Changes In Insulin Binding Research Articles

Hepatic Insulin Clearance in Regulation of Systemic Insulin Concentrations-Role of Carbohydrate and Energy Availability.

Hyperinsulinemia is the hallmark of insulin resistance in obesity, and the relative importance of insulin clearance, insulin resistance, and insulin hypersecretion has been widely debated. On the basis of recent experimental evidence, we summarize existing evidence to suggest hepatic insulin clearance as a major and immediate regulator of systemic insulin concentrations responding within days to altered dietary energy and, in particular, carbohydrate intake. Hepatic insulin clearance seems to be closely associated with opposite alterations in hepatic lipid content and glucose production, providing a potential mechanistic link to hepatic insulin sensitivity. The molecular regulation of insulin clearance in the liver is likely to involve changes in insulin binding and receptor internalization in response to the dietary alterations, the molecular mechanisms of which await further research.

Diet- and diabetes-induced change in insulin binding to the nuclear membrane in spontaneously diabetic rats is associated with change in the fatty acid composition of phosphatidylinositol

Insulin binding to the nucleus in vivo alters the binding of transcription factors to the promoter region of lipogenic genes, thereby changing expression of these genes. The present research was designed to investigate whether change in diet fat composition alters insulin binding to nuclear insulin receptors at various stages of onset of diabetes in spontaneously diabetic B/B rats. The fatty acid composition of lipids comprising the nuclear membrane was also examined. Weanling rats were fed a nonpurified diet (low-fat commercial rat chow) or a semipurified diet containing 20 g/100 g fat of either high (1.0) or low (0.25) polyunsaturated to saturated (P/S) fatty acid ratio. Insulin binding to liver nuclei was measured when the blood glucose level was 100 mg/dl and 400 mg/dl. No effect of diet treatment on age of onset of diabetes was found. Specific binding of insulin to nuclei from rats with a blood glucose level of 100 mg/dl did not differ from nondiabetic rats, and was higher than in diabetic rats with a blood glucose level of 400 mg/dl. Insulin binding was greater in rats fed a high P/S diet. The high versus low P/S diet treatment primarily altered the fatty acid composition of phosphatidylinositol in the nuclear membrane. Diabetic rats fed nonpurified diet showed a significant increase in levels of 18:2(n-6) and 22:6(n-3), whereas 20:4(n-6) decreased in the phosphatidylcholine fraction compared with control rats fed chow. As rats became diabetic, the level of monounsaturated fatty acids, 18:2(n-6) and 22:6(n-3) decreased, whereas the level of 20:4(n-6) increased in phosphatidylinositol. Change in the composition of these nuclear membrane components may be associated with transitions in insulin binding.

Stimulation of glucose transport by thyroid hormone in 3T3-L1 adipocytes: increased abundance of GLUT1 and GLUT4 glucose transporter proteins.

In 3T3-L1 adipocytes we have examined the effect of tri-iodothyronine (T(3)) on glucose transport, total protein content and subcellular distribution of GLUT1 and GLUT4 glucose transporters. Cells incubated in T(3)-depleted serum were used as controls. Cells treated with T(3) (50 nM) for three days had a 3.6-fold increase in glucose uptake (P<0.05), and also presented a higher insulin sensitivity, without changes in insulin binding. The two glucose carriers, GLUT1 and GLUT4, increased by 87% (P<0.05) and 90% (P<0. 05), respectively, in cells treated with T(3). Under non-insulin-stimulated conditions, plasma membrane fractions obtained from cells exposed to T(3) were enriched with both GLUT1 (3. 29+/-0.69 vs 1.20+/-0.29 arbitrary units (A.U.)/5 microg protein, P<0.05) and GLUT4 (3.50+/-1.16 vs 0.82+/-0.28 A.U./5 microg protein, P<0.03). The incubation of cells with insulin produced the translocation of both glucose transporters to plasma membranes, and again cells treated with T(3) presented a higher amount of GLUT1 and GLUT4 in the plasma membrane fractions (P<0.05 and P<0.03 respectively). These data indicate that T(3) has a direct stimulatory effect on glucose transport in 3T3-L1 adipocytes due to an increase in GLUT1 and GLUT4, and by favouring their partitioning to plasma membranes. The effect of T(3) on glucose uptake induced by insulin can also be explained by the high expression of both glucose transporters.

Insulin resistance in limb and trunk partial lipodystrophy (type 2 Köbberling-Dunnigan syndrome)

We studied insulin action in two patients with limb and trunk partial lipodystrophy with hirsutism and acanthosis nigricans. Glucose was normal in one of the patients and slightly above normal in the other during an oral glucose tolerance test (OGTT). An intravenous glucose tolerance test (IVGTT) was normal in both patients. Basal and glucose-stimulated insulin levels were elevated in both the OGTT and IVGTT in both patients. The response of plasma glucose to exogenously administered insulin was decreased. A euglycemic-hyperinsulinemic clamp performed in patient no. 2 indicated insulin resistance, which was not corrected by reducing the increased basal level of serum free fatty acids (FFAs). Binding of insulin to neck adipocytes was normal in both subjects, but glucose transport and oxidation in these cells was impaired. Insulin binding to abdominal adipocytes was increased in one patient whose adipocytes displayed higher glucose transport at low insulin concentrations. Glucose oxidation was decreased in abdominal adipocytes of both patients. We conclude that insulin resistance in Köbberling-Dunnigan type 2 partial lipodystrophy is not related to an alteration of the insulin molecule or to changes in insulin binding, but is more likely associated with a postreceptor defect, since glucose oxidation was impaired in adipocytes of the neck and abdomen.

Insulin receptor autophosphorylation and signaling is altered by modulation of membrane physical properties.

Many membrane functions are modulated by the bulk biophysical properties of the membrane. Various compounds which alter membrane physical properties were investigated for their ability to modulate insulin receptor autophosphorylation and signaling. Compounds which raise the bilayer to hexagonal phase transition temperature in model membranes, including carbobenzoxydipeptides, apolipoprotein A-I, acyl carnitines, and lysophosphatidylcholine, inhibited insulin stimulation of insulin receptor tyrosine phosphorylation of isolated receptors as well as in cells overexpressing human insulin receptor. For compounds of similar structure, the inhibition of insulin receptor tyrosine phosphorylation correlates well with their bilayer-stabilizing potency. Most of the compounds which inhibit tyrosine phosphorylation of the insulin receptor also inhibited glucose uptake in the same cells. Compounds which lower the bilayer to hexagonal phase transition temperature in model membranes enhanced insulin stimulation of autophosphorylation in isolated receptors, with no effect on insulin receptor activity in NIH 3T3 HIR 3.5 cells. The effects of cationic amphiphiles were not readily predictable from their membrane modulating activity. All of the compounds tested exert their effects independent of changes in insulin binding to the receptor or changes in the basal tyrosine kinase activity of the receptor. This provides evidence that mechanism of modulation of insulin signaling by these additives lies in their ability to alter the bulk physical properties of the membrane. The results suggest that membrane monolayer curvature strain is a factor contributing to the efficiency of insulin signal transduction.

Development of Ligand-Induced Down-Regulation and Endocytosis of Hepatic Insulin Receptors at Weaning in the Rat

In the present study, we have analyzed the changes in insulin binding to liver subcellular fractions which occur at weaning and assessed whether such changes are insulin-related. We have also examined, in suckling and weaned rats, the ability of an acute hyperinsulinemia to induce the endocytosis of the insulin receptor and its ligand

The inhibition of insulin action and glucose metabolism by porcine growth hormone in porcine adipocytes is not the result of any decrease in insulin binding or insulin receptor kinase activity

The present study was undertaken to determine the effects of porcine growth hormone (pGH) on glucose transport, to establish which lipogenic enzymes were affected by pGH, and to determine if changes in insulin binding or insulin receptor kinase activity contributed to the diminished insulin responsiveness of adipocytes from pigs treated with pGH. Pigs were treated with pGH daily (70 micrograms/kg body wt.) for 7 days. pGH treatment reduced the basal (non-insulin-stimulated) glucose transport rate by 62% and the insulin-stimulated transport rate by 47%. The decline in glucose transport rate was paralleled by a 64% decrease in fatty acid synthesis. The reduction in the lipogenic rate was associated with a marked decline in the activity of several lipogenic enzymes: glucose-6-phosphate dehydrogenase (50% decrease), 6-phosphogluconate dehydrogenase (11% decrease), malic enzyme (62% decrease) and fatty acid synthase (activity not detectable after pGH treatment). The pGH-dependent decline in insulin responsiveness was not associated with any change in the binding of insulin to intact adipocytes or to plasma membrane preparations. The insulin-stimulated tyrosine kinase activity of the wheat-germ agglutinin-purified receptors from pGH-treated adipocytes was not different from that in control adipocytes, except when high concentrations of insulin were employed. These findings establish that pGH elicits a number of metabolic effects in porcine adipocytes which collectively diminish the rate of lipid synthesis, and thereby contribute to the decrease in lipid deposition observed in pGH-treated pigs. Furthermore, the pGH-dependent impairment in insulin action appears to be mediated at some location distal to the receptor kinase step or in other signal pathway(s) which mediate the biological effects of insulin that are not dependent on activation of insulin receptor tyrosine kinase activity.

Effect of Metformin on Carbohydrate and Lipoprotein Metabolism in NIDDM Patients

The effect of metformin treatment on various aspects of carbohydrate and lipoprotein metabolism has been defined in 12 patients with non-insulin-dependent diabetes mellitus (NIDDM). Patients were studied before and after approximately 4 mo of metformin therapy. Treatment was initiated with a single dose of 500 mg/day, increased at weekly intervals, and maintained at a final dose of 2.5 g/day (given at divided intervals) for the last 3 mo of the treatment program. Results demonstrated that both fasting and postprandial glucose concentrations were significantly lower after metformin administration, with the greatest change seen after meals. As a result, the total incremental plasma glucose response above basal measured from 0800 to 1600 after metformin was less than 25% of that seen initially. The improvement in ambient plasma glucose concentration in association with metformin occurred despite a modest but statistically significant decrease in circulating plasma insulin concentration. In addition, insulin-stimulated glucose uptake measured during hyperinsulinemic clamp studies was similar before and after metformin treatment. Furthermore, changes in insulin binding and insulin internalization by isolated monocytes did not correlate with the improvement in glycemic control. Thus, the ability of metformin to lower plasma glucose concentration in NIDDM does not appear to be secondary to an improvement in insulin action. Finally, metformin treatment was associated with a significant (P less than 0.01) decrease in plasma triglyceride concentration and an increase in plasma high-density lipoprotein cholesterol concentration. These results indicate that metformin treatment of patients with NIDDM led to an improvement in both glycemic control and lipoprotein metabolism.

Carbohydrate metabolism in skeletal muscle: an update of current concepts.

We have reviewed carbohydrate metabolism in skeletal muscle with an emphasis on recent information. In this review a significant number of points have been made. These are summarized below. 1. CP and glycogen are concomitantly metabolized during short-term (less than or equal to 10 s) intense exercise. 2. Both epinephrine and contractile activity regulate glycogen use in muscle. 3. Glycogen sparing during exercise is promoted by fatty acids and probably glucose. 4. Glycogenesis increases during exercise in exercising muscle. 5. Ingested glucose is easily metabolized during exercise. 6. The heterogeneity in glucose uptake among muscles at rest and during exercise is likely not caused by blood flow differences per se. 7. Insulin binding, glucose transporters, glucose uptake, and glycogenesis are greater in ST than in FT muscles. 8. Acute changes in glucose metabolism are not always attributable to concomitant changes in insulin binding to its receptor. 9. Contractile activity alone will increase glucose uptake in muscle, and insulin is not required. 10. Insulin and contractile effects on glucose uptake are additive, suggesting that these stimuli mobilize different pools of glucose transporters. 11. Glycogen loss occurs in exercising and non-exercising muscle; therefore this substrate is not an appropriate index of muscle contractile activity. 12. Carbohydrate mobilization does not appear to be strictly determined by need for this substrate nor by the rate of muscle metabolism. 13. Glyconeogenesis from lactate occurs in ST and FT muscles and is regulated by pH.

Mechanism of Insulin Resistance Induced by Sustained Levels of Cytosolic Free Calcium in Rat Adipocytes*

We have recently provided evidence that elevated levels of cytosolic free Ca2+ ([Ca2+]i) decreased insulin-stimulated glucose uptake in isolated rat adipocytes. To investigate the mechanism of Ca2+ action, we examined the effects of elevated levels of [Ca2+]i on insulin binding, autophosphorylation, and tyrosine kinase activity (TKA) of insulin receptors as well as basal and insulin-stimulated cellular distribution of glucose transporters. The latter was assessed by cytochalasin-B binding to plasma membrane and cytosolic fractions. Elevated concentrations of [Ca2+]i were maintained by incubating adipocytes with a depolarizing concentration of K+ (40 mM). Basal nonstimulated glucose uptake was not altered by increased levels of [Ca2+]i. Adipocytes with higher [Ca2+]i (220 +/- 15 nM) showed 30% reduction in insulin-stimulated 2-deoxyglucose uptake compared with control cells ([Ca2+]i, 140 +/- 18 nM). Moreover, adipocytes with higher levels of [Ca2+]i demonstrated an approximately 10% reduction in autophosphorylation and TKA of insulin receptors without a change in insulin binding. Both basal and insulin-stimulated distributions of glucose transporters were unaffected by sustained levels of [Ca2+]i. The effects of elevated [Ca2+]i were not mimicked by protein kinase-C activation. These observations suggest that 1) elevated or sustained levels of [Ca2+]i impair insulin-stimulated glucose uptake; and 2) Ca2+-induced impairment appears to reside at the postbinding steps of insulin action and probably interferes with the TKA of insulin receptors and the intrinsic activity of glucose transporters.

Reduction of insulin binding in the arcuate nucleus of the rat hypothalamus after 6-hydroxydopamine treatment

Insulin receptors are present in the hypothalamus, but the cell types bearing them are unknown. In order to test the hypothesis that some insulin receptors in the hypothalamus are associated with catecholamine terminals, rats were injected with 50 μg or 75 μg doses (intracerebroventricular) of 6-hydroxydopamine (6-OHDA). Control rats received vehicle only. The animals were sacrificed 7 days after injection, and catecholamine and indolamine levels in the hypothalamus were measured by high performance liquid chromatography with electrochemical detection. Localization of specific binding sites for [ 125I]-insulin in the arcuate (ARC), dorsomedial (DMN) and ventromedial (VMN) nuclei were determined by quantitative film autoradiography. Treatment with 6-OHDA resulted in a 70% reduction in hypothalamic norepinephrine content as compared to vehicle-treated controls ( P < 0.01). A slight depletion of epinephrine, dopamine and indolamines was also detected. Computerized image analysis of the autoradiograms was used to determine radioactivity bound (DPM/mm 2) in each nucleus. Highest binding was in the ARC and DMN, with much lower binding in the VMN. Insulin binding in the ARC of the 6-OHDA-treated group was decreased by 25% compared to controls ( P < 0.01). No significant change in insulin binding was observed in the DMN or VMN. The 6-OHDA treatment had no significant effect on weight gain or on plasma insulin levels. The reduction of insulin binding in the ARC after 6-OHDA treatment supports the hypothesis that some insulin binding sites are located on catecholamine terminals in the arcuate nucleus.

Decreased insulin binding in Asian Indian women with gestational diabetes mellitus.

Insulin binding to erythrocyte insulin receptors was studied in 10 women with gestational diabetes and compared with 10 matched, normal, pregnant women and 10 normal, non-pregnant controls, with no family history of diabetes. Pregnant women had higher mean fasting and post-glucose plasma immunoreactive insulin (IRI) compared to non-pregnant controls (p less than 0.001). Women with gestational diabetes had higher mean fasting and post-glucose plasma glucose levels and a lower mean specific binding of insulin when compared with the other two groups (p less than 0.001). The decreased insulin binding was significant only at lower insulin concentrations (0.2-2 ng/ml) when compared with those of normal pregnant women (p less than 0.01), suggesting decreased receptor affinity with no change in receptor number. In addition, an increased mean ED50 value for 50% inhibition of maximal binding and a lower mean average affinity constant Ke (empty site) obtained in gestational diabetes in comparison to the other two groups also suggested de decreased affinity of the receptor. The finding that pregnancy with normal glucose tolerance was not accompanied by changes in insulin binding against decreased insulin binding and affinity observed in gestational diabetes suggested a pathogenetic role for impaired insulin binding as one of the factors responsible for insulin resistance and hyperglycemia in gestational diabetes.

Effects of a long-lasting hyperinsulinemia induced by insulin infusion on the subcellular distribution of liver insulin receptors in the rat.

Acute hyperinsulinemia in rats have been shown to cause enhanced endocytosis of liver insulin receptors with little or no change in the total receptor number. To determine whether a similar phenomenon occurs in long-lasting hyperinsulinemia, the subcellular distribution of liver insulin receptors has been studied in rats infused continuously with insulin (0.4 and 0.2 U/h) for 4 days. In rats in which plasma insulin concentration was maintained at 15-20 ng/ml, there was, from 3 to 24 h, a 2-fold decrease in insulin binding to plasma membranes (PM), along with 2 to 4-fold increase in insulin binding to the light (GEI), intermediate (GEi) and heavy (GEh) Golgi-endosomal fractions; concomitantly, there was a 10-fold increase in the insulin content of Golgi-endosomal fractions. After 24 h, the changes in insulin binding to PM and GEI were maintained, but the increase in both insulin binding activity and insulin content of GEi and GEh became progressively less marked, although plasma insulin concentration remained elevated. Throughout infusion, insulin binding to the total particulate fraction was unchanged. In rats, in which plasma insulin was maintained at 6-8 ng/ml, insulin binding to PM was decreased to a lesser degree and insulin binding to Golgi-endosomal fractions was unchanged (GEh) or decreased (GEI and GEi), although the insulin content of these fractions remained high. These results suggest that, while an enhanced receptor endocytosis accounts for the decrease in cell surface receptors observed at an early stage of the hyperinsulinemia, additional regulatory mechanisms are probably involved at a later stage.

Role of gestational hormones in the induction of insulin resistance.

Pregnancy is associated with insulin resistance. We studied insulin binding and postbinding function in isolated adipocytes from pregnant and nonpregnant rats. We also used a primary culture system for female virgin rat adipocytes to assess the effects of gestational hormones in vitro on insulin binding and postbinding function. Insulin binding to adipocytes was normal during pregnancy, but [14C]3-O-methylglucose transport was reduced. When hCG or estradiol was added to the culture medium, no change in maximum [14C]3-O-methylglucose transport was found; however, maximum insulin binding was increased with estradiol. Progesterone and cortisol both decreased maximum insulin binding and [14C]3-O-methylglucose transport. PRL and placental lactogen decreased maximum [14C]3-O-methylglucose transport, but did not change insulin binding. When these hormones were added concurrently no change in insulin binding was found, but maximum [14C]3-O-methylglucose transport was reduced. We conclude that the insulin resistance of pregnancy is associated with a postbinding defect in insulin action. Estradiol increased insulin receptor binding, but during pregnancy this effect may be offset by the reduction in insulin binding induced by progesterone and cortisol. The postbinding defect in insulin action during pregnancy is probably related to increasing amounts of progesterone, cortisol, PRL, and placental lactogen.

Dietary fat and the diabetic state alter insulin binding and the fatty acyl composition of the adipocyte plasma membrane

Control and diabetic rats were fed on semi-purified high-fat diets providing a polyunsaturated/saturated fatty acid ratio (P/S) of 1.0 or 0.25, to examine the effect of diet on the fatty acid composition of major phospholipids of the adipocyte plasma membrane. Feeding the high-P/S diet (P/S = 1.0) compared with the low-P/S diet (P/S = 0.25) increased the content of polyunsaturated fatty acids in membrane phospholipids in both control and diabetic animals. The diabetic state decreased the content of polyunsaturated fatty acids, particularly arachidonic acid, in adipocyte membrane phospholipids. The decrease in arachidonic acid in membrane phospholipids of diabetic animals tended to be normalized to within the control values when high-P/S diets were given. For control animals, altered plasma-membrane composition was associated with change in insulin binding, suggesting that change in plasma-membrane composition may have physiological consequences for insulin-stimulated functions in the adipocyte.

Developmental aspects of the rat brain insulin receptor: loss of sialic acid and fluctuation in number characterize fetal development.

In this study, I have investigated the structure of the rat brain insulin receptor during fetal development. There is a progressive decrease in the apparent molecular size of the brain alpha-subunit during development: 130K on day 16 of gestation, 126K at birth, and 120K in the adult. Glycosylation was investigated as a possible reason for the observed differences in the alpha-subunit molecular size. The results show that the developmental decrease in the brain alpha-subunit apparent molecular size is due to a parallel decrease in sialic acid content. This was further confirmed by measuring the retention of autophosphorylated insulin receptors on wheat germ agglutinin (WGA)-Sepharose. An inverse correlation between developmental age and retention of 32P-labeled insulin receptors on the lectin column was observed. Insulin binding increases 6-fold between 16 and 20 days of gestation [61 +/- 25 (+/- SE) fmol/mg protein and 364 +/- 42 fmol/mg, respectively]. Thereafter, binding in brain membranes decreases to 150 +/- 20 fmol/mg by 2 days after birth, then reaches the adult level of 63 +/- 15 fmol/mg. In addition, the degree of insulin-stimulated autophosphorylation closely parallels the developmental changes in insulin binding. Between 16 and 20 days of fetal life, insulin-stimulated phosphorylation of the beta-subunit increases 6-fold. Thereafter, the extent of phosphorylation decreases rapidly, reaching adult values identical with those in 16-day-old fetal brain. These results suggest that the embryonic brain possesses competent insulin receptors whose expression changes markedly during fetal development. This information should be important in defining the role of insulin in the developing nervous system.

Effect of exercise on insulin binding and glucose transport in adipocytes of normal humans.

Acute exercise increases insulin binding to its receptors on blood cells. Whether the enhanced insulin binding explains the exercise-induced increase in glucose uptake is unclear, since insulin binding and glucose uptake have not been measured simultaneously in a target tissue of insulin. In this study, we determined insulin binding and the rate of glucose transport in adipocytes obtained by needle biopsy from 10 healthy men before and after 3 h of cycle-ergometric exercise. During the exercise, plasma glucose (P less than 0.01) and insulin (P less than 0.001) fell and serum free fatty acid level rose 4.3-fold (P less than 0.001). 125I-insulin binding to adipocytes remained unchanged during exercise. The rate of basal glucose transport clearance fell from 28.1 +/- 5.7 fl.cell-1.s-1 to 22.9 +/- 5.6 fl.cell-1.s-1 (P less than 0.005), and the insulin-stimulated increase in glucose transport rate rose from 196 +/- 26 to 279 +/- 33% (P less than 0.025) during the exercise. Thus, in the adipocytes during exercise, the basal glucose transport rate and the responsiveness of glucose transport to insulin changed in the absence of alterations in insulin binding. These data indicate that the exercise-induced changes in insulin binding show tissue specificity and do not always parallel alterations in glucose transport.

Dissociation of in vitro sensitivities of glucose transport and antilipolysis to insulin in NIDDM.

It is unclear from previous studies whether qualitative or only quantitative differences exist in insulin action in adipocytes obtained from obese subjects with non-insulin-dependent diabetes mellitus (NIDDM) when compared with equally obese nondiabetic subjects. In addition, the role of changes in insulin binding as a cause of insulin resistance in NIDDM is still controversial. We compared the sensitivities of glucose transport and antilipolysis to insulin and measured insulin binding in abdominal adipocytes obtained from 45 obese nondiabetic (% fat, 41 +/- 1), 25 obese diabetic (% fat, 40 +/- 1), and 15 nonobese (% fat, 30 +/- 1) female southwestern American Indians. Compared with the nonobese group, the sensitivities of glucose transport and antilipolysis were reduced in both the obese nondiabetic and obese diabetic groups. Compared with the obese nondiabetic subjects, the ED50 for stimulation of glucose transport was higher in the obese patients with NIDDM (171 +/- 38 vs. 92 +/- 10 pM, P less than 0.005). In contrast, the ED50s for antilipolysis were similar in obese diabetic patients (32 +/- 6 pM) and obese nondiabetic subjects (27 +/- 3 pM). No difference was found in insulin binding in patients with NIDDM when compared with the equally obese nondiabetic subjects. These data indicate 1) the mechanism of insulin resistance differs in NIDDM and obesity, and 2) the selective loss of insulin sensitivity in NIDDM precludes changes in insulin binding as a cause of insulin resistance in this disorder.

Effect of cyclic AMP-dependent protein kinase on insulin receptor tyrosine kinase activity.

To explain the insulin resistance induced by catecholamines, we studied the tyrosine kinase activity of insulin receptors in a state characterized by elevated noradrenaline concentrations in vivo, i.e. cold-acclimation. Insulin receptors were partially purified from brown adipose tissue of 3-week- or 48 h-cold-acclimated mice. Insulin-stimulated receptor autophosphorylation and tyrosine kinase activity of insulin receptors prepared from cold-acclimated mice were decreased. Since the effect of noradrenaline is mediated by cyclic AMP and cyclic AMP-dependent protein kinase, we tested the effect of the purified catalytic subunit of this enzyme on insulin receptors purified by wheat-germ agglutinin chromatography. The catalytic subunit had no effect on basal phosphorylation, but completely inhibited the insulin-stimulated receptor phosphorylation. Similarly, receptor kinase activity towards exogenous substrates such as histone or a tyrosine-containing copolymer was abolished. This inhibitory effect was observed with receptors prepared from brown adipose tissue, isolated hepatocytes and skeletal muscle. The same results were obtained on epidermal-growth-factor receptors. Further, the catalytic subunit exerted a comparable effect on the phosphorylation of highly purified insulin receptors. To explain this inhibition, we were able to rule out the following phenomena: a change in insulin binding, a change in the Km of the enzyme for ATP, activation of a phosphatase activity present in the insulin-receptor preparation, depletion of ATP, and phosphorylation of a serine residue of the receptor. These results suggest that the alteration in the insulin-receptor tyrosine kinase activity induced by cyclic AMP-dependent protein kinase could contribute to the insulin resistance produced by catecholamines.

Effect of chlorpropamide on glucose transport in rat adipocytes in the absence of changes in insulin binding and receptor-associated tyrosine kinase activity

In an attempt to elucidate the cellular mechanism(s) by which sulfonylureas exert their extrapancreatic hypoglycemic effects, various parameters of insulin action were examined in vitro, using rat adipocytes maintained in a biochemically defined medium. Cells were maintained for 20 hours in the absence or presence of 175 μg/mL chlorpropamide and insulin binding, hexose transport, glucose metabolism, and insulin receptor tyrosine kinase activity were compared. Chlorpropamide treatment had no effect on insulin binding, altering neither receptor number nor affinity. However, the sulfonylurea did enhance 2-deoxyglucose transport in both the absence (17%, P < .01) and presence (20%, P < .01) of insulin. Furthermore, glucose metabolism as measured by the conversion of glucose (0.2 mmol/L) to CO 2 and total lipids was also significantly increased by chlorpropamide treatment in both the absence (30%, P < .01) and presence (31%, P < .05) of insulin. Potentiation of insulin-stimulated transport or metabolism was not explained by an increase in the basal state alone because the incremental responses to 40 ng/mL insulin were potentiated by 19% ( P < .01) and 25% ( P < .05), respectively. Activity of the insulin receptor kinase was unchanged as evaluated by autophosphorylation of partially purified receptors, phosphorylation of an artificial substrate and by phosphorylation of the receptor in situ. These studies demonstrate that the sulfonylurea, chlorpropamide, stimulates glucose transport and potentiates insulin's effect on this process by acting at a site(s) beyond insulin receptor binding and phosphorylation.