Decrease in A1 adenosine receptors in adipocytes from spontaneously hypertensive rats

Decrease

in A, Adenosine

Receptors in Adipocytes Hypertensive Rats

Allan Green, Jeff L. Johnson,

From Spontaneously

and Donald J. DiPette

We have investigated whether the insulin resistance reported to occur in hypertension is due to decreased insulin receptors or to adenosine receptors in adipocyte membranes. Membranes were isolated from adipocytes from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKy) rats and essayed for insulin receptors and equally, but in contrast the SHR membranes A, adenosine receptors. The two groups of membranes bound ‘?-insulin adenosine], an A, adenosine receptor agonist) bound approximately 25% less ‘261-HPIA ([(-)-N’-phydroxyphenylisopropyl than the WKy (P < .005). Scatchard analysis demonstrated that this was due to a lower number of receptors in the SHR. The affinity of the receptor for HPIA wes approximately 0.7 nmol/L in both groups. 5’-Nucleotidase activity was approximately 40% higher in membranes from SHR than WKy (P < .OOl1, indicating that adipocytes from SHR have a higher capacity for adenosine production. This may cause increased adenosine concentrations in the SHR adipose tissue, leading to adenosine receptor down-regulation. Since we have previously demonstrated that adenosine receptor down-regulation can lead to insulin resistance, these findings may partly account for the insulin resistance of hypertension. Q 1990 by W.B. Saunders Company.

T HAS BECOME increasingly evident over the last few years that hypertension is often accompanied by insulin resistance.’ This insulin resistance is evident even after correction for confounding factors such as obesity or noninsulin-dependent (type II) diabetes. Insulin resistance has also been reported to occur in the spontaneously hypertensive rat (SHR), an animal model of hypertension most closely thought to represent human essential hypertension.2-4 Both in man and in the animal model, the mechanism by which insulin resistance occurs in hypertension has not been established. Isolated adipocytes are an excellent model for studying insulin action in vitro. Recent studies have demonstrated that adipocytes from SHR are resistant to insulin, but have normal insulin binding.4 This suggests that the insulin resistance of these cells is not due to an alteration at the level of the insulin receptor, raising the question of the mechanism of insulin resistance in these cells. We have reported that there appears to be a relationship between the number of A, adenosine receptors on adipocytes and their sensitivity to insulin.5 Adenosine is released from adipocytes, and is thought to be an important autocrine regulator of adipose tissue metabolism, both in rats6-* and in man.‘.” Thus, adenosine markedly increases the insulin sensitivity of adipocytes, and it seems that a decrease in the number of adenosine receptors causes a decrease in insulin sensitivity. In view of the finding that adipocytes from SHR are markedly resistant to insulin,4 these observations led us to investigate the A, adenosine receptors of adipocytes from these hypertensive rats. I

METHODS Animals Eight to 9-week-oldmale SHR and control Wistar-Kyoto (WKy) rats were purchased from Harlan (Houston, TX). They were maintained in the institutional animal care facility in climate and humidity-controlled and light-cycled rooms. They were fed standard rat chow and given water ad libitum. During the next 5 to 7 days, the animals were allowed to acclimatize to the animal care facility. Following this acclimatization, tail cuff systolic blood pressures were determined noninvasively in nonheated rats by photoelectric sensor (Model 59 pulse amplifier, IITC, Woodland Hills, CA) daily for 7 to 10 days as we have previously described.” The daily rat blood pressure was the average of at least three readings, and the baseline blood pressure was the average of the last three daily blood pressures. Isolation

of Adipocytes

Rats were killed by cervical dislocation, and adipocytes were isolated from epididymal fat pads by the method of Rodbell.‘* The cells were washed three times in 137 mmol/L NaCI, 5 mmol/L KCI, 4.2 mmol/L NaHCO,, 1.3 mmol/L CaCI,, 0.5 mmol/L KH,PO,, 0.5 mmol/L MgCl,, 0.5 mmol/L MgSO,, 20 mmol/L Hepes, pH 7.4, plus 1% bovine serum albumin. Membrane

Measurement From the Departments of Internal Medicine and Pharmacology. University of Texas Medical Branch, Galveston, TX. Supported in part by Grant No. R-01 DK 40061 from the National Institutes ofHealth. Address reprint requests to Allan Green, PhD, Division of Endocrinology, Metabolism and Hypertension, Department of Internal Medicine, Route E-68, University of Texas Medical Branch, Galveston, TX 77550. @ 1990 by W.B. Saunders Company. 0026-0495/90/3912-0020$03.00/0 1334

Isolation

Adipocytes were isolated as described above, homogenized, and a crude plasma membrane fraction was isolated as previously described.5 The membranes were suspended in 154 mmol/L NaCl, 10 mmol/L MgCl,, 50 mmol/L Hepes, pH 7.6. Protein concentration of the membrane suspensions was determined by the method of Bradford,” adjusted to 1 mg/mL, and the membranes were frozen at - 7ooc. of A, Adenosine

Receptors

Adenosine receptors were quantified by binding of “‘1-HPIA, an A, adenosine receptor agonist14.16 ((- ) -N6-p-hydroxyphenylisopropyl adenosine), to the crude membrane preparations as previously described.5 Briefly, membranes (20 pg protein) were incubated with adenosine deaminase (10 pg/mL) and 0.2 nmol/L ‘Z51-HPIA. After 2.5 hours at 37”C, the membranes were precipitated with polyethylene glycol and counted in a y-counter. Nonspecific binding was determined in the presence of 10 rmol/L HPIA, and was typically about 10% of total binding (1% of total radioactivity). All data have been corrected for nonspecific binding. Metabolism, Vol39,No

12 (December),1990:~~ 1334-1338

A, ADENOSINE RECEPTOR IN HYPERTENSIVE RATS

1335

Insulin binding

Table 2. Insulin and Adenorine

Insulin binding was measured as follows. Membranes (30 pg) were incubated with ‘zSI-insulin (0.3 ng/mL, iodinated as previously described’7.‘8) in a final volume of 500 pL. The buffer was the same as that described above for lipolysis measurements, but the pH was 7.8. The membranes were incubated for 3 hours at 16°C. and then bound radioactivity was precipitated by adding 20 FL of y-globulin (12.5 mg/mL), followed by 500 aL of 20% (wt/vol) polyethylene glycol (moll wt, approximately 8,000). The tubes were centrifuged (30 minutes, 2,000 x g), the supernatant was aspirated, and the pellet was washed once with 10% (wt/vol) polyethylene glycol and counted in a y-counter.

S-Nucleotidase

activity

S-Nucleotidase activity was determined by following conversion of [‘HIAMP to [‘Hladenosine.” Sodium /3-glycerophosphate (20 mmol/L) was used to inhibit nonspecific phosphatases.“’

Adenylate Cyclase Activity Adenylate cyclase activity was determined using 100 pg of membranes for measurement of basal activity, or using 25 pg for -stimulated rates. Assays contained measurement of forskolin/Mr?’ 0.5 mmol/L ATP, 10 pg/mL adenosine deaminase, 0.5 mmol/L methyl-isobutylxanthine, 50 mmol/L Hepes, pH 7.4, 5 mmol/L MgCI,, 1 mg/mL bovine serum albumin plus, as indicated, 100 pmol/L forskolin and 10 mmol/L MnCl,. After 20 minutes at 37OC, the assays were terminated by boiling for 3 minutes. The samples were centrifuged (2,000 x g, 10 minutes) and cyclic AMP formation was measured on the supernatants using a competitive protein binding assay (Amersham). Except where otherwise indicated, all values are mean f SD of determinations performed on five animals (each measurement performed in duplicate). Statistical comparisons were performed using Student’s t test. RESULTS

The body weights, blood pressures, and weights of the epididymal fat pads of the SHR and WKy rats are listed in Table 1. As expected, the systolic blood pressures of the SHR were markedly higher than in the WKy. Neither the body weights nor the epididymal fat pad weights were significantly different between the two groups of animals. Adipocytes were isolated from the SHR and WKy, homogenized, and a crude membrane fraction was isolated by differential centrifugation. Insulin receptors were evaluated by binding of ‘251-insulin, and A, adenosine receptors by binding of ‘*‘I-HPIA (Table 2). Both ligands were used at “tracer” concentrations, so that a difference in either receptor number of affinity would result in a difference in binding. ‘*‘I-insulin was bound equally by the two groups of membranes, but in contrast, the membranes from the SHR bound significantly less ‘251-HPIA than those from the WKy. These findings indicate that insulin receptor number and affinity are equal between the hypertensive and normotensive ani-

of the Animals MY

Systolic blood pressure (mm Hg)

152 f 6

Body weight (g)

237 f 33

Weight of epididymal fat pads (g) 2.23

+ 0.48

SHR 197 + 8

P 1.001

255i12

NS

1.94 f 0.13

NS

Binding

WV

SHR

P

‘251-insulinbound (pg/mg protein)

108 f 13

108 + 14

NS

‘251-HPIA bound (fmol/mg protein)

123 + 13

87 + 23

<.0005

NOTE. Adipocytes membranes ware isolated from WKy and SHR, and assayed for insulin and adenosine receptor binding using “tracer” concentrations of the labeled ligands. as described in Methods. The results are means f SD (n = 8 for both groups).

mals but 1251-HPIA binding is significantly lower in the hypeitensive animals. To determine whether the lower ‘*‘I-HPIA binding ability of the membranes from the SHR is due to a lower number of receptors, or to an affinity change, membranes were incubated with 0.2 nmol/L ‘251-HPIA plus 0 to 10 nmol/L unlabeled ligand. The ability of the unlabeled ligand to compete for binding was determined, and the results are presented in Fig 1 in the form of a Scatchard plot. This analysis revealed that the affinity of the receptor is equal in the two groups of membranes, since the slopes of the plots are almost identical. The K,, calculated from the slopes, is about 0.7 nmol/L in both groups of animals. However, the B,,,, calculated from the intercept of the plots on the abscissa, is approximately 350 fmol/mg in the WKy membranes, but only 280 in the SHR. This finding indicates that the SHR have fewer A, adenosine receptors than the WKy. We have previously reported that adenosine receptors can be down-regulated by prolonged incubation of adipocytes with an adenosine receptor agonist, PIA. Therefore, we considered the possibility that the decreased adenosine receptor number in the SHR may be secondary to increased interstitial adenosine concentrations. Since most tissue adenosine is protein-bound,*’ direct measurement of adenosine would probably not be valuable. Instead, we measured the

0.15

ki E

0.10

2 5 is

0.05

0.00 0

Table 1. Characteristics

Receptor

20

40 BOUND

60 (PM)

80

100

Fig 1. Scatchard plot of adenosine receptor binding data. Membranes from WKy and SHR adipocytes were incubated with ‘ssl-HPIA and 0 to 10 nmol/L HPIA. and binding was determined after 2.5 hours as described in Methods. Each plot represents the mean of eight different plots, each performed in duplicate.

1336

GREEN, JOHNSON, AND DIPETTE

activity of S-nucleotidase, which is an ectoenzyme that hydrolyzes AMP to adenosine, and is thought to be important in regulation of extracellular adenosine concentration. As shown in Table 3, the activity of this membrane bound enzyme is approximately 40% higher in membranes from SHR than WKY. If this results in increased extracellular adenosine, this may account for the decreased adenosine receptor binding in the SHR. 5’-Nucleotidase is often used as a marker enzyme for the plasma membrane. Therefore, the finding that the activity of this enzyme is higher in the membranes from the hypertensive animals could be due to a different population of membranes being isolated from these rats. Therefore, we felt that it was important to determine the activity of a second marker for the plasma membrane, namely adenylate cyclase. The enzyme was maximally stimulated with a combination of forskolin and manganese ions, which are thought to act on the catalytic subunit of the enzyme. This should negate any differences in stimulatory or inhibitory G proteins or receptors. When measured under these conditions, adenylate cyclase activity was equal between the two groups, suggesting that the population of membranes isolated from the two groups of rats were equivalent. Interestingly, however, the basal activity of adenylate cyclase was significantly higher in the membranes from the SHR. Although the reason for this difference is not clear, this may be partly due to decreased inhibitory input as a result of the lower number of adenosine receptors. Further analysis of the adenosine receptor binding data revealed an interesting relationship between blood pressure and adenosine receptor binding in both the SHR and the WKy (Fig 2). The data for 15 SHR and 16 WKy rats were pooled and plotted against the systolic blood pressure for each animal. This analysis revealed a positive correlation between blood pressure and adenosine receptor binding, both in the WKy and the SHR. However, the regression lines for the two groups of rats were clearly different, such that for any given blood pressure the adenosine receptor binding was lower in the SHR than in the WKy. This finding suggests that the low adenosine receptor binding in the SHR is not a secondary effect of their hypertension, since a higher blood pressure by itself seems to be associated with increased adenosine receptor binding. Furthermore, since young SHR are normotensive, this finding would suggest that the biggest Table 3. 5’-Nucleotidase

and Adenylste

Cyclase in Membranes

From SHR and WKy Rats SHR

P

61 f 7

<.OOl

WV

5’-Nuclaotidase activity (pmollminlmg)

43.0

f 6.0

Adenylata cyclase (pmol/min/mg) Basal

12.3 f 3.4

Stimulated

924

f

174

49.6 964+

f 7.7

<.05

102

NS

NOTE. Adipocyte membranes from WKy and SHR were assayed for 5’-nucleotidase and adenylate cyclase as described in Methods. Stimulated adenylate cyclase activity was measured in the presence of forskolin and manganese. Values are means f SD (n = 8 for both groups).

/

I

r=0.74

0

/

(P(O.001)

//

I

0.

//0

a

:a A

A

A

AA

A

r=0.65

A A

(P(O.01) A AA

I

100

I

I

150

200

250

SYSTOLIC BLOOD PRESSURE(mmHg) Fig 2. Correlation of adenosine receptor binding with blood pressure. Data from several experiments were pooled. To correct for variation between the experiments, the data were normalized as follows. In each experiment. the mean ‘?-HPIA binding (measured in the absence of unlabeled HPlAl was calculated by combining the data from WKy and SHR. For each rat, binding was then calculated as a percent of the mean for that experiment. Hence, data for the WKy average higher than 100%. while those from SHR average less than lOD%.O. WKy rats; A. SHR.

difference in adenosine receptor binding in the SHR precedes the development of hypertension in these animals. DISCUSSION

We have demonstrated that adipocyte membranes from SHR have approximately 20% to 25% fewer A, adenosine receptors than membranes from WKy rats. This was a specific loss of these receptors, as opposed to a general change in the plasma membrane, since insulin receptors and maximally stimulated adenylate cyclase activity were equivalent between the two groups of rats. The reason for the lower number of adenosine receptors in the cells from the hypertensive rats is not clear, but may be related to the increased activity of S-nucleotidase we observed. Thus, we have previously demonstrated that adipocyte A, adenosine receptors can be down-regulated by chronic incubation with a suitable agonist.5 While the role of 5’-nucleotidase in adenosine formation is controversial,‘9~22it has been suggested that the activity of this enzyme plays a part in regulation of extracellular adenosine concentrations.23.24 Therefore, it is likely that the elevated 5’-nucleotidase activity in the SHR adipocytes leads to increased adenosine formation, resulting in higher extracellular adenosine concentrations and consequently lower adenosine receptor numbers. Adenosine has many diverse biological actions in a variety of tissues.” Adenosine has several effects on isolated adipocytes. First, adenosine is a potent inhibitor of lipolysis.9*26.27 Second, adenosine markedly potentiates the effects of insulin, both by a direct effect, increasing the sensitivity of the cells to the hormone,6.28,29and also probably by an indirect effect, where adenosine prevents hormones such as glucagon and catecholamines from antagonizing insulin action.‘.* Therefore, a decrease in the adenosine receptor content of adipocytes might be expected to result in insulin resistance, as we have previously demonstrated experimentally.5 The

1337

A, ADENOSINE RECEPTOR IN HYPERTENSIVE RATS

SHR has been demonstrated to show insulin resistance.‘” Furthermore, it has been demonstrated that adipocytes from SHR are resistant to insulin, and that the abnormality is distal to the level of insulin binding to the cells.4 Since we were also unable to demonstrate a difference in insulin receptor binding in adipocytes from the SHR, the decrease in A, adenosine receptors may play a role. The decrease in A, adenosine receptors, whether secondary to increased S-nucleotidase activity or not, could be either a result of hypertension or genetically determined in the SHR. Although further studies are clearly necessary, our findings of a significant positive correlation between the blood pressure and A, adenosine receptor binding within both the SHR and WKy rats indicate that the mechanism may be primary and therefore genetically determined in the SHR. Thus, for any given level of the blood pressure, the SHR would be expected to have reduced numbers of A, receptors. If the increase in A, adenosine receptors is secondary to a decrease in adenosine production, this would suggest that adenosine production may decrease as the blood pressure increases. As previously stated, adenosine has multiple biologic effects, one of which is vasodilation mediated by the A, receptor. If similar effects occur in other tissues such

as vascular smooth muscle, a reduction in a potent and potentially compensatory vasodilator would further exacerbate the blood pressure elevation, especially in the SHR. However, this reduction does not appear to be specific for the SHR, since a similar relationship is seen in the WKy rats. On the other hand, the decreased A, adenosine receptor binding, coupled with an increase in 5’-nucleotidase activity, indicates enhanced adenosine production for any given level of blood pressure in the SHR compared with the WKy. This enhanced adenosine production could therefore be beneficial by partially offsetting the vasoconstriction in the SHR. Similar studies in differing pathophysiologic models of hypertension appear warranted. In conclusion, we have demonstrated that adipocyte membranes from SHR have fewer A, adenosine receptors than those from WKy rats, but normal insulin receptor binding. Furthermore, the reduction in A, adenosine receptors in the SHR may be due to increased adenosine production as a result of increased 5’-nucleotidase activity. In view of our previous report that adenosine receptor down-regulation can lead to insulin resistance, the decrease in A, adenosine receptor number may partly account for the insulin resistance of these hypertensive animals.

REFERENCES

1. Ferrannini E, Buzzigoli G, Bonadonna R, et al: Insulin resistance in essential hypertension. N Engl J Med 317:350-357, 1987 2. Horl WH, Schaefer RM, Heidland A: Abnormalities of carbohydrate metabolism in spontaneously hypertensive rats. Klin Wochenschr 66:924-927,198s 3. Mondon CE, Reaven GM: Evidence for abnormalities of insulin metabolism in rats with spontaneous hypertension. Metabolism 37:303-305, 1988 4. Reaven GM, Chang H, Hoffman BB, et al: Resistance to insulin-stimulated glucose uptake in adipocytes isolated from spontaneously hypertensive rats. Diabetes 38:1155-l 160, 1989 5. Green A: Adenosine receptor down-regulation and insulin resistance following prolonged incubation of adipocytes with an Al adenosine receptor agonist. J Biol Chem 262: 15702-l 5707, 1987 6. Schwabe U, Schonhofer PS, Ebert R: Facilitation by adenosine of the action of insulin on the accumulation of adenosine 3’:5’monophosphate, lipolysis, and glucose oxidation in isolated fat cells. Eur J Biochem 46:537-545, 1974 7. Green A: Glucagon inhibition of insulin-stimulated 2-deoxyglucase uptake by rat adipocytes in the presence of adenosine deaminase. B&hem J 212:189-195,1983 8. Green A: Catecholamines inhibit insulin-stimulated glucose transport in adipocytes, in the presence of adenosine deaminase. FEBS Lett 152:261-264, 1983 9. Kather H, Bieger W, Michel G, et al: Human fat cell lipolysis is primarily regulated by inhibitory modulators acting through distinct mechanisms. J Clin Invest 76:1559-1565, 1985 10. Kather H: Role of “local” hormones in regulation of lipolysis. Prostaglandins 33:831-836, 1987 11. DiPette DJ, Greilich PE, Kerr NE, et al: Systemic and regional hemodynamic effects of dietary calcium supplementation in mineralocorticoid hypertension. Hypertension 13:77-821989 12. Rodbell M: Metabolism of isolated fat cells. I. Effects of hormones on glucose metabolism and lipolysis. J Biol Chem 239:375380,1964

13. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal Biochem 72:248-254, 1976 14. Linden J: Purification and characterization of (-) [ ‘*‘I] hydroxphenylisopropyladenosine, an adenosine R-Site agonist radioligand and theoretical analysis of mixed stereoisomer radioligand binding. Mol Pharmacol26:414-423, 1984 15. Schwabe U, Lenschow V, Ukena D, et al: [?]N6-phydroxyphenylisopropyladenosine, a new ligand for Ri adenosine receptors. Naunyn Schmiedebergs Arch Pharmacol321:84-87, 1982 16. Munshi R, Baer HP: Radioiodination ofp-hydroxyphenylisopropyladenosine: Development of a new ligand for adenosine receptors. Can J Pysiol Pharmacol60: 1320- 1322, 1982 17. Green A: The insulin-like effect of sodium vanadate on adipocyte glucose transport is mediated at a post-insulin-receptor level. Biochem J 238:663-669, 1986 18. Green A, Bustillos DP, Misbin RI: @-Hydroxybutyrate increases the insulin sensitivity of adipocyte glucose transport at a postreceptor level. Diabetes 33: 1045- 1050, 1984 19. Newby AC, Luzio JP, Hales CN: The prcperties and extracellular location of 5’-nucleotidase of the rat fat-cell plasma membrane. Biochem J 146:625-633,1975 20. Stanley KK, Edwards MR, Luzio JP: Subcellular distribution and movement of 5’nucleotidase in rat cells. Biochem J 186:59-69, 1980 21. Sparks HV Jr, Bardenheuer H: Regulation of adenosine formation by the heart. Circ Res 58:193-201, 1986 22. Sasaki T, Abe A, Sakagami T: Ecto-5’nucleotidase does not catalyze vectorial production of adenosine in the perfused rat liver. J Biol Chem 258:6947-6951, 1983 23. Newsholme EA, Blomstrand E, Newell J, et al: Maximal activities of enzymes involved in adenosine metabolism in muscle and adipose tissue of rats under conditions of variations in insulin sensivity. FEBSLett 181:189-192, 1985 24. Arch JRS, Newsholme EA: Activities and some properties of S-nucleotidase, adenosine kinase and adenosine deaminase in tissue

1338

from vertebrates and invertebrates in relation to the control of the concentration and the physiological role of adenosine. Biochem J 174:965-977, 1978 25. Arch JRS, Newsholme EA: The control of the metabolism and the hormonal role of adenosine. Essays Biochem 1482-123, 1978 26. Ebert R, Schwabe U: Studies on the antilipolytic effect of adenosine and related compounds in isolated fat cells. Naunyn Schmiedebergs Arch Pharmacol278:247-259,1973

GREEN, JOHNSON, AND DIPElTE

27. Schwabe U, Ebert R, Erbler HC: Adenosine release from fat cells: Effect on cyclic AMP levels and hormone actions. Adv Cyclic Nucleotide Res 5569-583, 1975 28. Wieser PB, Fain JN: Insulin, prostaglandin El, phenylisopropyladenosine and nicotinic acid as regulators of fat cell metabolism. Endocrinology96:1221-1225, 1975 29. Green A, Newsholme EA: Sensitivity of glucose uptake and lipolysis of white adipocytes of the rat to insulin and effects of some metabolites. Biochem J 180:365-370, 1979