Tissue-specific effects of in vivo adenosine receptor blockade on glucose uptake in Zucker rats

^a Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033–0850, USA

	ABSTRACT

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Previous studies have shown that treatment of obese Zucker rats with the adenosine receptor antagonist 1,3-dipropyl-8-(p-acrylic) phenyl xanthine (BWA1433) improves intraperitoneal glucose tolerance. In this study, a euglycemic hyperinsulinemic clamp was performed on obese (fa/fa) and lean (Fa/fa) Zucker rats that had been treated orally with BWA1433 or vehicle for 1 wk. A constant infusion of [³H]glucose was initiated in fasted animals to measure basal whole body glucose kinetics. No differences in glucose concentration or rates of glucose production/disappearance were observed between lean or obese animals with or without BWA1433. During the euglycemic hyperinsulinemic clamp, whole body glucose disposal in obese Zucker rats was only 22% of that observed in lean animals. BWA1433 treatment increased glucose disposal by 88% in obese Zucker rats. At the end of the clamp, [¹⁴C]-2-deoxyglucose was injected to determine tissue-specific differences in glucose uptake. Gastrocnemius, soleus, heart, and liver of untreated obese animals had significantly lower glucose uptake than lean controls under hyperinsulinemic conditions. BWA1433 treatment of obese animals increased glucose uptake in gastrocnemius and soleus muscles by 44 and 47%, respectively. Conversely, BWA1433 treatment decreased glucose uptake in adipose tissue by 54 and 49% in obese and lean Zucker rats, respectively. In summary, BWA1433 improves glucose tolerance by increasing glucose uptake in skeletal muscle while decreasing glucose uptake by adipose tissue. This study suggests that insulin resistance in obese Zucker rats is tissue specific and that signaling from adenosine receptors may be a factor contributing to tissue-specific insulin resistance.—Crist, G. H., Xu, B., LaNoue, K. F., Lang, C. H. Tissue-specific effects of in vivo adenosine receptor blockade on glucose uptake in Zucker rats. FASEB J. 12, 1301–1308 (1998)

Key Words: obesity • euglycemic hyperinsulinemic clamp • 2-deoxyglucose • glucose disposal • insulin resistance • muscle • adipose tissue

	INTRODUCTION

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OBESITY IS FREQUENTLY associated with insulin resistance and type II diabetes (1, 2). Although the mechanisms for this insulin resistance are likely to be multifactorial, recent data has implicated an involvement of adenosine receptors. Adenosine receptors are widely distributed in mammalian tissues and are part of a highly homologous family of G-protein-linked cell membrane receptors (3). The adenosine receptors are divided into four major classes, designated A₁, A_2a, A_2b, and A₃, all of which have been isolated, cloned, and expressed (4, 5). These subtypes differ in their influences on cell metabolism and their tissue distribution (3).

Activation of A₁ adenosine receptors (A₁ AR)² in skeletal muscle has been shown to lower insulin sensitivity, thereby increasing the concentration of insulin needed to stimulate glucose and amino acid transport into muscle (6, 7). Conversely, adenosine receptor activation has the opposite effect in isolated adipocytes, where it increases insulin sensitivity (8–10).

Studies of A₁AR in adipose tissue of obese (fa/fa) Zucker rats have suggested that adenosine receptors are more active in adipocytes of obese than lean animals (11, 12). This enhanced activity in isolated adipocytes is not due to high receptor numbers or to excess adenosine in the medium surrounding the isolated cells, but rather to a high tonic activity of the receptors. Moreover, although skeletal muscle of obese Zucker rats is insulin resistant (13), isolated adipocytes from young (<20 wk old) obese Zucker rats are more sensitive to insulin than adipose tissue of lean animals (14–16). We previously studied the influence of whole body adenosine receptor blockade on insulin action in genetically obese rats as assessed by an intraperitoneal glucose tolerance test (17). A₁AR antagonism was accomplished using the A₁AR antagonist, 1,3-dipropyl-8-(p-acrylic) phenylxanthine (BWA1433), which does not penetrate the blood–brain barrier (18). Acute and chronic oral administration of the antagonist significantly lowered both glucose and insulin levels during the intraperitoneal glucose tolerance test in obese animals. However, because of differences in the prevailing glucose and insulin concentrations between groups, we cannot definitively conclude that the A₁AR antagonist improved insulin action or differentiate between the effect of BWA1433 on hepatic glucose production and/or peripheral glucose uptake. Therefore, the purpose of this study was to determine whether BWA1433 could reverse or attenuate the impairment in insulin-stimulated whole body glucose uptake observed in obese Zucker rats and, if so, to determine which tissues were affected.

	MATERIALS AND METHODS

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Animal model
The Zucker rat colony used is maintained in the animal facility of The Milton S. Hershey Medical Center, The Pennsylvania State University College of Medicine. Breeding pairs used to initiate the colony were obtained from Marcelle Lavau, Inserm U177, Unite de Recherches sur la Physiolpathologie de la Nutrition, Paris, France. In the present experiments, 6- to 8-wk-old lean and obese female animals were used. Each group received either vehicle or vehicle plus BWA1433 (12 mg/kg every 12 h) for 1 wk prior to the start of the experimental protocol (17). The final dose of BWA1433 or vehicle was administered approximately 1 h before the start of the experimental protocol. All procedures used in connection with these animals were approved by The Milton S. Hershey Medical Center Institutional Animal Care and Use Committee. These studies adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

On the day before the start of the experimental protocol, animals were anesthetized with an intramuscular injection of ketamine and xylazine (90 and 9 mg/kg body weight, respectively) and sterile surgery was performed to implant catheters in the carotid artery and jugular vein (19). After surgery, animals were housed in individual cages and provided water ad libitum. Animals were fasted overnight.

Basal glucose kinetics and hyperinsulinemic clamp
The next morning, a primed, constant intravenous infusion of [3-³H]glucose (high-performance liquid chromatography purified; DuPont-New England Nuclear, Boston, Mass.) was initiated to determine basal glucose kinetics (19, 20). A 7 µCi bolus injection of labeled glucose was administered, followed by a continuous infusion at a rate of 0.083 µCi/min for the next 2 h. Arterial blood samples were collected at 100 and 120 min (0.3 ml each) after the start of the tracer infusion. Blood was collected in heparinized syringes, centrifuged, and the plasma glucose concentration and glucose specific activity were determined on each sample.

After samples were obtained for basal metabolic determinations, a euglycemic hyperinsulinemic clamp was performed. Regular human insulin (Eli Lilly, Indianapolis, Ind.) was infused intravenously at a rate of 100 mU·min^-1·kg^-1 for 3 h. This infusion rate had previously been determined by our laboratory to result in steady-state plasma insulin concentrations of ~5000 µU/ml (19–21). This insulin concentration has previously been demonstrated to maximally stimulate glucose disposal by the whole body and skeletal muscles with different fiber type composition (21). Tritiated glucose was not infused during the hyperinsulinemic clamp, because preliminary studies indicated that this insulin infusion rate completely suppressed endogenous glucose appearance in both obese and lean animals (data not shown).

Tissue glucose uptake
In vivo glucose uptake by individual tissues was determined using [¹⁴C]-labeled 2-deoxyglucose (2-DG), as described previously by our laboratory (19–22). Tissue-specific glucose uptake was determined between 140 and 180 min after the start of the euglycemic hyperinsulinemic clamp. A tracer amount of 2-DG (8 µCi/rat; Amersham, Arlington Heights, Ill.) was injected intravenously and tissues were obtained 40 min later. Before tissue collection, serial arterial blood samples (0.2 ml) were withdrawn into heparinized syringes, plasma deproteinized with perchloric acid (PCA), and ¹⁴C-radioactivity was determined. Thereafter, animals were anesthetized with sodium pentobarbital, exsanguinated, and selected tissues were excised to determine the intracellular accumulation of phosphorylated 2-DG.

Analytical procedures
During the hyperinsulinemic clamp, blood glucose concentrations were determined at 10 min intervals using the YSI glucose analyzer (Yellow springs, Ohio). Glucose specific activity was determined on neutralized supernatant of deproteinized plasma (20). Tissue samples (600–900 mg wet wt) were immersed in ice-cold 0.5 N PCA, homogenized, and centrifuged. The concentration of phosphorylated 2-DG in tissues was calculated as the difference between total ¹⁴C-radioactivity of the neutral extract and the ¹⁴C-radioactivity remaining after Somogyi treatment (21–23). Previously it had been demonstrated that 2-DG is incorporated into muscle glycogen under euglycemic hyperinsulinemic conditions (24). However, more recent work by O'Doherty et al. (25) indicates that the Somogyi extraction, which is used in the present study, removes both free intracellular 2-deoxyglucose-6-phosphate and any 2-DG incorporated into glycogen. Hence, the calculated value provides an estimate of glucose uptake by the tissue but does not differentiate between glucose entry into the glycolytic pathway or glycogen synthesis.

Calculations
Rates of whole body glucose appearance (Ra) and disappearance (Rd) were calculated using the steady-state equations of Steele (26). The glucose metabolic clearance rate (MCR) was calculated by dividing the glucose Rd by the prevailing glucose concentration. Since the prevailing insulin levels during the hyperinsulinemic clamp completely suppressed endogenous glucose production, the rate of whole body glucose disposal equals the exogenous glucose infusion rate. The increment in insulin-stimulated glucose uptake (IMGU) for each animal was calculated by subtracting the basal endogenous glucose Rd from the measured rate of glucose disposal determined during the last 40 min of the clamp. In vivo glucose uptake for each tissue examined was calculated based on the accumulation of phosphorylated 2-DG by a respective tissue, the integrated 2-DG:glucose ratio in the plasma during the 40 min labeling period, and the lumped constant as described previously (23).

Experimental values are presented as means ±SEM. The number of animals per group is indicated in the figure and table legends. Data were analyzed using analysis of variance, followed by Student-Newman Keuls test to determine treatment effect (Glantz, McGraw-Hill, N.Y.). Statistical significance was set at P < 0.05.

	RESULTS

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Treatment of rats with BWA1433 for 1 wk did not produce significant changes in body weight in either the lean or obese animals ( Table 1). As expected, the body weight of obese animals was 36% greater than that of their lean littermates.

Basal carbohydrate metabolism and whole body insulin action
The basal postabsorptive arterial glucose concentration, glucose Ra/Rd, and MCR did not differ significantly between lean and obese rats ( Table 1). Moreover, the administration of the adenosine receptor antagonist did not produce detectable alterations in any parameter of whole body glucose metabolism in either group under basal conditions ( Table 1).

The rate of glucose infusion in each group was stable during the final 60 min of the 3 h euglycemic hyperinsulinemic clamp (data not shown). Furthermore, the average plasma glucose concentration during the final hour of the clamp was not different among the four groups. Glucose concentrations (mM) averaged 5.5 ± 0.1 for lean, 5.4 ± 0.1 for lean BWA1433-treated, 5.6 ± 0.2 for obese, and 5.5 ± 0.1 for the obese BWA1433-treated rats. Plasma insulin concentrations were not determined during the clamp, because previous studies have reported that the insulin infusion rate used in the current study achieved circulating levels of insulin (i.e., >1000 U/ml) that are maximally stimulating for glucose uptake (19–21). Based on the exogenous glucose infusion rate, whole body glucose disposal in lean control animals was 450% greater than in time-matched obese control rats ( Fig. 1, top). Administration of BWA1433 to lean animals resulted in a small (16%) but statistically significant decrease in whole body glucose disposal. In contrast, BWA1433 increased the rate of whole body glucose uptake in obese rats by 88% compared to values in untreated obese rats ( Fig. 1, top). Despite the ability of the adenosine receptor antagonist to increase glucose uptake in obese animals, the rate was still ~50% lower than that observed in lean control animals.

View larger version (18K): [in this window] [in a new window]   Figure 1. Top panel: Rate of whole body glucose disposal in lean and obese rats treated with vehicle or the adenosine receptor antagonist BWA1433 for 1 wk. Values are means ±SEM; n = 6, 6, 5, and 5, respectively. Where absent, standard error bars are within the symbol. Groups with different letters (a–d) are significantly different from each other (P<0.05). Bottom panel: Insulin-mediated glucose uptake (IMGU) in lean and obese rats treated with vehicle or BWA1433. IMGU was calculated as the difference between whole body basal glucose disappearance determined under basal conditions and glucose uptake determined under maximally stimulating euglycemic hyperinsulinemic conditions. The increment in total glucose utilization by the whole body provides a reliable estimate of IMGU (20). Values are means ±SEM; n = 6, 6, 5, and 5, respectively. Groups with different letters (a,b,c) are significantly different from each other (P<0.05).

Under basal conditions, most of the glucose uptake by peripheral tissues occurs via noninsulin-mediated pathways, and the contribution of this non-IMGU (NIMGU) to the overall rate of whole body glucose disposal may obscure changes in IMGU (23). To account for potential differences in NIMGU among groups, the insulin-dependent increment in glucose disposal was calculated for each animal ( Fig. 1, bottom). Whole body IMGU was depressed by 95% in obese control animals compared to lean controls. Treatment of obese rats with BWA1433 increased IMGU by more than sixfold compared to values in vehicle-treated obese animals. No change in IMGU was observed in lean Zucker rats treated with BWA1433.

Tissue glucose uptake
Under euglycemic hyperinsulinemic conditions, glucose uptake by gastrocnemius, soleus, and heart was lower (50, 54, and 67%, respectively) in obese control animals than in lean control rats ( Fig. 2). Similarly, glucose uptake by whole liver was also lower (52%) in untreated obese animals. One week treatment of obese rats with BWA1433 significantly increased glucose uptake in gastrocnemius and soleus by 44 and 47% compared to vehicle-treated obese rats. However, there was no significant change in glucose uptake in heart or liver in BWA1433-treated obese rats. The adenosine antagonist also altered insulin-stimulated glucose uptake in lean animals. BWA1433 resulted in a small, albeit statistically significant, decrease in glucose uptake by gastrocnemius and soleus (16 and 12%, respectively) in lean rats. Glucose uptake under hyperinsulinemic conditions was unaffected by BWA1433 in heart and liver of lean animals.

View larger version (26K): [in this window] [in a new window]   Figure 2. In vivo glucose uptake by various tissues from lean and obese rats treated with vehicle or BWA1433. Values are means ±SEM; n = 6, 6, 5, and 5, respectively. Groups with different letters (a–d) are significantly different from each other (P<0.05). Glucose uptake was determined during the final 40 min of a 3 h euglycemic hyperinsulinemic clamp in which insulin was infused at a rate sufficient to maximally stimulate whole body and tissue glucose uptake.

Glucose uptake was similar in adipose tissue of obese and lean Zucker rats when normalized to wet weight of tissue (33.7 ± 1.7 nmol/min/g and 39.2 ± 3.7 nmol·min^-1·g^-1; respectively) ( Fig. 3A). In contrast to its effects on skeletal muscle, a dramatic reduction in glucose uptake of adipose tissue took place in obese (54%) and lean (49%) Zucker rats in response to BWA1433. Since glucose uptake can occur only in the aqueous portion of an adipocyte, differences in the intracellular aqueous volumes of lean and obese adipocytes skew wet weight comparisons and do not accurately reflect the intracellular glucose uptake. Obese adipocytes contain approximately 12-fold the volume of lean adipocytes, but only 3-fold more aqueous volume (11). When differences in density of lean vs. obese adipose tissue were taken into account, a correction factor could be calculated that accounted for the differences in aqueous volume (see Discussion below). Aqueous volume per gram wet weight of adipose tissue was calculated to be 17.5 µl/g for obese and 60.5 µl/g for lean Zucker rats. Hence, basal glucose uptake by adipose tissue from obese rats was ~3.5-fold greater than uptake in lean rats when data are expressed as glucose uptake per intracellular aqueous volume ( Fig. 3B). Moreover, BWA1433 treatment reduced adipose tissue glucose uptake in obese animals to values that were not statistically different from vehicle-treated lean animals.

View larger version (18K): [in this window] [in a new window]   Figure 3. In vivo glucose uptake by epididymal fat from lean and obese rats treated with vehicle or BWA1433. Values are means ±SEM; n = 6, 6, 5, and 5, respectively. Group with different letters (a,b,c) are significantly different from each other (P<0.05). Top panel: Data calculated per gram wet weight of adipose tissue. Bottom panel: Glucose uptake in adipose tissue where wet weight data are corrected for lean vs. obese differences in adipose tissue aqueous volume. Data calculated per µl/intracellular adipose tissue aqueous volume. Aqueous volume per gram wet weight of adipose tissue was calculated to be 60.5 µl/g wet weight and 17.5 µl/g wet weight in lean and obese rats, respectively.

	DISCUSSION

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In this study we used the adenosine receptor antagonist BWA1433 to study the influence of endogenous interstitial adenosine on glucose metabolism of specific tissues. BWA1433 has previously been shown to be an effective antagonist of A₁ adenosine receptors in rats (3). Competition binding studies using rat brain particulate fractions indicate that the xanthine derivative is about 50-fold more selective for A₁ than for A_2a (3). However, the relative specificity of BWA1433 may be species-specific. In Chinese hamster ovary cells in which cloned sheep or human A₃ receptors have been expressed, the K_i is estimated to be 21 nM and 53 nM, respectively, which is similar to the K_i of binding to A₁ receptors in rat (15 nM). However, BWA1433 exhibits a much lower affinity for the cloned rat A₃ adenosine receptor (15 µM) and A₃ receptors in membranes isolated from rabbit brain (25 mM) (27). No information is available about BWA1433 binding to A_2b adenosine receptors.

Our previous study (17) indicated that BWA1433 effectively antagonized A₁ adenosine receptor stimulation by an A₁-selective agonist. At serum concentrations ranging from 6 to 10 µg/ml, BWA1433 prevented the A₁ selective agonist N⁶-(L-2-phenylisopropyl) adenosine from inhibiting adipose tissue lipolysis. However, there are questions concerning which receptor mediates the response to BWA1433 in skeletal muscle. Little information is available about which adenosine receptor subtypes are expressed in muscle. The receptor numbers are too low to be measured by the traditional ligand binding techniques. Studies of the functional effects of various specific adenosine receptor agonists and antagonists on insulin-stimulated glucose uptake suggest that A₁ adenosine receptors mediate the observed changes in insulin responsiveness (6, 7). However, more recent investigation of the tissue distribution of adenosine receptor mRNA using PCR technique indicated that no A₁ or A₃ adenosine receptors are expressed in muscle, whereas low levels of A_2a and A_2b receptor mRNA could be detected (28). Since our data indicate that BWA1433 has an opposite affect on glucose metabolism in muscle and adipose tissue, it is possible that this effect is mediated by different receptors, e.g., A₁ in adipose tissue and A_2b or A_2a in muscle.

Data presented in this investigation show that the glucose Ra, which represents hepatic glycogenolysis and gluconeogenesis, was not different in lean and obese rats under basal fasting conditions. Likewise, since all animals were in a steady state, the rate of whole body glucose disappearance also did not differ between fasted lean and obese rats. Although plasma insulin levels were not determined in the present study, numerous studies have demonstrated that fasting blood insulin concentrations are approximately threefold higher in obese than in lean rats (2, 17). A normal rate of glucose production and utilization with concomitant hyperinsulinemia is consistent with the presence of both peripheral and hepatic insulin resistance in obese Zucker rats (13, 29). Moreover, the twice daily oral administration of BWA1433 also did not significantly influence basal rates of glucose production and/or utilization in either lean or obese animals. This finding suggests that under in vivo conditions, endogenous adenosine has little measurable influence on basal glucose homeostasis.

Under euglycemic hyperinsulinemic conditions, whole body glucose uptake in lean rats was 450% greater than that in obese rats. A similar degree of peripheral insulin resistance has been reported previously (30, 31). Obese animals treated with the adenosine antagonist demonstrated an 88% greater rate of glucose uptake during the hyperinsulinemic clamp compared to vehicle-treated obese animals. These data support previous findings demonstrating that BWA1433 improves whole body glucose tolerance (17). IMGU was also estimated in these studies as the difference between the rate of glucose utilization under hyperinsulinemic conditions and the basal fasting glucose Rd. IMGU was depressed by 95% in obese control animals compared to lean controls. Treatment of obese animals with BWA1433 increased IMGU by nearly sixfold compared to obese control values. However, IMGU in the obese-BWA1433 group was still depressed by 69% compared to values in lean control animals. Paradoxically, BWA1433-treated lean animals exhibited a small (16%) decrease in glucose uptake during the clamp. There was, however, no difference in the calculated rate of IMGU between lean animals receiving BWA1433 and those administered vehicle.

Radiolabeled 2-DG was injected at the end of the clamp to determine tissue-specific differences in glucose uptake. As anticipated, obese Zucker rats exhibited impaired glucose uptake into skeletal muscle and heart under hyperinsulinemic conditions. BWA1433 increased glucose uptake in both gastrocnemius (primarily fast-twitch fibers) and soleus (primarily slow-twitch fibers) muscles of obese animals by 45–50%. However, skeletal muscle glucose uptake was still moderately depressed in obese BWA1433-treated animals compared to lean control values. In contrast, the ability of BWA1433 to partially reverse the defect in insulin-stimulated glucose uptake was not evident in liver or cardiac muscle. These changes in tissue glucose uptake in response to BWA1433 are unlikely to be due to alterations in tissue perfusion, since A₂ adenosine receptors predominate in the vasculature and BWA1433 does not block adenosine receptor agonist-induced vasodilation (32, 33).

The response of the adipose tissue to insulin-stimulation and the adenosine antagonist differed markedly from that seen in skeletal muscle. In agreement with in vitro studies (14–16), we found that glucose uptake by adipose tissue was not impaired in the obese animals under in vivo conditions. The relationship between rates of glucose uptake by adipose tissue from lean and obese rats, however, is probably not accurately depicted when normalized to wet weight because obese cells contain more lipid and less aqueous volume per total tissue volume. Since glucose uptake can occur only into the aqueous portion of an adipocyte, intracellular adipose tissue aqueous volumes were used to normalize data in Fig. 3, bottom panel. Values for intracellular aqueous volume per gram weight of tissue were then used to convert the data.

To determine the conversion factor we used published data (11) for adipocyte volumes of lean and obese Zucker rats. These volumes were calculated from measured diameters assuming the cells are spheres. The volume of aqueous space per adipocyte was determined in the same report (11) using data obtained by flotation, through silicone oil, of a known number of adipocytes suspended in ³H₂O. The volume of lean and obese adipose tissue per gram wet weight was measured as the inverse of tissue density (1.22 and 1.27 ml, respectively). From this we subtracted the extracellular space of the tissue, which was estimated as 0.123 ml/g (34). This provided a total intracellular volume per gram wet weight of tissue of 1.10 ml for lean and 1.14 ml for obese animals. When this number is multiplied by the percent of intracellular volume that is aqueous, a conversion factor is obtained for lean (60.5 µl/g wet) and obese (17.5 µl/g wet) rats. Therefore, when data were expressed per aqueous volume, adipose tissue glucose uptake was markedly elevated in obese rats compared to lean animals under hyperinsulinemic conditions. This finding agrees with previous reports that under in vitro conditions adipose tissue is hypersensitive to insulin in young obese Zucker rats (14–16). Moreover, inhibition of glucose uptake by the adenosine receptor antagonist is also in agreement with previous in vitro studies which show that A₁ adenosine receptors stimulate insulin-dependent glucose uptake in adipose tissue (8–10).

These data suggest that the genetic lesion causing the obesity and whole body insulin resistance results in a tissue-specific change in insulin action that favors glucose uptake, and thereby fat storage by adipose tissue, and impairs glucose uptake by muscle during hyperinsulinemia. The mutation in the obese Zucker rat has been mapped to the gene encoding the receptor for leptin (35). The mechanism by which this single mutation results in tissue-specific alterations in glucose handling is unclear, and this issue has been the focus of studies in animal models as well as humans with noninsulin-dependent diabetes mellitus (NIDDM). Obese humans with NIDDM tend to have high levels of circulating leptin (36), suggesting that they may also be leptin resistant, like the obese Zucker rat. Mounting evidence also indicates that individuals with NIDDM demonstrate tissue-specific insulin resistance (1). However, leptin does not appear to directly affect glucose transport in skeletal muscle or adipocytes (37).

Several hypotheses have been suggested to explain the link between obesity and insulin resistance. Tumor necrosis factor (TNF-) is known to induce insulin resistance (19), and Spiegelman and co-workers (30, 31, 38) have proposed that overexpression of this cytokine in adipose tissue may be a precipitating factor in the development of insulin resistance. Our present results make it unlikely that TNF- induces whole body insulin resistance directly. Since TNF- is generated in the adipose tissue and exported from there to other tissues, one would expect the level of TNF- to be highest in adipose tissue and thereby have a more pronounced effect on insulin action in adipose tissue than in muscle. Our data show, however, that although muscle is insulin resistant, adipose tissue is not.

It seems likely that serum free fatty acids contribute to the insulin resistance in obese Zucker rats. Fatty acids can block insulin-stimulated glucose uptake in muscle (39) and serum free fatty acids are elevated in the obese rats (31, 40). However, the influence of BWA1433 on insulin-stimulated glucose uptake is unlikely to result from a decrease in serum fatty acids. This conclusion is based on the observation that the A₁AR inhibits lipolysis in adipose tissue by lowering cAMP levels. Therefore, the antagonist would be expected to have the reverse effect, i.e., to raise free fatty acids. Our previous studies (17) showed that after 1 wk of oral administration of BWA1433, animals developed resistance to the influence of the antagonist on lipolysis, as judged by serum glycerol levels. Hence, there is no reason to suspect fatty acid levels in the serum would be elevated by BWA1433 administration, especially since glycerol levels were not (17).

In summary, our data indicate that the marked reduction in whole body glucose uptake in obese Zucker rats is due to a decreased ability of insulin to stimulate glucose uptake in skeletal muscle, but not in adipose tissue. Moreover, in the obese animal, BWA1433 was able to improve whole body glucose disposal by selectively increasing glucose uptake in skeletal muscle. In vivo glucose uptake into adipose tissue was the same or higher in obese rats, and BWA1433 treatment decreased glucose uptake in adipose tissue from both lean and obese animals. The data support the conclusion that insulin resistance in the obese Zucker rat is tissue specific. Furthermore, the marked divergence in the effects of BWA1433 on lean vs. obese Zucker rats suggests that differences in adenosine receptors may play a role in the pathogenesis of the tissue-specific insulin resistance.

	ACKNOWLEDGMENTS

 
The A₁ adenosine receptor antagonist, BWA1433, was generously supplied by Dr. Susan Daluge, Glaxo Wellcome Corporation.

	FOOTNOTES

 
¹ Correspondence: Department of Cellular and Molecular Physiology, The Pennsylvania State University, College of Medicine, PO Box 850, H166, Hershey, PA 17033–0850, USA. E-Mail: clang@PSU.EDU

² Abbreviations: AR, adenosine receptor(s); 2-DG, 2-deoxyglucose; PCA, perchloric acid; MCR, metabolic clearance rate; IMGU, insulin-stimulated glucose uptake; NIDDM, noninsulin-dependent diabetes mellitus; TNF, tumor necrosis factor; Ra, whole body glucose appearance rate; Rd, whole body glucose rate of disappearance.

Received for publication February 4, 1998. Accepted for publication May 5, 1998.

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