Extrapancreatic effects of sulfonylureas

Extrapancreatic Effects of Sulfonylureas Potentiation of insulin Action Through Post-Binding Mechanisms

DEAN H. LOCKWOOD, M.D. BRUCE

L. MALOFF,

Ph.D.

SUZANNE

M. NOWAK,

Ph.D.

MICHAEL

L. f&CALEB,

Ph.D.

Rochester, New York

From the Department of Medicine, EndocrineMetabolism Unit, University of Rochester School of Medicine and Dentistry, Rochester, New York. This study was supported by U.S. Public Health Service research grant AM-20129 and a grant from the Upjohn Company, Kalamazoo, Michii. Requests for reprints should be addressed to Dr. Dean H. Lockwood, Department of Medicine, Endocrine-Metabolism Unit, University of Rochester Medical Center, Rochester, New York 14642.

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Previous studies have suggested that sulfonylureas exert at least some of the hypoglycemic effects at extrapancreatic sites. We have recently used an organ culture system for adipose tissue in an attempt to determine what parameters of insulin action, if any, are directly influenced by the sulfonylureas, tolazamide and glyburide. We measured Insulin binding, basal and insulin-stimulated hexose uptake, and glucose metabolism in adipocytes from the cultured tissue. These studies demonstrate that sulfonylureas in vitro potentiate insulin action beyond the binding portion of the receptorprimarily at the level of insulin-stimulated glucose transport. The capability of the sulfonylureas to lower blood sugar in type IInoninsulindependent diabetes mellitus (NIDDM)-has been recognized for the past three decades. It was originally believed that the sulfonylureas exerted their hypoglycemic effects by stimulating insulin secretion in this type of diabetes [ 11, which is characterized by a relative rather than absolute insulin deficiency. However, a number of studies have indicated that with chronic usage of these drugs, an improvement in glucose tolerance is achieved in the absence of an increase in plasma insulin levels [2-41. These latter observations are compatible with the hypothesis that sulfonylureas potentiate the actions of endogenous circulating insulin on target tissues. Consequently, the attention of a number of studies has been focused on the mechanism by which sulfonylureas exert their extrapancreatic hypoglycemic effects. Several parameters of insulin action have been evaluated in cells or tissues obtained from humans or animals after drug treatment. Most of these studies have demonstrated an increase in insulin binding after sulfonylurea therapy [3-51 and another has shown the potentiation of insulin-stimulated hexose transport in muscle [6]. However, the relationship between altered insulin binding and hormonal response was not explored in any of these studies. Recently we have used an organ maintenance system for adipose tissue to characterize better those parameters of insulin action that are influenced by hormones, substrates, and drugs. We believe that studies with this system offer advantages over studies in target cells derived from treated animals in that direct effects of pharmacologic agents can be assessed in a target tissue maintained in a biochemically defined medium. In this report, we will review our findings of the in vitro effects in adipocytes of the sulfonylurea, tolazamide [7] and glyburide on insulin binding to receptors, on down-regulation of insulin binding to receptors, and on basal and insulin-stimulated glucose transport and utilization.

The American Journal of Medicine

DIABETES SYMPOSIUM-LOCKWOOD

METHODS Adipose Tissue Culture and Preparation of Adipocytes. Male Sprague-Dawley rats (150 to 225 g) were maintained on a diet of Purina laboratory chow and water ad libitum. Under sterile conditions epididymal fat pads were removed, minced, and placed in Parker medium 199 containing 1 percent (weight/volume) bovine serum albumin and 0.3 mg/ml Hepes, pH 7.4 [8]. Approximately 20 pieces, each weighing about 10 mg, were placed in a culture dish and incubated at 37’C for 20 to 44 hours under an atmosphere of 95 percent oxygen and 5 percent carbon dioxide. During this period the fat tissue was incubated in either the absence of or presence of 0.003 to 0.30 mg/ml of tolazamide (sodium salt) or 1.O pg/ml of glyburide. These concentrations were chosen because they include the therapeutic plasma levels observed in man. The sulfonylureas were present throughout the incubation period and during subsequent cell isolation and assessment of insulin action. After the incubation, the minced tissue was washed with Krebs-Ringer phosphate buffer, pH 7.4, which contained 3 percent (weight/volume) bovine albumin; the isolated adipocytes were prepared by collagenase digestion [ 91. Aliquots from the same cell suspension were used to assay insulin binding, hexose transport, and glucose metabolism. There was no degradation of sulfonylurea during culture as determined by gas-liquid chromatography [lo]. Table I shows that neither culture alone nor exposure to sulfonylureas had any effect on morphology or distribution spaces [ 1 I] when cultured and treated cells were compared to freshly prepared adipocytes. insulin Binding. Monoiodinated 1251insulin (1.0 Ci/pmol) was prepared by the chloramine-T method and purified on talc [ 121. Adipocytes (~2 X lo5 cells) were incubated with 0.6 ng/ml ‘*? insulin and zero to 5,000 ng/ml native insulin at room temperature for one hour. Cells were separated from the medium by centrifugation through oil at the end of the incubation [ 131. The amount of labeled hormone remaining bound at a native hormone concentration of 5,000 ng/ml was used as a correction for nonspecific binding. Hormone degradation was determined by precipitation in 5 percent (weight/volume) trichloroacetic acid and did not exceed 10 percent of the labeled hormone used. Hexose Uptake. The rates of uptake of 2deoxy-D[l3H]-glucose (0.1 mM) and 3-O-[i4C]methyl-Dglucose (0.1 mM) were used to assess the activity of the glucose transport

TABLE I

system. Aliquots (“2 X lo5 ceils) of the adipocyte suspension were incubated with zero to 1,000 ptJ insulin/ml at 37% under a humidified atmosphere of 95 percent oxygen and 5 percent carbon dioxide for one hour. 2-Deoxyglucose was then added for one minute at 37%, and the incubation was terminated by centrifugation through oil [ 131. Alternatively, the cells were exposed to labeled 3-Omethylglucose for 10 to 20 seconds before termination of transport by addition of cytochalasin b (final concentration 50 PM), which specifically blocks the glucose transport system. Extracellular trapping of the glucose analogs by the fat cell pellet was corrected by parallel incubations with either inulin or cytochalasin b. The fat cell pellets were dissolved in 10 percent Triton X-100 before scintillation counting. Glucose Metabolism. Glucose conversion to triglycerides and carbon dioxide was examined using D-[U-14C]glucose over a concentration range of 0.1 to 50 mM. Aliquots of cells (~2 X lo5 adipocytes) were gassed with 95 percent oxygen and 5 percent carbon dioxide and incubated for two hours at 37’C in the absence or presence of 1,000 /.JUinsulin/ml. The resultant radiolabeled metabolites were measured according to standard techniques, as described in Reference 7. RESULTS Hypoglycemic Effects of Sulfonylureas Beyond Insulin Unlike most published studies, Binding to Receptors. which have shown that in vivo administration of sulfo-

nylureas enhances insulin binding in adipocytes, our studies have shown that exposure of fat tissue in vitro to tolazamide for periods of up to 44 hours has no effect on the binding of 1251insulin to receptors on the adipocytes [ 71. Also, tolazamide in vitro does not influence the ability of unlabeled insulin to compete for binding with the labeled hormone to the cells. Scatchard anal-

ysis of binding reveals no difference between treated and untreated cells in the number of receptors or their affinity for insulin. Insulin degradation is also unaffected. Our inability to demonstrate a direct effect of tolazamide on insulin binding in adipocytes in vitro has been confirmed with other sulfonylureas. Vigneri et al. [ 141 found no effect of tolbutamide or glyburide on insulin binding in four cell lines in culture. More recently our

Effects of Organ Culture and Toiazamide on Ceil Morphology and Distribution Spaces

Cultured for 20 Hours with No Drug

Cultured lor 20 Hours with Tolaramide (0.30 mglml)

48.6 f 1.3’ 9.8 f 0.4

49.3 f 1.4 9.1 f 0.3

49.4 f 1.1 9.7 f 0.4

0.36 f 0.02 0.22 f 0.01

0.34 f 0.02 0.21 f 0.01

0.37 f 0.19 f

Freshly Prepared Cells

Cell diameter (PM) Lipid content (mg/105 cells) Distribution space (@lo5 cells) 3-0-Methylglucose lnulin (extracellular * Means

f

(total water space) space)

0.02 0.01

SEM of 4 to 10 experiments.

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O--T-q--

15

lz%Labeled Insulin Bound (fm) Figure 1. The effect of tolazamide (0.3 mglml) on insulin binding and on down regulation by insulin (60 nglml) is depicted in this figure. These curves were generated by Scarchard analysis of the specific binding of 125I insulin to ceils prepared from insulin-treated tissue in the absence (0) or presence (0) of tolazamide and from tissue not treated with insulin in the absence (0) or presence (A) of tolazamide. The total and specific amounts of hormone bound in the presence of insulin (0.6 to 10.6 nglml) were significantly different from each of the insulin-treated conditions versus those not treated with insulin (p < 0.05 by paired-t analysis), but they were not significantly different for the corresponding tolazamidetreated conditions versus those not treated with tolazamide. The results represent the mean of three separate experiments. Reproduced with permission from ref. [7].

laboratory also has been unable to demonstrate an effect of glyburide (1 pg/ml) on insulin binding to adipocytes after exposure to the drug for 20 hours. In contrast to our finding in adipose tissue, Prince and Olefsky [ 151 recently reported that glyburide (1 pg/ml) slightly increased insulin binding to nontransformed human fibroblasts in tissue culture. A more striking finding of these investigators was the demonstration that glyburide inhibits the process of insulin-induced receptor loss in these cells. The magnitude of this inhibition of receptor loss was approximately 34 percent. As shown in Figure 1, we were unable to demonstrate that tolazamide influenced the down-regulation of insulin binding by insulin (60 ng/ml) as we had previously demonstrated in our adipose tissue maintenance system [ 161. In more

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recent unpublished studies, we have found that glyburide also does not affect the insulin-induced downregulation process. Prince and Olefsky [ 151 have suggested that the increase in insulin binding to receptors by sulfonylureas in vitro could have occurred through inhibition of internalization of the hormone-bound receptor. This hypothesis is supported by studies showing that tolbutamide diminishes the internalization of some proteins, presumably through its ability to inhibit the activity of transglutaminase, a membrane-associated enzyme which is thought to play a role in the internalization process [ 171. Further work with human fibroblasts in Olefsky’s laboratory has also supported this hypothesis, with the demonstration that transglutaminase was present in abundant amounts in fibroblasts and that dansylcadaverine, a potent inhibitor of transglutaminase activity, prevented insulin-induced down-regulation of receptors [ 181. However, these investigators showed that this mechanism did not seem to be operative in IM-9 lymphocytes in which down-regulation could also be demonstrated. We have recently completed some in vivo studies that further support our proposal that sulfonylureas exert their hypoglycemic effects primarily at sites other than the binding portion of the insulin receptor. Male Sprague-Dawley rats were fed either no drug or tolazamide (75 mg/kg/day) for a period of 10 days. Following drug treatment, six-hour fasting glucose levels in the tolazamide-treated rats were markedly reduced (42 mg/dl) as compared to those in the control group (96 mg/dl). Plasma insulin levels in the rats treated with tolazamide had not changed, which suggested an extrapancreatic hypoglycemic effect of the sulfonylurea. However, the activity of transglutaminase in the postnuclear fraction of liver homogenates and fat homogenates was the same in both groups of animals. Interestingly, the specific binding of 1251insulin to purified liver membranes in the tolazamide-treated rats was unchanged. This latter observation suggests that sulfonylurea-induced hypoglycemia can be caused in vivo without changes in insulin levels or in insulin binding to receptors. Enhanced Hexose Transport by Sulfonylureas in the Perhaps an explanation for the Presence of Insulin. hypoglycemic effect of the sulfonylureas is provided by our studies of hexose transport in the adipose tissue culture system [7]. Exposure of adipose tissue to tolazamide (0.30 mg/ml) for 20 to 44 hours did not result in a significant change from control when transport of 2-deoxyglucose (a glucose analog not metabolized beyond phosphorylation) was assessed in the absence of insulin (basal transport). However, insulin-stimulated 2-deoxyglucose transport was significantly potentiated

DIABETES SYMPOSIUM-LOCKWOOD

at submaximal and maximally effective concentrations of the hormone. The average increase in insulin-stimulated transport of 2deoxyglucose was approximately 30 percent after in vitro treatment of adipose tissue with tolazamide (0.30 mg/ml) for 20 hours. Potentiation of insulin-stimulated hexose transport was dependent on the tolazamide concentration, and this effect of tolazamide was not observed when an analog, lacking hypoglycemic activity in vivo, was tested. Although the insulin response was greater in sulfonylurea-treated cells, the concentrations of insulin that caused 50 percent maximal uptake were the same. As shown in Figure 2, exposure of the cells to tolazamide for 20 hours resulted in increased insulin-stimulated 3-O-methylglucose uptake but did not influence basal transport. 3-0-Methylglucose is a glucose analog that is transported through the glucose transport system but is not metabolized by fat cells: hence, it is a pure indicator of the uptake phenomenon. We have also recently shown that glyburide treatment of adipose tissue for 20 hours potentiates insulin-stimulated hexose uptake but does not influence 2-deoxyglucose uptake in the absence of insulin. Furthermore, treatment of adipose tissue with either tolazamide or glyburide results in potentiation of hexose transport in the presence of the insulin mimickers, hydrogen peroxide and vitamin Kg. These insulin mimickers are oxidants which are known to stimulate glucose uptake to the same degree as does insulin, but they do so without interacting with the binding portion of the insulin receptor [ 191. We think that the potentiation of hexose transport by these agents provides further evidence for the postreceptor locus of sulfonylurea action in adipocytes. Glucose Metabolism Not Altered by Sulfonylureas. The influence of sulfonylureas on adipocyte glucose metabolism has also been investigated under conditions in which glucose transport is rate limiting and under conditions in which the metabolic capacity of the fat cell is tested [ 71. As shown in Figure 3, the influence of tolazamide on hexose transport was reflected by glucose conversion to carbon dioxide and total lipids at glucose concentrations (0.1 to 5.0 mM) in which transport was rate limiting. In contrast, pretreatment with tolazamide showed no apparent effect under conditions in which transport is no longer rate limiting (50 mM glucose). Also at each glucose concentration, basal levels of metabolism were not significantly altered by the drug, and the proportions of metabolites that were formed were unaltered by tolazamide treatment. Culturing of adipose tissue in the presence of glyburide produced similar results. These observations provide additional evidence that the sulfonylureas exert their effects primarily at the level of glucose transport rather than by causing alterations in metabolic pathways or

Basal (Control) Basal (Tolazamlde, 1OOObU lnsulh

0 30 mg ‘ml)

ml (Control)

1OOOp.U lnsul~n ml (Tolazamide

”

[

P- 0 05 by Palred-

Tesl

Tolazamlde-Treated

vs

Non-Treated

0 30 mg/ml)

Cells

60

10

15

20

Time in Seconds This graft depicts the uptake of 3-O-methyl-DFigure 2. glucose in adipocytes prepared from tissue cultured in the presence or absence of tofazamide. The values represent the mean f SEM of these experiments.

enhancing metabolic capacities. It should be noted that in all of our studies, sulfonylureas have been found to be incapable of altering insulin-stimulated glucose transport and metabolism within two hours of treatment. Potentiation of Gpogenesls in Hepatocytes by Sulfonylureas in the Presence of Insulin. Of additional interest are the studies of Salhanick et al. [ 201, in which they investigated the in vitro effects of tolazamide on insulin action in primary cultures of rat hepatocytes. Preliminary results of these studies indicated that tolazamide, by itself, did not stimulate lipogenesis. However, when hepatocytes were exposed to a combination of insulin and tolazamide for a period of 16 hours, there was marked potentiation of insulin’s action of lipogenesis as compared to cells cultured in the presence of insulin alone. These investigators found that tolazamide had no effect on insulin binding in most of their studies. There was no shift of the dose-response curve for insulin and lipogenesis after exposure of the cells to the sulfonylurea. This finding has provided further evidence that in the liver, which is a major target

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DIABETES SYMPOSIUM-LOCKWOOD

50 -

40 z= 2 .p!

q Basal (Control) q Basal (Tolazamlde, q 1000 pU Insulin/ml

0.30 mg/ml)

H

(Tolazamide,

1000 pU Insulin/ml

(Control) 0 30 mg/ml)

30 -

20.

lo-

O_

Glucose, mM

organ for insulin, potentiation of insulin action occurs through postreceptor mechanisms. Identical studies in hepatocytes obtained from streptozotocin-induced, nonketotic diabetic rats showed the same results. COMMENTS

It appears from the findings in a multitude of in vivo and in vitro studies, that sulfonylureas can produce hypo-

Sulfonylureas

/\ t Insulin Secretion

Potentiate Insulin Action [Postreceptor Mechanism(s)]

Frequent Reduction in Insulin Levels

f Insulin &ding

(Up Regulation),tlnsulin

Sensitivity

Figure 4. The proposed mechanism of the hypoglycemic actions of solfonylureas.

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Figure 3. Shown here is glucose utilization in adipocytes prepared from tissue cultured in the presence or absence of tolazamide (0.30 mglml) for 20 hours. Conversion of glucose to carbon dioxide (clear portion of bar) and total lipids (filled portion of bar) was determined. The values represent the mean f SEM of three experiments. Asterisks denote statistically significant differences in total glucose utilization between the control and tolazamide-treated cells by the paired-t test at p < 0.05. Reproduced with permission from ref. [7].

glycemia through several mechanisms. In all instances, however, insulin must be present. This finding explains why these drugs do not work in a setting of total insulin deficiency, for example, type I, insulin-dependent diabetes mellitus (IDDM). It is now recognized that In type II, noninsulin-dependent diabetes mellitus (NIDDM), where sulfonylurea therapy is effective, multiple abnormalities exist which contribute to carbohydrate intolerance. At the level of the endocrine pancreas, there is a relative insulin deficiency that is primarily due to a reduction in the rapidly releasable insulin pool [21]. This abnormality is responsible for the reduced initial insulin response to a glucose challenge. At the same time, there is insulin resistance in target tissues in patients with NIDDM [ 221. Recent studies have demonstrated that receptor and postreceptor defects contribute to this clinical state of insulin resistance [ 231. Of course, these problems are aggravated when obesity coexists with NIDDM. Obesity is well known as a condition of insulin resistance. Studies of cellular alterations have clearly demonstrated that it is a state of receptor and postreceptor defects [24-261. The recognition that sulfonylureas seem to be capable of either directly or indirectly influencing those abnormalities known to be responsible for the carbohydrate intolerance in noninsulin-dependent diabetes mellitus is of pharmacologic interest. For example, sulfonyiureas can stimulate insulin secretion [l] and can alleviate the insulin-resistant component by po-

DIABETES

tentiation of insulin action. A proposal with regard to the relative importance of each pharmacologic action of the sulfonylureas is presented in Figure 4. Sulfonylureas may initially exert their hypoglycemic effects through stimulation of insulin secretion and potentiation of insulin action in target tissues via postreceptor mechanisms. As the target tissues become more responsive to circulating insulin, the demand for the hormone decreases, and a reduction in insulin levels may occur. If insulin levels become significantly reduced, then the phenomenon of “up-regulation” of the insulin receptor may occur, which could account for increased insulin binding. Increased insulin binding would be considered a secondary or indirect action: nevertheless, it would contribute to increased insulin sensitivity in target tissues. Clearly, additional studies must be carried out, especially in patients with NIDDM to evaluate the relative beneficial effects of this interesting class of hypoglycemic agents. SUliMARY

Pieces of rat epididymal fat tissue were maintained in a biochemically defined medium for 20 to 44 hours in either the absence or presence of a sulfonylurea at levels known to be effective in humans. Prolonged exposure of adipocytes to sulfonylureas did not influence the number of insulin receptors or their affinity to insulin

7.

6.

9.

10.

Grodsky GM, Epstein GH, Fanska R, Karam JH: Pancreatic action of the sulfonylureas. Fed Proc 1972; 36: 27142719. Duckworth WD, Solomon SS, Kitabchi AE: Effect of chronic sutfonylwea therapy on plasma insulinand proinsulin levels. J Clin Endocrinol Metab 1972; 35: 585-591. Beck-Nielsen t-t. Pedersen 0. Lindskov HO Increased insulin sensitivity and cellular insulin binding in obese diabetics following treatment with glibenclamide. Acta Endocrinol 1979; 90: 451-462. Olefsky JM. Reaven GM: Effects of sulfonylurea therapy on insulin binding to mononuclear leukocytes of diabetic patients. Am J Med 1976; 60: 89-95. Feinglos MN. Lebovii HE: Sulfonylueas increase the nwnber of insulin receptors. Nature 1978; 276: 184-185. Feldman JM. Lebovitz HE: An insulin dependent effect of chronic tolbutamide administration on the skeletal muscle carbohydrate transport system. Diabetes 1969; 18: 8495. Maloff BL, Lockwood DH: In vitro effects of a sulfonylwea on insulin action in adipocytes: potentiation of insulin-stimulated hexose transport. J Clin Invest 1981; 68: 85-90. Smith U: Studies of human adipose tissue in culture. I. Incorporation of glucose and release of gfycerol. Anat Ret 1972: 172: 597-602. Rodbell MI: Metabolism of isolated fat cells. I. Effects of horm0ne.son glucose metabolism and lipolysis. J Biol Chem 1964; 239: 375-380. Aggarwal V. Sunshine I: Determination of sulfonylureas and metabolites by pyrolysis gas chromatography. Clin Chem 1974; 29: 200-224.

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or the ability of insulin to induce receptor loss (downregulation). Also, the sulfonylureas did not influence the basal uptake of the D-glucose analogs 2deoxyglucose and 3-O-methylglucose. However, exposure to these drugs resulted in a potentiation of the stimulatcry effects of insulin on hexose transport at submaximal and maximally effective concentrations of insulin. The average potentiation was N 30 % . In addition, sulfonylureas enhanced stimulation of hexose uptake by the insulin mimickers, hydrogen peroxide and vitamin Kg. These oxidants are known to manifest insulin-like actions subsequent to insulin binding. Under conditions in which glucose transport was rate limiting, the conversion of glucose to carbon dioxide and the total lipids mirrored the findings of hexose uptake. However, at a glucose concentration of 50 mM, at which hexose transport is no longer rate limiting, sulfonylureas did not potentiate metabolism in the absence or presence of insulin. These results may help to explain the hypoglycemic action of the drug in view of the recent finding that a postreceptor deficit is present in noninsulindependent diabetes mellitus. ACKNOWLEDGMENT We thank Lorraine Drake for the excellent assistance she gave us during our studies.

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technical

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sulin-sensitive lipogenesis by a sulfonylurea in rat hepatocytes. Diabetes 1982; 31 (suppl 2): 54A. Perley MJ, Kipnis DM: Plasma insulin responses to oral and intravenous glucose: studies in normal and diabetic subjects. J Clin Invest 1967; 46: 1954-1982. Reaven GM, Bernstein R, Davis B, Olefsky JM: Nonketotic diabetes mellitus: insulin deficiency or insulin resistance? Am J Med 1978; 60: 80-88. Kolterman OG. Gray RS, Griffin J, et al.: Receptor and postreceptor defects contribute to the insulin resistance in noninsulindependent diabetes mellitus. J Clin Invest 1981;

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