Adenosine modulation of fat cell responsiveness to insulin and oxytocin

Regulator), Peptides, 16 (1986) 125-134

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Elsevier RPT 00528

Adenosine modulation of fat cell responsiveness to insulin and oxytocin H. J o s e p h G o r e n a'*, K h a w a r H a n i f b, R i c h a r d D u d l e y a, M o r l e y D. H o l l e n b e r g b a n d K a r l Lederis b Endocrine Research Group, aDepartment of Medical Biochemistry, and bDepartment of Pharmacology and Therapeutics, University of Calgary, 3330 Hospital Drive Northwest, Calgary, Alberta, Canada T2N 4N1

(Received 18 March 1986; revised manuscript received 16 July 1986; accepted for publication 2 September 1986)

Summary We have investigated the effects of adenosine on the stimulation of glucose oxidation and lipogenesis by oxytocin and insulin in rat epididymal adipocytes. The addition of adenosine deaminase (1 U/ml) to the assay medium reduced the maximal oxytocin response (glucose oxidation and lipogenesis) to between 25 and 50% of the maximum response in control cells. The maximal response to insulin was not appreciably affected under these conditions. The addition of adenosine (10 or 30 pM) increased the cell sensitivity to oxytocin by elevating the maximum rate of oxytocin-stimulated glucose metabolism. Adenosine also increased the cell sensitivity to insulin by decreasing its EDso. A change in EDso, however, was observed only when control or adenosine-treated cells were compared to adenosine deaminase-treated cells; but not when control and adenosine-treated cells were compared. On its own, adenosine also caused an appreciable increase in both glucose oxidation and lipogenesis (EDso ~ 3 ~tM adenosine). The difference in the effect of adenosine on oxytocin action, compared with the effect on insulin action, points to differences in the mechanisms by which insulin and oxytocin stimulate glucose metabolism in adipocytes. insulin; adipocytes; glucose oxidation; lipogenesis

* To whom correspondenceshould be addressed. Telephone: 403-220-6870. 0167-0115/86/$03.50 ~ 1986 ElsevierSciencePublishers B.V. (BiomedicalDivision)

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Introduction

Adenosine, which is released spontaneously from isolated rat adipocytes [1], can increase the response of fat cells to insulin under certain experimental conditions, whereas the addition of adenosine deaminase to an adipocyte suspension decreases insulin-stimulated responses by shifting the dose-response curve to the right [2-4]. The ability of adenosine to potentiate the metabolic effect of insulin in adipocytes may be explained in part by the observation that adenosine can augment insulinstimulated glucose transport activity [5-8]. Oxytocin, by interacting with oxytocin-specific receptors, exhibits insulin-like activity (stimulation of glucose oxidation and lipogenesis; inhibition of lipolysis) in rat adipocytes [9,10]. Some postreceptor events appear to be shared by insulin and oxytocin. For example, both hormones increase pyruvate dehydrogenase activity [10,11]. However, oxytocin, in contrast to insulin, does not stimulate glucose transport in isolated adipocytes [10]. Since oxytocin and insulin action share some biochemical pathways, we were interested to examine the effects of adenosine and adenosine deaminase on oxytocin-stimulated and insulin-stimulated glucose oxidation and lipogenesis, and to compare these effects to determine the role, if any, that endogenous adenosine has in oxytocin action. The effects of increasing adenosine concentrations on adipocyte glucose oxidation, and lipogenesis were also examined.

Materials and Methods

Reagents Porcine zinc insulin (Lot 491.4) was a gift from Dr. J. Clement, Connaughat Laboratories (Toronto, Canada), and highly purified synthetic oxytocin (486 + 22 U/mg [12]) was generously supplied by Dr. M. Manning (Medical College of Ohio, Toledo, Ohio). [U-~4C]Glucose (13.9 Ci/mol) was purchased from New England Nuclear Corporation, bovine serum albumin (BSA; Fraction V, Lot T13904, Armour Pharmaceutical Company; Lot 123F-0574) from Sigma Chemical Company, collagenase Type I (Lot 48M201 and 43S5208) from Worthington Biochemical Corporation, and adenosine and adenosine deaminase from Sigma Chemical Company.

Adipocyte glucose oxidaton and lipogenesis Fat cells were prepared by collagenase digestion of epididymal fat pads from l10-300-g Sprague-Dawley rats [13,14]. The final cell suspension yielded a homogeneous population of adipocytes, as determined by light microscopy. Adipocyte glucose oxidation activity was monitored by measuring the conversion of [U14C]glucose to 14CO2, as previously described [14]. Each assay bottle contained 0.2 ml fat cell suspension (0.04 1.2 × 105 cells), insulin or oxytocin and/or adenosine or adenosine deaminase and 1.7 ml substrate solution (0.28 mM D-glucose, 0,12 /~Ci/ml D-[u-lgC]glucose) in Krebs-Ringer bicarbonate/2% BSA buffer (pH 7.5) equilibrated with 95% 02/5% CO:. Adenosine deaminase was generally 1 U/ml or greater in the incubations, a concentration in excess of the amount needed to convert

127 all the adipocyte-secreted adenosine to inosine [15]. All suspensions were incubated at 37°C for 60 min. The assay bottles for measurement of triglyceride synthesis contained the same solutions as the glucose oxidation vessels. [14C]Acylglycerol content was measured as previously described [9]. All metabolic data were expressed as nmol of glucose converted to CO2 or triacylglycerol per h per l0 s cells. Adipocyte number was estimated gravimetrically [9,16]. The addition of adenosine or adenosine deaminase changed the basal rate and maximal hormone-stimulated rates of glucose metabolism. To compare data, normalized dose-response curves (percent response versus hormone concentration) were constructed using the following equation: %R(H); = [ R ( H ) g - B] × 100%/[M(H) - B] where %R(H)i was the percent response of a hormone, H, at concentration i, R(H)i was the hormone-stimulated rate of glucose metabolism, B was the basal rate and M(H) was the maximal rate for the hormone under the same assay conditions. A stimulation index (SI) was also calculated, where SI = M(H)/B. Response curves, based on the data points and their associated error bars ( + S.E.M.) were drawn visually.

Results

The addition of adenosine deaminase (1 U/ml) lowered the basal rate of adipocyte glucose oxidation and lipogenesis (Fig. 1). In control cells, the addition of oxytocin increased glucose metabolism in a concentration-dependent manner, but in the presence of adenosine deaminase the increase was markedly reduced; that is, in control cells 100 nM oxytocin increased the glucose oxidation rate above basal approximately 0.4 nmol/h per 105 cells and in adenosine deaminase-treated cells approximately 0.1 nmol/h per 105 cells. Oxytocin-stimulated lipogenesis was similarly affected by adenosine deaminase; oxytocin increased the lipogenesis rate above basal about 2.0 nmol/h per l0 s cells in untreated cells and only about 1.1 nmol/h per 105 cells in adenosine deaminase-treated cells. The data in Fig. 1 suggested that oxytocin action depended on adipocyte-secreted adenosine. To support this hypothesis, and to determine whether adenosine affects oxytocin action differently from insulin action, we compared insulin-stimulated and oxytocin-stimulated glucose oxidation and lipogenesis in the presence of adenosine deaminase or in the presence of exogenous adenosine (Fig. 2). The oxytocin-stimulated dose-response curves (Fig. 2B,D) appear similar to the curves in Fig. 1, where in the presence of adenosine deaminase the response with 100 nM oxytocin was significantly reduced from the 100 nM oxytocin-response in the presence of 10 pM (Fig. 2B) or 30/xM (Fig. 2D) adenosine. In contrast, the shape of the insulin concentration curves and the optimal insulin responses were similar in the presence of adenosine (10 or 30 pM) or adenosine deaminase (Fig. IA,C). The data of Fig. 2 were normalized and as previously reported [1,3] the EDso

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(concentration of hormone required to produce 50% of its maximal response) of the insulin response in the presence of adenosine was less than in the presence of adenosine deaminase; EDso (adenosine) ~ 0.05 nM, EDso (adenosine deaminase) ,~ 0.3 nM (Fig. 3). In contrast to the normalized insulin dose-response curves, the normalized oxytocin dose-response curves were unaffected by the addition of adenosine and adenosine deaminase, and the ED~o was the same in the presence of the nucleoside or the enzyme, EDso ~ 1 nM (Fig. 3). The effect of adenosine (10 or 30/~M), on the maximum glucose metabolic rates above basal, on oxytocin and insulin EDso, and on the stimulation indexes of insulin and oxytocin in adipocytes, has been compared with untreated and adenosine deaminase (1 U/ml) treated adipocytes. Whereas the addition of adenosine deaminase to an adipocyte suspension increased insulin EDs0 above the EDso of untreated adipocytes, the addition of adenosine to a fat cell suspension did not decrease the insulin EDso; EDso (adenosine) = EDso (untreated) ~ 0.05 nM, EDso (adenosine deaminase) ~ 0.3 nM. The normalized oxytocin dose-response curves, as previously dis-

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cussed, did not suggest changes in the EDs0, with the addition of either adenosine or adenosine deaminase (Fig. 3). Fig. 1 indicated that the addition of adenosine deaminase to an adipocyte suspension impaired the ability of oxytocin to stimulate glucose oxidation and lipogenesis. Conversely, one would expect that the addition of adenosine would increase the oxytocin rate of glucose metabolism. The incremental rate increases of oxytocinstimulated glucose metabolism above basal rates, however, did not increase further with the addition of adenosine; e.g. oxytocin increased the lipogenesis rate above the basal rate 2.9 nmol/h per 10 5 cells in untreated fat cells and 2.5 nmol/h per 10 5 cells in adenosine treated cells (Fig. 8 of ref. 17). The stimulation by oxytocin, relative to the basal rate of metabolism as reflected by the stimulation indexes, were generally insensitive to the presence of adenosine; e.g. for lipogenesis SI (adenosine) = 1.5, SI (untreated) = 1.6, and SI (adenosine deaminase) = 1.4. In contrast, the insulin-stimulated rates of glucose metabolism which were also increased with the addition of adenosine (Fig. 8 of ref. 17), demonstrated decreased stimulation indexes with adenosine; e.g. for lipogenesis SI (adenosine) = 6, SI (untreated) = 6 and SI (adenosine deaminase) = 14. The decreased

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Fig. 2. Effect of adenosine and adenosine deaminase on insulin-stimulated and oxytocin-stimulated lipogenesis and glucose oxidation. Isolated adipocytes were incubated in the presence of adenosine, 10/tM (A,B) or 30 #M (C,D) (open symbols), or adenosine deaminase, 1 U/ml (filled symbols), and in the presence of insulin (A,C) or oxytocin (B,D). Triacylglycerol synthesis (A,B) or glucose oxidation (C,D) was measured as described in Methods. Results are the means ± S.E.M. of 3 assays at each concentration.

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stimulation indexes were a consequence of increased basal activity without an equivalent increase in maximal insulin-stimulated rates (Fig. 2). As indicated in Figs. 1 and 2A and B, the addition of adenosine deaminase to adipocyte suspensions lowered the basal rate of glucose metabolism supporting previous observations of a metabolic effect of endogenously produced adenosine [3,4,6,18-20]. The effect of adding adenosine to unstimulated adipocytes has previously been investigated in a systematic manner for the stimulation of 2-deoxyglucose uptake [7] and for the stimulation of [1-~4C]glucose oxidation in the presence of phenazine methosulfate [5]. Our data indicate that added adenosine can also increase both lipogenesis (Fig. 4A) and glucose oxidation (Fig. 4C). Maximal stimulation was observed upon adding 30 #M adenosine to the incubation medium. The stimulation of glucose metabolism by 30 #M adenosine was about 1.4-fold, compared to 3.9-fold for insulin-stimulated lipid synthesis (Fig. 4B) and 9-fold for insulin-stimulated glucose oxidation (Fig. 4D). The effect of endogenous adenosine was also observed under conditions where assays were performed with very low and high fat cell concentrations; that is, dilution of fat cells was equivalent to the addition of adenosine deaminase [1]. Accordingly, the basal rate of lipid synthesis increased with increasing fat cell concentration; with 8500 cells/ml, the rate was 6.6 + 0.5 nmol/h per 105 cells and with 42500 cells/ml,

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the rate was 9.2 + 0.5 nmol/h per l05 cells. In addition, with increasing cell concentration, the stimulatory effects of oxytocin (10 nM) and insulin (0.17 nM) on li]~ogenesis were also increased. The increases, however, were parallel to the increase seen in the basal rate of lipogenesis: the oxytocin-stimulated rate was 8.3 + 0.3 nmol/h per 105 cells at a cell concentration of 8500 cells/ml and 10.6 ± 0.4 nmol/h per 105 cells at a cell concentration of 42500 cells/ml. The insulin-stimulated rate was 11.0 + 0.7 nmol/h per l0 s cells and 13.7 + 0.6 nmol/h per 105 cells, respectively, for the two concentrations of adipocytes.

Discussion

U p o n binding to its receptor on the fat cell [21-23], oxytocin stimulates glucose oxidation and lipogenesis via biochemical pathways that appear to share some, but

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not all steps, with those regulated by insulin. Like insulin, oxytocin stimulates pyruvate dehydrogenase activity [10,11], but unlike insulin, oxytocin does not stimulate glucose transport [10]. The effects of adenosine and adenosine deaminase on oxytocin sensitivity, described in this report, illustrate yet further similarities and differences between the glucoregulatory actions of oxytocin and insulin in rat adipocytes. Adenosine sensitized the adipocytes to the stimulatory actions of both oxytocin and insulin, as reflected by a leftward shift in the normalized dose-response curves for insulin, and by increased stimulation of maximum rates by oxytocin. The effect of adenosine on the insulin and oxytocin dose-response curves was apparent only if the data obtained in the presence of adenosine were compared with results obtained in the presence of adenosine deaminase. The addition of adenosine deaminase caused a pronounced decrease of the response of adipocytes to oxytocin; the maximum stimulation by oxytocin in the presence of adenosine deaminase was only about 25 to 50% of the stimulation observed in the absence of the enzyme. In contrast, our findings, in accord with previous work [3-5,7], indicate that adenosine deaminase did not alter the maximum cellular response to insulin. Thus, a clear difference in the mechanism by which adenosine modulates the cell sensitivity to insulin and oxytocin has been established; adenosine increases fat cell sensitivity to insulin by decreasing its EDso, and increases fat cell sensitivity to oxytocin by increasing the maximum response to the hormone. Adenosine can inhibit adipocyte adenylate cyclase by interacting with either a ribose-specific receptor or a purine-specific receptor [24] and consequently decrease intracellular 3',5'-adenosine monophosphate (cAMP) [4]. A decrease in intracellular cAMP has been suggested as one mechanism to stimulate glucose transport [25]. Since adenosine stimulates glucose transport [5 7], it follows that the insulin-like effects of adenosine, increased glucose oxidation and lipogenesis, may result from the increased rate of glucose uptake. Insulin stimulates glucose transport and enzyme activity associated with glucose metabolism [24]. In contrast, oxytocin cannot stimulate glucose transport in adipocytes, but does increase the activity of pyruvate dehydrogenase [10,11]. In the absence of adenosine (with the addition of adenosine deaminase) the maximum stimulation by insulin may be unaffected in adipocytes because insulin can still stimulate glucose uptake. However, unlike insulin, oxytocin cannot stimulate glucose transport and therefore in the absence of adenosine, stimulation of glucose metabolism may be restricted to the stimulation by oxytocin of intracellular enzymes. Thus, the ability of adenosine to stimulate glucose transport may be sufficient to explain the strong dependence of oxytocin on adenosine in the regulation of glucose metabolism. It is important, however, to determine whether intracellular enzymes stimulated by insulin and oxytocin are also affected by adenosine.

Acknowledgements This research was supported by the Medical Research Council of Canada (Grant MA 7271 to H.J.G, Grant MT 3911 to K.L. and Grant MT 6859 to M.D.H.). K.L.

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is a Career Investigator of the Medical Research Council of Canada. R.D. was an Alberta Heritage Foundation for Medical Research Summer Student.

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