis and lipogenesis in mouse adipose tissue — Interactions with insulin

European Journal of Pharmacology 597 (2008) 92–101

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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r

Endocrine Pharmacology

Adenosine A1 receptors regulate lipolysis and lipogenesis in mouse adipose tissue — Interactions with insulin Stina M. Johansson a,⁎, Eva Lindgren a, Jiang-Ning Yang a, Andreas W. Herling b, Bertil B. Fredholm a a b

Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden Aventis Deutschland, Frankfurt am Main, Germany

a r t i c l e

i n f o

Article history: Received 18 December 2007 Received in revised form 10 August 2008 Accepted 21 August 2008 Available online 31 August 2008 Keywords: Adenosine A1 receptor Knock out mouse Adipocyte 2-chloroadenosine Free fatty acids

a b s t r a c t Adenosine acting at adenosine A1 receptors is considered to be one major regulator of adipose tissue physiology. We have examined the role of adenosine and its interactions with insulin in adipose tissue by using A1R knock out (−/−) mice. Removal of endogenous adenosine with adenosine deaminase caused lipolysis in A1R (+/+), but not A1R (−/−) adipocytes. The adenosine analogue, 2-chloroadenosine, inhibited noradrenalinestimulated lipolysis and cAMP accumulation in A1R (+/+), but not in A1R (−/−) adipocytes. Insulin reduces lipolysis and cAMP via another mechanism than adenosine and acted additively, but not synergistically, with adenosine. Plasma levels of free fatty acids, glycerol and triglycerides were significantly lower in A1R (+/+) than in A1R (−/−) mice after administration of an adenosine analogue. 2-chloroadenosine induced lipogenesis in presence of insulin in A1R (+/+), but not in A1R (−/−) adipocytes. There were no changes in mRNA levels for several genes involved in fat synthesis in adipose tissue between genotypes. Body weight was similar in young A1R (+/+) and A1R (−/−) mice, but old male A1R (−/−) mice were heavier than wild type controls. In conclusion, adenosine inhibits lipolysis via the adenosine A1 receptor and other adenosine receptors play no significant role. Adenosine and insulin mediate additive but not synergistic antilipolytic effects and 2-chloroadenosine stimulates lipogenesis via adenosine A1 receptors. Thus deletion of adenosine A1 receptors should increase lipolysis and decrease lipogenesis, but in fact an increased fat mass was observed, indicating that other actions of adenosine A1 receptors, possibly outside adipose tissue, are also important. © 2008 Elsevier B.V. All rights reserved.

1. Introduction There is excellent evidence that adenosine can regulate several aspects of adipose tissue function including lipolysis (Fredholm, 1978; Schwabe et al., 1974), blood flow and neurotransmitter release (Hedqvist and Fredholm, 1976; Sollevi et al., 1981). Adenosine is released from adipose tissue e.g. during sympathetic nerve activation (Fredholm, 1976; Fredholm and Sollevi,1981) and acts on four G protein-coupled receptors: the adenosine A1 receptor, the adenosine A2A receptor, the adenosine A2B receptor and the adenosine A3 receptor (Fredholm et al., 2001a,b). There is pharmacological evidence that the receptor responsible for the antilipolytic effect is the adenosine A1 receptor (Fatholahi et al., 2006; Schoelch et al., 2004; Xu et al., 1998) and already basal levels of endogenous adenosine are sufficent to cause inhibition of lipolysis (Lönnroth et al., 1989). Adenosine is considered to be a major endogenous antilipolytic factor and it was therefore surprising that elimination of the adenosine A1 receptor did not cause any compensatory changes of other antilipolytic agents acting on G protein-coupled receptors (Johansson et al., 2007b). ⁎ Corresponding author. Department of Physiology and Pharmacology, Karolinska Institutet, S-171 77 Stockholm, Sweden. Tel.: +46 8 524 8 7937; fax: +46 8 34 12 80. E-mail address: [email protected] (S.M. Johansson). 0014-2999/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2008.08.022

The possibility remains that the antilipolytic capacity of other agents, notably insulin, could rely on adenosine A1 receptor signaling, even though the mechanisms of antilipolysis are quite different (Carmen and Víctor, 2006). Indeed, it has been reported that long-term activation of adenosine A1 receptors leads to a desensitization of both adenosine and insulin effects (Green, 1987). There is also evidence that at least some aspects of insulin signaling are enhanced by activation of adenosine A1 receptors (Cheng et al., 2000; Takasuga et al., 1999). The possibility that adenosine and insulin interact with each other in adipose tissue has potential clinical relevance in view of the growing concern for type 2 diabetes secondary to obesity (Bray, 2004; Wild et al., 2004). The data in the literature are, however, not completely coherent. It has, on the one hand, been reported that both adenosine A1 receptor overexpression (Dong et al., 2001) and an adenosine A1 receptor agonist improve insulin sensitivity (Schoelch et al., 2004). In line with this is the finding that removal of adenosine or addition of an adenosine receptor antagonist decreases insulin-stimulated glucose transport (Steinfelder and PethöSchramm, 1990). On the other hand, it has also been reported that an adenosine receptor antagonist having A1 selectivity would improve glucose tolerance (Xu et al., 1998). There is also considerable evidence that long-term coffee consumption is associated with a decreased risk for type 2 diabetes, possibly via antagonistic actions of caffeine on adenosine A1 receptors (van Dam and Feskens, 2002; van Dam and Hu, 2005).

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We recently showed by using A1R (−/−) mice (Johansson et al., 2001) that insulin and glucagon levels in plasma were significantly higher after glucose administration in A1R (−/−) mice than in A1R (+/+) mice (Johansson et al., 2007a), but we found no major differences in glucose tolerance or glucose uptake in isolated skeletal muscle between the genotypes. Given the markedly altered insulin secretion in these mice (Johansson et al., 2007a) we thought that there might be alterations in insulin sensitivity in another major insulin target. In the present study, we have therefore further studied these A1R (−/−) mice and in particular we tested the hypothesis that adenosine via the adenosine A1 receptor in the adipose tissue interacts with insulin, and that deletion of the A1 receptor would lead to adaptive changes in the insulin responses. 2. Material and methods 2.1. Materials Bovine serum albumin fraction V (BSA), L-noradrenaline hydrochloride, 2-chloroadenosine, adenosine 5′-triphosphate (ATP), β-nicotinamide adenine nucleotide (NAD), insulin, adenosine 3′,5′-cyclic monophosphate (cAMP), prostaglandin E2, 2,5-diphenyloxazole, 1,4 bis (4-methyl-5phenyl-2-oxazoyl)benzene, 3-isobutyl-1-methylxanthine (IBMX), N6cyclopentyladenosine (CPA) and tylose were from Sigma (St Louis, MO). 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) was from Research Biochemicals International (Natick, MA). Collagenase (type 1, CLS) was from Worthington (Lakewood, NJ). Glycerol, toluene and heptane were from VWR International Ltd. (Dorset, UK). Ready Safe scintillation liquid was from Beckman (Fullerton, CA). Hydrazinium hydroxide and L-cysteine were from Merck (Darmstadt, Germany). Adenosine deaminase, glycerol kinase, glycerol-3-phosphate dehydrogenase, triglyceride GPD-PAP kit and cholesterol CHOD-PAP kit were from Roche Diagnostics GmbH (Mannheim, Germany). Amrinone was a kind gift from Dr. T Kenakin. [2, 8-3H]-adenosine 3′,5′-cyclic phosphate was from PerkinElmer (Boston, MA). D-[3-3H]-glucose was from GE Healthcare (Buckinghamshire, UK). RNeasy Lipid Tissue kit and QIAquick PCR Purification kit were from Qiagen GmbH (Hilden, Germany). CyScribe First-Strand cDNA Labeling kit was from Amersham Pharmacia Biotech (Piscataway, NJ). High Capacity cDNA Archive kit, TaqMan Gene Expression Assays for real-time RT-PCR, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and TaqMan Universal PCR Master Mix No AmpErase UNG were from Applied Biosystems (Foster City, CA). NEFAC kit was from WAKO Chemicals GmbH (Neuss, Germany). Glycerol kit was from Randox (Krefeld, Germany). 2.2. Animals Mice lacking the adenosine A1 receptor (A1R (−/−)), heterozygote (A1R (+/−)) and wild type (A1R (+/+)) mice were used in this study. The mice were generated as previously described (Johansson et al., 2001). These mice, with C57BL/6 background, had been back-crossed to be congenic (by 140 genomic markers) by Jackson Laboratory (Bar Harbor, ME). The mice were bred and housed in the animal facility in a room with controlled temperature (22–23 °C). Littermates were genotyped using PCR (Turner et al., 2003). The animals were kept on a 12:12 h light:dark cycle and standard pellet food and water were provided ad libitum. The mice were 3–10 months old in the experiments. The exact age of the mice is stated in each described experiment, see below. Before sacrifice by decapitation the mice were anesthetized with carbon dioxide. The experiment procedures were approved by the Regional Animal Ethics Committees in Stockholm or Frankfurt am Main. 2.3. Isolation of adipocytes Mouse adipocytes were isolated as described elsewhere (Johansson et al., 2007b) using modifications of established protocols for rat adipocytes (Fredholm, 1985; Rodbell, 1964). Briefly, epididymal fat

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pads were isolated from male mice (older than 5 months unless otherwise is stated in the Results section) and treated with collagenase (CLS type 1, 1 mg/ml) for 45–60 min in a Krebs' Ringer bicarbonate buffer containing 25 mM HEPES, 5 mM glucose and 3% BSA (lipolysis/ cAMP accumulation) or in a Krebs' Ringer phosphate buffer containing 4% BSA (lipogenesis), pH 7.4. After the collagenase treatment, the adipocytes were filtered through a nylon mesh, washed, counted and diluted. The isolated adipocytes were used immediately for lipolysis, cAMP accumulation or lipogenesis experiments. 2.4. Lipolysis experiments Lipolysis experiments were performed as previously described (Johansson et al., 2007b) and glycerol release was measured as an index of lipolysis (Laurell and Tibbling, 1966). In brief, aliquots of the cell suspension (approximately 150,000 adipocytes/ml) were placed into separate tubes (1.9 ml to each tube) and incubated with or without adenosine deaminase (0.1 U/ml) for 15 min in a shaking water bath at 37 °C. Insulin (10–1000 µU/ml) and/or 2-chloroadenosine (0.003–3 µM) were added to the adipocytes and after 5-min incubation, noradrenaline (1–300 nM) was added. Incubation was stopped after another 60 min and the glycerol release was measured. 2.5. cAMP measurements The adipocytes were aliquoted into separate tubes (0.9 ml in each tube) and incubated in the presence or absence of adenosine deaminase (0.1 U/ml) in a shaking water bath at 37 °C for 15 min. Different concentrations of insulin (0.01–10,000 µU/ml) and/or 2chloroadenosine (0.00001–1 µM) were added to the adipocytes. Noradrenaline (3 µM) was added 5 min later. The reactions were terminated after 20 min of noradrenaline-stimulation by transferring 450 μl of the adipocyte suspension into tubes containing 50 μl 4 M perchloric acid on ice (Fredholm, 1985). The samples were neutralized with 4 M KOH/1 M Tris–HCl and after centrifugation (1500 g for 15 min at 4 °C), the cAMP content in the supernatant was determined by using the [3H]-cAMP protein binding assay (Nordstedt and Fredholm, 1990). 2.6. Lipogenesis experiments Lipogenesis was measured as previously described (Arner and Engfeldt, 1987) as incorporation of D-[3-3H]-glucose in toluene-extractable lipids. Isolated adipocytes were incubated in the presence or absence of adenosine deaminase (0.1 U/ml) in a Krebs' Ringer phosphate buffer containing 2% BSA for 20 min at 37 °C in a shaking water bath and then aliquoted into separate tubes containing D-[3-3H]-glucose (5 × 106 cpm/ml), unlabelled glucose (1 μM), different concentrations of insulin (1–10,000 µU/ml) and/or 2-chloroadenosine (0.00001–100 µM). The total volume in each tube was 0.5 ml. The adipocytes were incubated for 2 h at 37 °C in the shaking water bath with air as the gas phase. The incubation was stopped by rapidly chilling of the incubation vials on ice and adding 50 μl 6 M H2SO4. Thereafter 4 ml of toluene-based scintillation liquid containing 2,5-diphenyloxazole (PPO) and 1,4 bis (4-methyl5phenyl-2-oxazoyl) benzene (POPOP) was added. The vials were left overnight at room temperature and then radioactivity was measured by liquid scintillation counting (Moody et al., 1974). The toluene-based scintillation liquid is hydrophobic and therefore only the D-[3-3H]-glucose that is incorporated in the fat cells will be determined. The Ready Safe scintillation liquid is less hydrophobic and was used for counting the total amount of D-[3-3H]-glucose in the samples. Background radioactivity was measured in adipocyte suspension incubated with 6 M H2SO4 for 2 h. To correct for variations between samples, the lipogenesis results were normalized to the total lipid content in the adipocyte suspension. Organic extraction was used for measuring the lipid content in the adipocyte suspension. The lipids (0.3 ml) were extracted by using

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an extraction mixture (5 ml) consisting of isopropanol/heptane/H2SO4 (1 M) (40:10:1) mixed with heptane (3 ml) and water (3 ml). The lipids were washed twice with heptane during the extraction process. The upper organic phase from the extraction was saved for determining the lipid content. 2.7. In vivo experiments The mice (males and females, 5–7 months) were starved overnight with free access to water. Groups of n = 8 mice were used for the experiment. CPA was given to the mice orally in a dose of 1 mg/kg suspended in 1% tylose; the control mice received the vehicle only. Blood samples were taken 2 h after dosing by retrobulbar bleeding during short-term isoflurane anesthesia. Blood metabolic parameters were determined enzymatically on a Hitachi 912 using commercially available kits for free fatty acids (NEFAC kit), glycerol, triglycerides (GPD-PAP) and cholesterol (CHOD-PAP). 2.8. RNA and cDNA preparation Epididymal adipose tissue from male and female mice (3–7 months) was dissected out and rapidly frozen in liquid nitrogen. RNeasy Lipid tissue kit (Qiagen) was used for RNA isolation according to the manufacturer's instructions. The quality of the RNA was controlled with an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) before microarray analysis. For the microarray analysis, cDNA was synthesized and labeled as previously described by using CyScribe First-Strand cDNA Labeling kit (Amersham Pharmacia Biotech) and QIAquick PCR Purification kit (Qiagen) and then the cDNA was spotted on mouse 15K EST microarrays (Johansson et al., 2007b). For the realtime reverse transcription polymerase chain reaction (RT-PCR) analysis, cDNA synthesis was carried out with the High Capacity cDNA Archive kit (Applied Biosytem) with random primers and multiscribe reverse transcriptase enzyme according to the manufacturer's instructions. 2.9. Real-time reverse transcription polymerase chain reaction (RT-PCR) Gene expression levels were detected by using TaqMan Gene Expression Assays (Applied Biosystems), where each assay consists of two sequence-specific PCR primers and a TaqMan Assay FAM DyeLabeled MGB probe. The TaqMan Gene Expression Assay IDs were: Mm00514571_m1 for 7-dehydrocholesterol reductase (Dhcr7), Mm00662319_m1 for Fatty acid synthase (Fasn), Mm00516030_m1 for Isocitrate dehydrogenase 1 (NADP+) soluble (Idh1), Mm0049 9674_m1 for Isocitrate dehydrogenase 3 (NAD+) alpha (Idh3a), Mm007823 80_s1 for Malic enzyme supernatant (Mod 1), Mm00485951_g1 for Stearoyl-Coenzyme A desaturase 2 (Scd2) and Mm013523 66_m1 for Succinate dehydrogenase complex, subunit A, flavoprotein (Fp) (Sdha). Real-time RT-PCR reactions were run as previously described (Johansson et al., 2007b). In brief, TaqMan Gene Expression Assay (containing primers and probe), cDNA and TaqMan Universal PCR No AmpErase UNG Master Mix were added to each well in the plate and run in an ABI Prism 7500 Sequence Detector System (Applied Biosystems, Foster City, CA). GAPDH was used as endogenous control. The data were expressed as change in gene expression at RNA level in adipose tissue in A1R (−/−) mice relative to A1R (+/+) mice or as Δct values (ct = cycle at threshold).

Network (UHN) of Toronto, Canada (http://www.microarrays.ca) were used as a starting point for studies of gene expression (Johansson et al., 2007b) and analyzed with Axon GenePix Pro 4.0 software (Molecular Devices Corporation, Union City, CA) and Gene Traffic Duo 2.5 microarray analysis/database package (Stratgene, La Jolla, CA). Statistical analyses were performed in GraphPad Prism 5 (GraphPad, San Diego, CA). Values are presented as means ± S.E.M. unless otherwise stated. 3. Results 3.1. Body weight is increased in old A1R (−/−) mice The body weight was measured in the A1R (−/−) mice since it has been reported that adenosine A1 receptors are deficient in genetically obese rodents (LaNoue and Martin, 1994). The results show that the body weight was not significantly different in young male A1R (−/−) versus A1R (+/+) mice, but that older male A1R (−/−) mice (≥5 months) had a significantly increased body weight, 7–8.5% higher than their A1R (+/+) mice counterparts (Fig. 1). 3.2. Adenosine mediates its antilipolytic effects via the adenosine A1 receptor in the mouse The lipolytic response of mouse adipocytes was examined by measuring the glycerol accumulation in the medium. Noradrenaline caused a concentration-dependent increase in lipolysis over the range 1–300 nM (data not shown), in agreement with earlier results in mice (Johansson et al., 2007b; Liu et al., 2003). As expected, the efficacy of antilipolytic agents, including insulin (Fig. 2A) and 2-chloroadenosine (Fig. 2B) was greatest at submaximal levels of catecholamine stimulation, and since the focus of this study was on antilipolysis, in subsequent studies we used 3 or 10 nM noradrenaline to stimulate lipolysis. In agreement with previous data from adipocytes of rat (Hjelmdahl et al., 1976; Schwabe et al., 1974) and mouse (Johansson et al., 2007b), we found that the basal and noradrenaline-stimulated rate of lipolysis in adipocytes from A1R (+/+) mice was increased by adding adenosine deaminase (Table 1; data from Fig. 1B in Johansson et al., 2007b), which removes endogenous adenosine by converting it to inosine. DPCPX also increased 3 nM noradrenaline-stimulated lipolysis approximately 3-fold. Since DPCPX at a concentration of 30 nM is a selective adenosine A1 receptor antagonist, this suggested a role of endogenous adenosine acting at the adenosine A1 receptor. Adenosine deaminase was also included in most of the subsequent experiments in order to minimize variation in the levels of endogenous adenosine. We then stimulated the adenosine receptors on the adipocytes by adding 2-chloroadenosine. This compound is resistant

2.10. Data analysis Statistical analyses were performed with t-test or ANOVA followed by Turkey's post hoc test or Bonferroni post test where appropriate. Sigmoidal dose–response curves were calculated by using non-linear regression. Data obtained from the mouse 15K EST microarrays (spotted cDNA, duplicate spots of each gene), University Health Care

Fig. 1. The body weight was significantly increased in male A1R (−/−) mice ≥ 5 months, n = 12–45 mice of each genotype. ⁎P b 0.05, ⁎⁎P b 0.01 between groups. Each value represents mean ± S.E.M.

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Fig. 2. Dose-dependent inhibition of lipolysis by insulin (A) and 2-chloroadenosine (B). Panel A, lipolysis was stimulated in adipocytes from A1R (+/+) mice by addition of 3 or 10 nM noradrenaline (NA) together with adenosine deaminase (ADA; 0.1 U/ml). The IC50 value of insulin was 73 µU/ml (95% confidence interval; 10–548 μU/ml) when 10 nM noradrenaline was added and 72 µU/ml (47–111 μU/ml) when 3 nM noradrenaline was added. Each value represents mean ± S.E.M. of three to four separate experiments using adipocytes from four mice for each experiment. 100% corresponds to a value between 123.8 and 1478.3 μmol/h/105 cells glycerol and 0% corresponds to a value between 39.5 and 761.4 µmol/h/105 cells glycerol. Panel B, lipolysis was stimulated with 3 or 30 nM noradrenaline. In adipocytes from A1R (+/+) mice, the IC50 value of 2-chloroadenosine was 11 nM when lipolysis was stimulated with 3 nM noradrenaline and 52 nM when 30 nM noradrenaline was used. 2-chloroadenosine had no effect on lipolysis on adipocytes from A1R (−/−) mice. The graph shows one representative experiment and 100% corresponds to a value of 44.8 µmol/h/105 cells glycerol and 0% corresponds to a value of 9.70 µmol/h/105 cells glycerol.

to metabolism by adenosine deaminase and has a similar receptor binding profile as the endogenous compound (Fredholm et al., 2001a, b) and we found that that 2-chloroadenosine had no effect on lipolysis in A1R (−/−) adipocytes (Fig. 2B). Systemic administration of the A1 receptor-selective adenosine analogue CPA at 1 mg/kg to A1R (+/+) mice caused a clear-cut reduction of the plasma levels of free fatty acids (Fig. 3A), glycerol (Fig. 3B) and triglycerides (Fig. 3C), whereas cholesterol levels (Fig. 3D) were unchanged. Similar results were obtained in A1R (+/−) mice. In contrast, even under control conditions (without CPA) the A1R (−/−) mice showed a tendency toward elevated free fatty acid levels, indicating a tonic suppressive effect of adenosine on in vivo lipolysis. In addition, and in line with the notion of a functional role for adenosine A1 receptors, the antilipolytic effect of CPA was significantly attenuated in A1R (−/−) mice.

Fig. 3. Plasma levels of free fatty acids (A), glycerol (B), triglycerides (C) and cholesterol (D) in control or CPA (1 mg/kg) treated A1R (+/+), (+/−) and (−/−) mice. The results are given in mean ± S.E.M. ⁎P b 0.05, ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001; each versus corresponding A1R (+/+) animal, n = 8 mice of each genotype.

Table 1 Adenosine is an antilipolytic factor in adipocytes from A1R (+/+) mice Treatment

Glycerol (µmol/h/105 cells)

Basal 39 ± 5 Adenosine deaminase (0.1 U/ml) 285 ± 46 Noradrenaline (10 nM) 374 ± 75 Noradrenaline (10 nM) + Adenosine deaminase (0.1 U/ml) 719 ± 81 Each value represents mean ± S.E.M. of 5–12 separate experiments.

3.3. Spare adenosine A1 receptors exist The effects on cAMP accumulation of adenosine in the adipocytes were also studied. In these experiments much higher levels of noradrenaline had to be used, in agreement with previous results (Hjemdahl and Fredholm, 1976).

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3.5. Lipogenesis can be increased by an adenosine A1 receptor ligand The role of the adenosine A1 receptor and its interaction with insulin in lipogenesis was also studied. Lipogenesis was measured as an incorporation of D-[3-3H]-glucose into lipids. There was no significant difference in basal glucose incorporation between non-treated adipocytes from A1R (+/+) and (−/−) mice (basal glucose incorporation was 53 ± 7 pmol/g lipid/2 h in A1R (+/+) adipocytes and 47 ± 3 pmol/g lipid/2 h in A1R (−/−) adipocytes). This suggests that endogenous adenosine acting on the adenosine A1 receptor has a minimal effect on basal glucose incorporation into lipids. Indeed, adenosine deaminase (0.1 U/ml) caused

Fig. 4. cAMP accumulation was stimulated with noradrenaline (3 μM) and adenosine deaminase (0.1 U/ml) and inhibited with increasing concentrations of 2-chloroadenosine in adipocytes from A1R (+/+), (+/−) and (−/−) mice. The IC50 value of 2chloroadenosine in adipocytes from A1R (+/+) mice was 1.2 nM (95% confidence interval; 0.72–2.0 nM) and the IC50 value in adipocytes from the A1R (+/−) was 3.1 nM (2.1–4.5 nM). 2-chloroadenosine had no effect in adipocytes from A1R (−/−) mice. Each value represents mean ± S.E.M. of two to three separate experiments using adipocytes from four mice for each experiment.

The EC50 value of noradrenaline was 0.77 μM. To evoke a sufficiently robust response, cAMP accumulation was stimulated with 3 μM noradrenaline in these experiments. 2-chloroadenosine caused a concentration-dependent inhibition of cAMP accumulation in A1R (+/+) adipocytes (Fig. 4). No effect at all was observed in A1R (−/−) adipocytes. Interestingly, the dose–response curve was shifted to the right in A1R (+/−) adipocytes and these results indicate that spare adenosine A1 receptors exist (see also Discussion). 3.4. Adenosine and insulin mediate additive effects Insulin was antilipolytic (Fig. 5A) in agreement with the large literature base. The antilipolytic effect of insulin (1000 μU/ml) was essentially eliminated in the presence of the phosphodiesterase inhibitors, amrinone and IBMX (Table 2). In contrast, the ability of 2chloroadenosine (1 μM) or prostaglandin E2 (1 μM) to decrease noradrenaline- (3 nM) and adenosine deaminase- (0.1 U/ml) stimulated lipolysis was much less affected by the phosphodiesterase inhibitors (Table 2). Obviously IBMX could not be used together with 2-chloroadenosine as it is not only a phosphodiesterase inhibitor but also an adenosine receptor antagonist (Fredholm et al., 2001a,b). Next, we examined if there were interactions between adenosine and insulin. Addition of 2-chloroadenosine did not influence the potency (IC50 value) of insulin (Fig. 5A), nor did the addition of DPCPX (Fig. 5B), even through the absolute level of lipolysis varied. Similarly, there was no difference in the potency of insulin in A1R (+/+) and (−/−) adipocytes (Fig. 5C). The lipolysis data presented above suggest that both adenosine, acting on adenosine A1 receptors, and insulin regulate noradrenalinestimulated lipolysis, but provided no evidence for a direct interaction between the two agents. We wanted to confirm this conclusion by examining cAMP accumulation instead. Insulin inhibited cAMP accumulation with similar potency in the absence or presence of 2chloroadenosine, since there was no significant difference in the IC50 value of insulin with or without 2-chloroadenosine treatment of the adipocytes from A1R (+/+) mice (Fig. 6A). Conversely, the potency (IC50 value) of 2-chloroadenosine was essentially identical in the absence and presence of insulin (Fig. 6B). However, the maximal inhibition of noradrenaline- and adenosine deaminase-stimulated cAMP response increased when both insulin and 2-chloroadenosine were added together (Fig. 6A) even though the potency of insulin (Fig. 6A) and 2chloroadenosine (Fig. 6B) were not altered.

Fig. 5. Insulin inhibits lipolysis in a dose-dependent manner in the absence or presence of 2-chloroadenosine, DPCPX or the adenosine A1 receptor. Panel A, lipolysis was stimulated with noradrenaline (10 nM) and adenosine deaminase (0.1 U/ml). The IC50 value of insulin was 80 μU/ml (95% confidence interval; 34–191 μU/ml) and 71 µU/ml (9–553 μU/ml) when 2-chloroadenosine (3 nM) was added to the A1R (+/+) adipocytes. Panel B, lipolysis was stimulated with noradrenaline (10 nM). The IC50 value of insulin was 139 μU/ml (95% confidence interval; 23–839 μU/ml) and 52 μU/ml (23–117 μU/ml) when DPCPX (30 nM) was added to the A1R (+/+) adipocytes. Panel C, the IC50 value of insulin was 72 μU/ml (95% confidence interval; 47–111 μU/ml) in adipocytes from A1R (+/+) mice and 77 µU/ml (32–181 μU/ml) in adipocytes from A1R (−/−) mice when lipolysis was stimulated with noradrenaline (NA; 3 nM) and adenosine deaminase (ADA; 0.1 U/ml). The IC50 value was 139 μU/ml (23–839 μU/ml) (10 nM) in A1R (+/+) adipocytes and 166 μU/ml (31–906 µU/ml) in A1R (−/−) adipocytes when lipolysis was stimulated with noradrenaline. Each value represents mean ± S.E.M. of two to five separate experiments using adipocytes from four mice for each experiment.

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Table 2 Effects of insulin, 2-chloroadenosine and prostaglandin E2 on lipolysis stimulated with noradrenaline (3 nM) and adenosine deaminase (0.1 U/ml) in the absence or presence of phosphodiesterase inhibitors (amrinone and IBMX) in adipocytes from A1R (+/+) mice Treatment

Antilipolytic effect (%)

Insulin (1000 µU/ml) Insulin (1000 µU/ml) + Amrinone (1 mM) Insulin (1000 µU/ml) + IBMX (10 µM) 2-chloroadenosine (1 µM) 2-chloroadenosine (1 µM) + Amrinone (1 mM) Prostaglandin E2 (1 µM) Prostaglandin E2 (1 µM) + IBMX (10 µM)

37 ± 4 4±2 9±3 73 ± 4 55 ± 5 69 ± 5 62 ± 2

Each value represents mean ± S.E.M. of three separate experiments with duplicate determinations in each experiment.

only a non-significant trend towards lower incorporation (glucose incorporation was 38 ± 2 pmol/g lipid/2 h when adenosine deaminase (0.1 U/ml) was added to A1R (+/+) adipocytes). Adenosine deaminase had no effect in A1R (−/−) adipocytes (glucose incorporation was 51 ±4 pmol/g lipid/2 h). The ability of insulin to stimulate lipogenesis was also not significantly affected by adenosine deaminase in either A1R (+/+) or A1R (−/−) adipocytes (Fig. 7). Addition of 2-chloroadenosine slightly enhanced the insulin effect in A1R (+/+) adipocytes but not in the A1R

Fig. 7. Dose–response curves of insulin on D-[3-3H]-glucose incorporation in A1R (+/+) adipocytes (A) and A1R (−/−) adipocytes (B) in the absence or presence of adenosine deaminase (ADA; 0.1 U/ml) and/or 2-chloroadenosine (10 μM). The IC50 values are given in the text. Each value represents mean ± S.E.M. of two to five separate experiments using adipocytes from two mice for each experiment.

Fig. 6. Dose-dependent inhibition of noradrenaline- and adenosine deaminase-induced cAMP accumulation by insulin (A) and 2-chloroadenosine (B). Panel A, dose–response curves of insulin in the absence or presence of 2-chloroadenosine. The IC50 value of insulin was 0.54 µU/ml (95% confidence interval; 0.1–2.1 µU/ml) and 0.50 µU/ml (0.1–3.0 μU/ml) when 2-chloroadenosine (0.3 nM) was added to adipocytes stimulated with noradrenaline (3 μM) and adenosine deaminase (0.1 U/ml). Each value represents mean ± S.E.M. of three separate experiments using adipocytes from four mice for each experiment. Panel B, dose–response curves of 2-chloroadenosine in the absence or presence of insulin in adipocytes from A1R (+/−) mice (similar results were obtained from A1R (+/+) adipocytes; data not shown). The IC50 of 2-chloroadenosine was 3.1 nM (95% confidence interval; 2.0–4.9 nM), 2.7 nM⁎ (1.5–4.7 nM) in the presence of 100 μU/ml insulin and 7.0 nM⁎ (3.1– 15.7 nM) in the presence of 10,000 μU/ml insulin in adipocytes stimulated with noradrenaline (3 μM) and adenosine deaminase (0.1 U/ml). Each value represents mean ± S.E.M. of two to four separate experiments using adipocytes from four mice for each experiment. The mice were older than 5 months in all experiments except in one where the age of the mice was 3.5 months. However, the age of the mice did not affect the results when comparing the experiments separately.

(−/−) adipocytes (Fig. 7). The EC50 values of insulin were 672 μU/ml (95% confidence interval; 500–902 μU/ml) in control A1R (+/+) adipocytes, 732 μU/ml (578–926 μU/ml) in adenosine deaminase-treated A1R (+/+) adipocytes and 286 μU/ml (231–355 μU/ml) when 2-chloroadenosine (10 μM) was added to adenosine deaminase-treated A1R (+/+) adipocytes. The EC50 values were 655 μU/ml (95% confidence interval; 482–892 μU/ ml) in control A1R (−/−) adipocytes, 804 μU/ml (615–1050 μU/ml) in adenosine deaminase-treated A1R (−/−) adipocytes and 679 μU/ml (484– 952 μU/ml) when 2-chloroadenosine was added to the adenosine deaminase-treated A1R (−/−) adipocytes. The apparent shift in the dose– response curve for insulin could mean that adenosine A1 receptor stimulation potentiates the effect of insulin. However, it could also mean that activation of the adenosine A1 receptor does lead to stimulation of lipogenesis, but only when insulin is present. As shown in Fig. 8, lipogenesis was only stimulated at an excessively high concentration of 2-chloroadenosine (100 μM) in the absence of insulin in A1R (+/+) adipocytes. When 100 μU/ml insulin was added to the A1R (+/+) adipocytes, 2-chloroadenosine increased lipogenesis already at 1 μM and when 1000 μU/ml insulin was added, 2-chloroadenosine had an effect on lipogenesis at 0.01 μM (Fig. 8). Therefore, our conclusion is that insulin needs to be present if adenosine via the adenosine A1 receptor should affect lipogenesis. In addition, Fig. 8 also shows that the effects of 2-chloroadenosine at 0, 100 and 1000 μU/ml insulin was abolished in the A1R (−/−) adipocytes, which suggests that the adenosine A1 receptor is the only adenosine receptor that is of importance in lipogenesis. 3.6. Gene expression is not altered in adipose tissue from A1R (−/−) mice Lipogenesis (in the absence of 2-chloroadenosine and in the presence of 1000 μU/ml insulin) was, as shown in Fig. 8, higher in adipocytes from A1R (−/−) mice than from A1R (+/+) mice. This could be a compensatory effect due to the lack of adenosine A1 receptors. In

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4. Discussion

Fig. 8. Dose–response curves of 2-chloroadenosine on D-[3-3H]-glucose incorporation in A1R (+/+) and A1R (−/−) adipocytes in the absence or presence of insulin (100 or 1000 µU/ml). ⁎P b 0.05, ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001; each versus corresponding non-treated adipocytes. ##P b 0.01; control A1R (+/+) versus control A1R (−/−) adipocytes when adenosine deaminase (0.1 U/ml) and insulin (1000 μU/ml) were added. Each value represents mean ± S.E.M. of two to five separate experiments using adipocytes from two mice for each experiment.

order to further explore compensatory changes in the adipose tissue from the A1R (−/−) mice at a genetic level, the gene expression of metabolically important genes in adipose tissue was investigated by using the cDNA microarray gene chip technique. We found more than 1.5-fold up-regulation of mRNA for 287 genes in adipose tissue from the A1R (−/−) mice compared with A1R (+/+) mice, and mRNA for 56 genes was down-regulated to less than half (significance P b 0.001 in both cases; data not shown). Genes with known functions in aspects of lipid synthesis that showed altered expression on the microarray chips in the adipose tissue from the A1R (−/−) (see Table 3) were selected for real-time RT-PCR verification. However, concordance results from the real-time RT-PCR analysis did not verify the microarray data (Table 3) and we conclude that no real expression differences in these genes in adipose tissue between A1R (−/−) and A1R (+/+) mice exist.

The present study gives additional strong evidence that adenosine mediates its antilipolytic effects via the adenosine A1 receptor. The fact that adenosine is a potent inhibitor of lipolysis has been shown in different species such as rat (Fredholm, 1978; Hjemdahl and Fredholm, 1976; Schoelch et al., 2004; Schwabe et al., 1974), dog (Fredholm and Sollevi, 1977; Sollevi and Fredholm, 1981), hamster (Castan et al., 1994) and human (Heseltine et al., 1995; Ohisalo, 1981). Although the level of receptor expression may vary (Kaartinen et al., 1991; Larrouy et al., 1991; Lohse et al., 1987), the antilipolytic effect of adenosine thus seems to be relative constant feature in mammalian adipose tissue (Castan et al., 1994). Previous studies used pharmacological tools, where selectivity is always a concern. Therefore, and because the antilipolytic effect of adenosine in the mouse is not well studied, the present data obtained using adenosine A1 receptor knock out mice not only confirm but also extend previous knowledge. The present findings that genetic ablation of the adenosine A1 receptor mimics the effects of adenosine deaminase and DPCPX in increasing basal lipolysis and eliminating the antilipolytic effect of a non-selective adenosine receptor agonist provide strong evidence that other adenosine receptors play a minor role in fat cells. The observation that the non-selective agonist 2-chloroadenosine was not able to affect cAMP accumulation in adipocytes from A1R (−/−) mice also further support this conclusion. In addition, treatment with an adenosine A1 receptor agonist decreased plasma levels of free fatty acids and glycerol in A1R (+/+) mice, which also suggest that adenosine A1 receptors are important in vivo in regulating lipolysis. We recently showed that there is a substantial adenosine A1 receptor reserve in adipose tissue (Johansson et al., 2007b), since the dose–response curve of 2-chloroadenosine on noradrenaline- and adenosine deaminase-stimulated lipolysis was shifted two-fold to the right in adipocytes from A1R (+/−) mice compared with adipocytes from A1R (+/+) mice. Here we show, examining cAMP accumulation, approximately a doubling of the IC50 value with 2-chloroadenosine in adipocytes from A1R (+/−) mice compared with A1R (+/+) adipocytes. This right shift in the curve suggests an adenosine A1 receptor reserve in adipose tissue. Previous studies in rats have also provided evidence for a substantial receptor reserve and a homogenous receptor population (Fatholahi et al., 2006; Liang et al., 2002; Lohse et al., 1986). A large receptor reserve implies so called spare receptors where a full agonist can cause a maximum response even when only a fraction of the receptors are occupied, and a parallel shift in a dose– response curve without decreased maximum response of a drug is an indication of spare receptors (Neubig et al., 2003). The high number of adenosine A1 receptors in fat cells explains why partial agonists can be quite selective (Fatholahi et al., 2006; Schoelch et al., 2004) and why the endogenous antilipolysis is physiologically more relevant than

Table 3 Comparison of gene expression at mRNA level in epididymal adipose tissue from A1R (−/−) and A1R (+/+) mice (n = 5–8 mice of each genotype) with cDNA microarray gene chip and real-time RT-PCR analysis UniGene name

7-dehydrocholesterol reductase (Dhcr7)1 Fatty acid synthase (Fasn)2 Isocitrate dehydrogenase 1 (NADP+), soluble (Idh1)3 Isocitrate dehydrogenase 3 (NAD+) alpha (Idh3a)4 Malic enzyme, supernatant (Mod1)2 Stearoyl-Coenzyme A desaturase 2 (Scd2)5, 6 Succinate dehydrogenase complex, subunit A, flavoprotein (Fp) (Sdha)7

UniGene cluster ID

cDNA array

Real-time RT-PCR

Gene chip ratio A1R (−/−)/(+/+)

Relative change ratio, A1R (−/−)/(+/+)

A1R (+/+) Δct mean, S.E.M. (C.I.)

A1R (−/−) Δct mean, S.E.M. (C.I.)

Mm.249342 Mm.236443 Mm.9925 Mm.279195 Mm.148155 Mm.193096 Mm.158231

1.74a 1.74a 1.66a 1.88a 2.20a 1.76a 1.45a

1.22n.s. 1.00n.s. 1.04n.s. 1.08n.s. 1.03n.s. 0.95n.s. 0.80n.s.

8.97 ± 0.28 (8.20–9.75) 1.22 ± 0.14 (0.83–1.62) 1.34 ± 0.16 (0.91–1.78) 1.90 ± 0.11 (1.59–2.22) 3.26 ± 0.10 (2.93–3.58) 3.55 ± 0.24 (2.89–4.21) 2.61 ± 0.10 (2.33–2.89)

8.69 ± 0.28 (7.79–9.59) 1.22 ± 0.21 (0.56–1.88) 1.28 ± 0.14 (0.85–1.72) 1.79 ± 0.10 (1.48–2.11) 3.22 ± 0.28 (2.32–4.11) 3.63 ± 0.27 (2.78–4.48) 2.93 ± 0.07 (2.72–3.15)

1. Waterham and Wanders, 2000. 2. Hillgartner et al., 1995. 3. Koh et al., 2004. 4. Chen and Plaut, 1963. 5. Kim and Ntambi, 1999. 6. Miyazaki et al., 2005. 7. Favier et al., 2005. 95% confidence intervals (C.I.) in parentheses. a P b 0.001, n.s. = not significant. (Δct = ctrelevant gene − ctGADPH).

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other adenosine A1 receptor effects in the same tissue (Sollevi et al., 1981). Noradrenaline appears to be more potent as a stimulus for lipolysis than for cAMP accumulation (Fredholm and Hjemdahl, 1976), because submaximal cAMP levels are sufficient for maximal lipolysis (Hjemdahl and Fredholm, 1976). This also explains why it is difficult to inhibit lipolysis when a high concentration of a stimulating agent is administrated. Given that there are spare adenosine A1 receptors, it may seem strange therefore that the adenosine analogue is apparently more potent in blocking cAMP accumulation (IC50 value — 1.2 nM) than at acting as an antilipolytic agent (IC50 value — 17 nM) (present data and Johansson et al., 2007b). However, the reason is that in order to reduce lipolysis by half, much more than a 50% reduction of cAMP accumulation is required. Although the adenosine A1 receptor clearly plays an important role as a regulator of both basal and stimulated lipolysis, the life-long elimination of these receptors has led to surprisingly little adaptive change. We showed previously that there is no alteration in the adenosine receptor expression or in relevant G proteins (Johansson et al., 2007b). The response to other G protein-coupled receptors was not significantly altered either. Here we show that the antilipolytic effect of insulin is similarly not markedly affected. Although it is known that insulin and adenosine meditate their antilipolytic effects by different signaling mechanisms – insulin by increasing cAMP breakdown and adenosine by reducing cAMP formation via an action on the Gi protein – it has been suggested that insulin might partly act via Gi proteins (Goren et al., 1985), but see Wesslau et al. (1993). The apparent discrepancy might be related to endogenous adenosine modulating the response to insulin. However, our data suggest that insulin and adenosine signaling do not interact directly since the antilipolytic potency of insulin (i.e. its IC50 value) was not affected by an adenosine receptor agonist or antagonist (2-chloroadenosine or DPCPX) or by genetic ablation of the adenosine A1 receptor (Fig. 5). Similarly the effect of insulin (i.e. its IC50 value; Fig. 6A) on cAMP accumulation was not affected by 2-chloroadenosine. These results are compatible with the antilipolytic effects being mediated via different independent mechanisms that do not directly interact with each other. It must however be noted that adenosine and insulin have additive effects on lipolysis and cAMP accumulation, since the maximum response is stronger if both agents are added together to the adipocytes (see for example Fig. 6A). Therefore, under some circumstances a blockade of the Gi protein can reduce the antilipolytic effect of insulin, by eliminating endogenous adenosine. Adenosine might interact with insulin signaling not only at the level of lipolysis but also on lipogenesis. We found that the effect of 2-chloroadenosine on glucose incorporation into lipids was dependent of the concentration of insulin present in the surroundings: glucose incorporation increased significantly even at a low concentration of 2-chloroadenosine when insulin levels were high, but much more of the adenosine analogue was needed for a significantly increased lipogenesis in the presence of less insulin. Although insulin increased the effect of 2chloroadenosine in A1R (+/+) adipocytes, the effect of insulin was apparently not dependent on adenosine since there were no significant differences in the EC50 values of insulin between in adenosine deaminasetreated and control adipocytes from A1R (+/+) and A1R (−/−) mice. Previous studies have shown that the lipogenesis assay that was used here reflects glucose transport since the rate-limiting step in lipogenesis, when glucose is used at micromolar concentration, is the transport of glucose into the cell (Arner and Engfeldt, 1987). At these low levels of glucose we are far from the capacity of the enzymes involved in lipogenesis. One advantage of using this method for assessing glucose transport is that less amount of tissue is required and the results obtained with this method are comparable with those that orginate from direct glucose transport measurements (Arner and Engfeldt, 1987; Nordenström et al., 1989). Hence, as our lipogenesis assay reflects indirect measurement of glucose transport (Arner and

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Engfeldt, 1987), we conclude that adenosine is able to stimulate glucose uptake in adipocytes and that this effect is mediated via adenosine A1 receptors. This finding is in agreement with previous pharmacological studies in adipose tissue showing a stimulating effect of adenosine on glucose uptake in different species (Joost and Steinfelder, 1982; Martin and Bockman, 1986; Schwabe et al., 1974). Endogenous adenosine had only a minor effect on lipogenesis/ glucose uptake. As an adenosine analogue stimulates lipogenesis, we expected lower lipogenesis in A1R (−/−) mice than in A1R (+/+) mice. However, lipogenesis (in absence of 2-chloroadenosine) was significantly higher in adipocytes from the A1R (−/−) mice when the adipocytes were stimulated with a high dose of insulin. This might be related to the increased body weight in older A1R (−/−) mice. Given that our lipogenesis assay mainly reflects glucose uptake and no clearcut changes in expression of genes involved in lipogenesis between A1R (−/−) and A1R (+/+) mice were found, indicating that no adaptations in lipogenetic enzymes had occurred, we suspect there is an increased glucose uptake in adipose tissue of these mice. This could be due to insulin mediated effects. We have previously shown a higher release of insulin in A1R (−/−) mice than in A1R (+/+) mice after glucose administration (Johansson et al., 2007a) and this finding can be related to the increased lipogenesis and fat mass in A1R (−/−) mice since higher insulin levels increase lipogenesis (Kersten, 2001). Since the fat mass is increased in the A1R (−/−) mice, it is also possible that the adipocytes in these mice are larger than those in the A1R (+/+) mice. Large fat cells usually exhibit lower rates of lipolysis, but in our experiments differences between A1R (+/+) adipocytes (in the presence of adenosine deaminase) and A1R (−/−) adipocytes were small indicating that size effects were not large. We therefore assume that a possible effect of size on glucose uptake/lipogenesis may similarly have provided only a limited confounding effect. Nevertheless, this awaits further evaluation. High levels of free fatty acids are associated with obesity and can cause insulin resistance in skeletal muscle and liver (Boden, 2003). A drug that lowers free fatty acids may therefore be used for treating obesity, type 2 diabetes or dyslipidemia. Acipimox (nicotinic acid analogue) is an antilipolytic drug which has been evaluated in diabetic and obese subjects, but its rebound effect, causing increased levels of free fatty acids, has limited its usefulness (Santomauro et al., 1999; Vaag and Beck-Nielsen, 1992). Interestingly, this rebound phenomenon does not occur when a partial adenosine A1 receptor agonist, CVT-3619, is used for decreasing the free fatty acid levels in rats (Dhalla et al., 2007). Promising results from rodent studies (Dhalla et al., 2007; Fatholahi et al., 2006), suggest that CVT-3619 has potential as a new free fatty acid-lowering drug. The adenosine A1 receptor reserve in the adipose tissue makes it possible for a partial agonist to elicit a maximum antilipolytic response with only minor effects on organs where spare receptors are not present. However, epidemiological studies have indicated that adenosine A1 receptor antagonists also can be of relevance for treatment of type 2 diabetes (van Dam and Feskens, 2002; van Dam and Hu, 2005). If an adenosine A1 receptor antagonist is utilized as an antidiabetic drug, we believe that pancreas is the primary target since we have previously shown that deletion of adenosine A1 receptors results in increased release of insulin (Johansson et al., 2007a). A theoretical side effect of an adenosine A1 receptor antagonist might then be a decreased antilipolysis in adipose tissue, resulting in insulin resistance. However, we find no direct evidence for decreased insulin sensitivity in adipose tissue (present data) or in skeletal muscle (Johansson et al., 2007a). Caffeine is a widely consumed beverage and antagonizes the adenosine A1 receptors, the adenosine A2A receptors and to lesser extent also the adenosine A2B receptors (Fredholm et al., 1999; Fredholm et al., 2001a,b; Schulte and Fredholm, 2000). Beneficial effects of long-term caffeine intake (if epidemiology is to be trusted) may not be due to the adenosine A1 receptor; instead blockade of adenosine A2A receptors and adenosine A2B receptors

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appears to be of relevance for the association, see e.g. Rüsing et al., (2006) where the antidiabetic potential of an adenosine A2B receptor antagonist has been discussed. In conclusion, adenosine mediates its antilipolytic effects only via the adenosine A1 receptor; spare adenosine A1 receptors exist in adipose tissue; adenosine and insulin mediate additive but not synergistic antilipolytic effects and insulin-dependent lipogenesis is increased by an adenosine analogue. Acknowledgements We thank Vanessa van Harmelen and Kerstin Wahlen for providing us with the technique used for studying lipogenesis. The study was supported by grants from the Swedish Research Council (Project No. 2553), Novo Nordisk fund and the Biovitrum fat fund. References Arner, P., Engfeldt, P., 1987. Fasting-mediated alteration studies in insulin action on lipolysis and lipogenesis in obese women. Am. J. Physiol. 253, E193–E201. Boden, G., 2003. 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