The effects of glucocorticoids on insulin-stimulated lipogenesis in primary cultures of rat hepatocytes.

We used primary cultures of rat hepatocytes to evaluate the effects of glucocorticoids on insulin-responsive hepatic lipogenesis. The data indicate that hepatocytes incubated for 20 h with dexamethasone (0.1 microM) alone are profoundly resistant to the ability of insulin to stimulate lipogenesis acutely. In contrast, primary cultures of hepatocytes incubated with dexamethasone plus insulin are hyper-responsive to the ability of insulin to stimulate lipogenesis chronically. This potentiation of insulin action by a glucocorticoid occurs at physiological concentrations of the two hormones. Exposure to dexamethasone plus insulin for more than 4 h is required for the two hormones to enhance insulin action either by overcoming the insulin resistance induced by dexamethasone alone or by stimulating insulin action induced by insulin alone. Despite the marked potentiation of insulin action, hepatocytes exposed to dexamethasone plus insulin are less sensitive to insulin, as demonstrated by a shift to the right in the dose-response curve for insulin-stimulated lipogenesis. The resistance of hepatocytes to the acute effects of insulin after exposure to dexamethasone alone and the potentiation of insulin action and decreased sensitivity to insulin after exposure to insulin plus dexamethasone are all mediated by post-insulin-binding events. These studies demonstrate potentiation of insulin action in the liver by physiological concentrations of glucocorticoids and may have physiological significance for the regulation of normal hepatic lipogenesis, for the hyperlipidaemia observed with the pharmacological use of glucocorticoids, and for disease states in man associated with hyperinsulinaemia and hypercortisolism.     

Effect of T3 on insulin action, insulin binding, and insulin receptor kinase activity in primary cultures of rat hepatocytes

In vivo studies have demonstrated that the liver is the main site of insulin resistance in hyperthyroidism. To further investigate the effect of thyroid hormone in the liver, we have incubated primary cultures of rat hepatocytes in the presence and absence of triiodothyronine (T3) 1 ng/ml and 5 ng/ml for 20 hr. Without affecting basal activity, T3 5 ng/ml decreased insulin-stimulated (1 x 10(-7) M) lipid synthesis but not insulin-stimulated alpha-aminoisobutyric acid uptake. These changes occur in the absence of any abnormalities in 125I-insulin binding, degradation, internalization or insulin receptors structure as determined by affinity-labeling methods. However, basal insulin receptor kinase activity using Glu4: Tyrl as phospho-acceptor was decreased by T3 without altering its insulin responsiveness. These results demonstrate the heterogeneity of T3's effects at the postinsulin binding level in the liver.     

Regulation of insulin binding and glycogenesis by insulin and dexamethasone in cultured rat hepatocytes

Regulation of insulin-binding and basal (insulin-independent) as well as insulin-stimulated glycogen synthesis from [14C]glucose, net glycogen deposition and glycogen synthase activation by insulin and dexamethasone were studied in primary cultures of adult rat hepatocytes maintained under chemically defined conditions. Insulin receptor number was increased in a dose-dependent fashion by dexamethasone added to the medium between 24 and 48 h of culture and reduced by insulin, whereas ligand affinity remained unaltered. Insulin-induced down-regulation of insulin receptors was not affected by the glucocorticoid. Although the changes in the sensitivity to insulin of glycogen synthesis from glucose and net glycogen deposition paralleled the modulation of the number of insulin receptors, postbinding events appear to be implicated also in the regulation of insulin-sensitivity. Alterations of the responsiveness of glycogen synthesis to insulin caused by the glucocorticoid and/or insulin and by variation between individual rats were inversely related to cellular glycogen contents, suggesting that hepatocellular glycogen content participates in the regulation of insulin-responsiveness of this metabolic pathway. Regulation of insulin-dependent glycogen synthesis were different. Since the effects of this 'physiological' increase in exogenous glucose were small compared to the acute action of insulin, insulin rather than portal venous glucose is considered to represent the prime stimulator of hepatic glycogen synthesis.     

Diacylglycerols modulate insulin action in rat adipocytes

Effect of 1,2-diacylglycerols on the insulin receptor function and insulin action in rat adipocytes was studied. 1,2-dioctanoylglycerol (100 micrograms/ml) did not alter insulin binding but it did stimulate phosphorylation of the beta-subunit of the insulin receptor as well as its tyrosine kinase activity. However, dioctanoylglycerol inhibited insulin-stimulated receptor autophosphorylation. This concentration of dioctanoylglycerol inhibited insulin-stimulated CO2 metabolism, lipogenesis and 3-O-methyl-glucose transport in a dose-dependent manner but did not alter any of these bioeffects in absence of insulin. While there was no direct link between diacylglycerol effect on tyrosine kinase activity of the insulin receptor and insulin action in rat adipocytes, the parallel inhibition of insulin-stimulated receptor autophosphorylation and insulin bioeffects by dioctanoylglycerol suggests its direct or indirect role in insulin signalling in rat fat cells.     

Development of insulin sensitivity in white adipose tissue during the suckling-weaning transition in the rat. Involvement of glucose transport and lipogenesis.

The changes of insulin responsiveness of white adipose tissue during the suckling-weaning transition in the rat were investigated in vitro on isolated adipocytes. Insulin binding, glucose transport and glucose metabolism in adipocytes from suckling rats and from rats weaned on to a high-carbohydrate (HC) or a high-fat (HF) diet were compared. Despite similar insulin binding, insulin-stimulated glucose transport rate is lower in adipocytes from suckling rats and HF-weaned rats than in adipocytes from HC-weaned rats. Moreover, whereas insulin markedly stimulates glucose metabolism in adipocytes from HC-weaned rats, glucose metabolism is totally unresponsive to insulin in adipocytes from suckling and HF-weaned rats. This insulin resistance is associated with a very low rate of lipogenesis and low activities of acetyl-CoA carboxylase, fatty acid synthase and pyruvate dehydrogenase.     

The adipokine sFRP4 induces insulin resistance and lipogenesis in the liver

Secreted frizzled-related protein (sFRP) 4 is an adipokine with increased expression in white adipose tissue from obese subjects with type 2 diabetes and non-alcoholic fatty liver disease (NAFLD). Yet, it is unknown whether sFRP4 action contributes to the development of these pathologies. Here, we determined whether sFRP4 expression in visceral fat associates with NAFLD and whether it directly interferes with insulin action and lipid and glucose metabolism in primary hepatocytes and myotubes. The association of sFRP4 with clinical measures was investigated in obese men with or without type 2 diabetes and with or without biopsy-proven NAFLD. To determine the impact of sFRP4 on metabolic parameters, primary human myotubes (hSkMC), or primary hepatocytes from metabolic healthy C57Bl6 and from systemic insulin-resistant mice, i.e. aP2-SREBP-1c, were used. Gene expression of sFRP4 in visceral fat from obese men associated with insulin sensitivity, triglycerides and NAFLD. In C57Bl6 hepatocytes, sFRP4 disturbed insulin action. Specifically, sFRP4 decreased the abundance of IRS1 and FoxO1 together with impaired insulin-mediated activation of Akt-signalling and glycogen synthesis and a reduced suppression of gluconeogenesis by insulin. Moreover, sFRP4 enhanced insulin-stimulated hepatic de novo lipogenesis (DNL). In hSkMC, sFRP4 induced glycolysis rather than inhibiting insulin signalling. Finally, in hepatocytes from aP2-SREBP-1c mice, sFRP4 potentiates existing insulin resistance. Collectively, we show that sFRP4 interferes with hepatocyte insulin action. Physiologically, sFRP4 promotes DNL in hepatocytes and glycolysis in myotubes. These sFRP4-mediated responses may result in a vicious cycle, in which enhanced rates of DNL and glycolysis aggravate hepatic lipid accumulation and insulin resistance.     

Insulin action and binding in isolated hepatocytes from fasted, streptozotocin-diabetic, and older, spontaneously obese rats

Insulin binding and basal and insulin-stimulated uptake of α-aminoisobutyric acid were measured in isolated hepatocytes from young control rats as well as from older spontaneously obese, 72h-starved, and nonketotic streptozotocin-diabetic rats. Isolated hepatocytes from older spontaneously obese rats are similar to those from younger smaller rats in size, maximal insulin responsiveness, the dose–response relationship for insulin-stimulated aminoisobutyrate uptake, and the number and affinity of insulin receptors. Hepatocytes from 72h-fasted rats have similar numbers of insulin receptors per cell as cells from young control animals, but are significantly smaller, have an enhanced basal rate of aminoisobutyrate uptake, and are insulin resistant with regard to maximal insulin-stimulated uptake of aminoisobutyrate at 0.1mm-aminoisobutyrate. Because of the decreased maximal response to insulin, the concentration of insulin that elicits a half-maximal response of aminoisobutyrate uptake is decreased. Hepatocytes from diabetic animals, like those from starved rats, have significantly greater basal rates of aminoisobutyrate uptake; whereas the maximal absolute insulin response is the same as control cells, the percentage response is smaller. These cells bind significantly more insulin than do control cells. The increase in insulin binding is reflected in a shift to the left of the dose–response curve for insulin-stimulated uptake of aminoisobutyrate. These studies indicate that there is no insulin resistance with regard to uptake of aminoisobutyrate in hepatocytes from older obese rats. Furthermore, the insulin resistance observed in hepatocytes from starved rats occurs despite an increase in the number of receptors per unit surface area and cannot be explained by alterations in the interaction between insulin and its receptor. The enhanced insulin binding per unit surface area, however, is reflected in the shift to the left of the dose–response curve for insulin. This is also true for hepatocytes from diabetic animals, in which insulin binding per cell is increased.     

Ethanol modulation of the hormonal and nutritional regulation of glucose 6-phosphate dehydrogenase activity in primary cultures of rat hepatocytes.

The hormonal and nutritional regulation of glucose 6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) was studied in primary cultures of rat hepatocytes maintained in a chemically defined medium. Inoculation of hepatocytes from starved rats into primary cultures resulted in a 4-5-fold increase in G6PDH activity in 48 h in the absence of hormones. Parallel cultures treated simultaneously with glucocorticoids and insulin exhibited a 12-15-fold increase during the same time. Glucocorticoids by themselves did not elevate G6PDH activity, whereas insulin alone significantly stimulated enzyme activity. Thus the glucocorticoids acted in a 'permissive' role to amplify the insulin stimulation of G6PDH. Elevated concentrations of glucose in the culture medium increased enzyme activity in both the control cultures and those treated with hormones. Ethanol was found to potentiate G6PDH activity in cultures treated with glucocorticoids and insulin. The effect of ethanol was time- and dose-dependent. These results establish that insulin, glucocorticoids, glucose and ethanol interact in some undefined manner to regulate hepatic G6PDH activity.     

Regulation of glycogen metabolism in primary cultures of rat hepatocytes. Restoration of acute effects of glucose in cells from diabetic rats involves protein synthesis

Defective acute regulation of hepatic glycogen synthase by glucose and insulin, caused by severe insulin deficiency, can be corrected in adult rat hepatocytes in primary culture by inclusion of insulin, triiodothyronine, and cortisol in a chemically defined serum-free culture medium over a 3-day period (Miller, T. B., Jr., Garnache, A. K., Cruz, J., McPherson, R. K., and Wolleben, C. (1986) J. Biol. Chem. 261, 785-790). Using primary cultures of hepatocytes isolated from normal and diabetic rats in the same serum-free chemically defined medium, the present study addresses the effects of cycloheximide and actinomycin D on the chronic actions of insulin, triiodothyronine, and cortisol to facilitate the direct effects of glucose on the short-term activation of glycogen synthase. The short-term presence (1 h) of the protein synthesis blockers had no effect on acute activation of glycogen synthase by glucose in primary hepatocyte cultures from normal rats. Normal cells maintained in the presence of cycloheximide or actinomycin D for 2 and 3 days exhibited unimpaired responsiveness to glucose activation of synthase. The protein synthesis inhibitors were effective at blocking the restoration of glucose activation of synthase in diabetic cells in media which restored the activation in their absence. Restoration of glycogen synthase phosphatase activity by insulin, triiodothyronine, and cortisol in primary cultures of diabetic hepatocytes was also blocked by cycloheximide or actinomycin D. These data clearly demonstrate that restoration of acute glycogen synthase activation by glucose and restoration of glycogen synthase phosphatase activity in primary cultures of hepatocytes from adult diabetic rats are dependent upon the synthesis of new protein.     

Regulation of insulin binding and glycogenesis by insulin and dexamethasone in cultured rat hepatocytes

Regulation of insulin-binding and basal (insulin-independent) as well as insulin-stimulated glycogen synthesis from [¹⁴C]glucose, net glycogen deposition and glycogen synthase activation by insulin and dexamethasone were studied in primary cultures of adult rat hepatocytes maintained under chemically defined conditions. (1) Insulin receptor number was increased in a dose-dependent fashion by dexamethasone added to the medium between 24 and 48 h of culture and reduced by insulin, whereas ligand affinity remained unaltered. Insulin-induced down-regulation of insulin receptors was not affected by the glucocorticoid. (2) Although the changes in the sensitivity to insulin of glycogen synthesis from glucose and net glycogen deposition paralleled the modulation of the number of insulin receptors, postbinding events appear to be implicated also in the regulation of insulin-sensitivity. (3) Alterations of the responsiveness of glycogen synthesis to insulin caused by the glucocorticoid and/or insulin and by variation between individual rats were inversely related to cellular glycogen contents, suggesting that hepatocellular glycogen content participates in the regulation of insulin-responsiveness of this metabolic pathway. (4) Regulation of insulin-independent glycogenesis in response to an increase from 5 to 10 mM glucose, and of insulin-dependent glycogen synthesis were different. Since the effects of this ‘physiological’ increase in exogenous glucose were small compared to the acute action of insulin, insulin rather than portal venous glucose is considered to represent the prime stimulator of hepatic glycogen synthesis.     

Reversibility of decreased insulin-stimulated glucose transport capacity in diabetic muscle with in vitro incubation. Insulin is not required

The mechanisms by which insulin deficiency affects muscle glucose transport were investigated. Epitrochlearis muscles from rats with streptozotocin-induced diabetes and from controls were incubated in vitro for 0.5-14 h. The incubation was shown not to impair muscle energy stores or tissue oxygenation. Diabetes decreased basal 3-O-methylglucose transport by 40% (p less than 0.01), and insulin-stimulated (20 milli-units/ml) glucose transport capacity by 70% (p less than 0.001). In vitro incubation gradually normalized insulin responsiveness (3.77 +/- 0.38 before versus 8.97 +/- 0.65 mumol X ml-1 X h-1 after 12 h of incubation). Basal glucose transport remained significantly reduced. The reversal of the insulin responsiveness did not require the presence of rat serum and, furthermore, took place even in the absence of insulin. In fact, insulin responsiveness was higher after incubation (14 h) with no insulin than with 100 microunits/ml insulin (9.85 +/- 0.59 versus 8.06 +/- 0.59 mumol X ml-1 X h-1, p less than 0.05). Glucose at 30 mM did not affect the normalization of the insulin-stimulated glucose transport capacity, whereas incubation in serum from diabetic rats resulted in a slightly (26%) blunted reversal (7.60 +/- 0.39 versus 8.89 +/- 0.45 mumol X ml-1 X h-1 with diabetic versus control serum for 14 h, p less than 0.05; before incubation the value was 3.87 +/- 0.40). Inhibition of protein synthesis by cycloheximide blocked the normalization by 80%. These results suggest the presence in diabetic serum of some labile factor that might inhibit the glucose transport system. The results indicate that the decreased insulin-stimulated glucose transport capacity, in the insulin-deficient diabetic muscle, is not a direct consequence of the lack of insulin or of high glucose concentrations.     

Different effects of IGF-I on insulin-stimulated glucose uptake in adipose tissue and skeletal muscle

The effect of insulin-like growth factor I (IGF-I) on insulin-stimulated glucose uptake was studied in adipose and muscle tissues of hypophysectomized female rats. IGF-I was given as a subcutaneous infusion via osmotic minipumps for 6 or 20 days. All hypophysectomized rats received L-thyroxine and cortisol replacement therapy. IGF-I treatment increased body weight gain but had no effect on serum glucose or free fatty acid levels. Serum insulin and C-peptide concentrations decreased. Basal and insulin-stimulated glucose incorporation into lipids was reduced in adipose tissue segments and isolated adipocytes from the IGF-I-treated rats. In contrast, insulin treatment of hypophysectomized rats for 7 days increased basal and insulin-stimulated glucose incorporation into lipids in isolated adipocytes. Pretreatment of isolated adipocytes in vitro with IGF-I increased basal and insulin-stimulated glucose incorporation into lipids. These results indicate that the effect of IGF-I on lipogenesis in adipose tissue is not direct but via decreased serum insulin levels, which reduce the capacity of adipocytes to metabolize glucose. Isoproterenol-stimulated lipolysis, but not basal lipolysis, was enhanced in adipocytes from IGF-I-treated animals. In the soleus muscle, the glycogen content and insulin-stimulated glucose incorporation into glycogen were increased in IGF-I-treated rats. In summary, IGF-I has opposite effects on glucose uptake in adipose tissue and skeletal muscle, findings which at least partly explain previous reports of reduced body fat mass, increased body cell mass, and increased insulin responsiveness after IGF-I treatment.     

Minimal requirement of the remnant pancreas for protein and DNA synthesis of cultured hepatocytes prepared from pancreatectomized rats

The incorporation of 3H-thymidine and 3H-leucine into the hepatocytes was studied, using cultured hepatocytes prepared from normal and pancreatectomized rats. (1) In the cultured hepatocytes prepared from 80% pancreatectomized rats, the incorporation of 3H-thymidine and 3H-leucine into hepatocytes remained unchanged compared with those of sham-operated controls. In contrast, in those from totally pancreatectomized rats, the incorporation of 3H-thymidine and 3H-leucine decreased to approximately 67% and 37% respectively of sham-operated controls. However, those returned to near normal in the cultured hepatocytes from totally pancreatectomized rats treated by 0.8 IU/kg of insulin. (2) The addition of insulin (10(-4) M) to the culture medium stimulated the incorporation of 3H-thymidine into cultured hepatocytes prepared from normal rats to 148% of controls. The insulin-stimulated incorporation was inhibited by the addition of glucagon to the culture medium. The combined addition of insulin and glucagon did not synergistically act on DNA synthesis. It is suggested that the portal blood insulin in the presence of more than 20% of the pancreas is imperative for maintaining spontaneous regeneration.     

Effect of oral treatment with a new hypoglycemic agent, AS-6, on the metabolic activities of adipocytes in db/db mice: a comparative study

The mechanism of a new hypoglycemic agent, AS-6, was comparatively studied using the adipocytes from AS-6 treated and untreated genetically obese diabetic mice, db/db. the db/db mice were treated for 1 week with a diet admixture of AS-6 (0.1%). The treatment resulted in the following alterations in metabolic activities; AS-6 treatment increased 125I-insulin binding by 1.4-3.3 fold over the insulin range of 1-1000 microU/ml, the treatment increased the basal activities in 2-deoxyglucose uptake, and in CO2 generation and lipogenesis from U-(14C)-glucose compared with the db/db controls, the treatment partially restored insulin responsiveness in 2-DG uptake and CO2 generation, and 1 mU/ml of insulin greatly stimulated lipogenesis by 5.6 fold above the basal in the control adipocytes while AS-6 treatment changed the lipogenic response less stimulative to the insulin. The results suggest that AS-6 treatment significantly increases insulin binding to the adipocytes associating with an enhancement in glucose metabolism under basal and physiological concentrations of insulin.     

Characterization of sulfonylurea receptors in isolated human pancreatic islets

Current information on pancreatic islet sulfonylurea receptors has been obtained with laboratory animal pancreatic β cells or stable β-cell lines. In the present study, we evaluated the properties of sulfonylurea receptors of human islets of Langherans, prepared by collagenase digestion and density-gradient purification. The binding characterisitics of labeled glibenclamide to pancreatic islet membrane preparations were analyzed, displacement studies with several oral hypoglycemic agents were performed, and these latter compounds were tested as for their insulinotropic action on intact human islets. [³H]glibenclamide saturable binding was shown to be linear at ≤0.25 mg/ml protein; it was both temperature and time dependent. Scatchard analysis of the equilibrium binding data at 25°C indicated the presence of a single class of saturable, high-affinity binding sites with a K_d value of 1.0 ± 0.07 nM and a Bmax value of 657 ± 48 fmol/mg of proteins. The displacement experiments showed the following rank order of potency of the oral hypoglycemic agents we tested: glibenclamide = glimepiride > tolbutamide > chlorpropamide ≫ metformin. This binding potency order was parallel with the insulinotropic potency of the evaluated compounds. J. Cell. Biochem. 71:182–188, 1998. © 1998 Wiley-Liss, Inc.     

Stimulation of hepatic lipogenesis and acetyl-coenzyme A carboxylase by vasopressin.

The effect of vasopressin on the short-term regulation of fatty acid synthesis was studied in isolated hepatocytes from rats fed ad libitum. Vasopressin stimulates fatty acid synthesis by 30-110%. This increase is comparable with that obtained with insulin. Angiotensin also stimulates fatty acid synthesis, whereas phenylephrine does not. The dose-response curve for vasopressin-stimulated lipogenesis is similar to the dose-response curve for glycogenolysis and release of lactate plus pyruvate. Vasopression also stimulates acetyl-CoA carboxylase activity in a dose-dependent manner. Vasopressin does not relieve glucagon-inhibited lipogenesis, whereas insulin does. The action of vasopressin on hepatic lipogenesis is decreased, but not suppressed, in Ca2+-depleted hepatocytes. The results suggest that vasopressin acts on lipogenesis by increasing availability of lipogenic substrate (lactate + pyruvate) and by activating acetyl-CoA carboxylase.     

Effects of hormones and pyruvate on the rates of secretion of very-low-density lipoprotein triacylglycerol and cholesterol by rat hepatocytes

The secretion of very-low-density lipoprotein (VLDL) triacylglycerol and cholesterol was determined under various conditions in hepatocytes prepared from rats maintained on a controlled lighting and feeding schedule. The rate of lipogenesis in hepatocytes prepared from rats during the feeding period was 2-3-fold higher than that in cells prepared immediately before the animals had access to food. However, there were no corresponding changes in the rates of secretion of triacylglycerol and cholesterol. Pyruvate alone stimulated triacylglycerol secretion but had no effect on the secretion of cholesterol. Despite its stimulation of lipogenesis, insulin suppressed the secretion of both triacylglycerol and cholesterol. This effect on triacylglycerol secretion was more pronounced when lipogenesis was enhanced in the presence of pyruvate. Thus, insulin may act to alleviate hypertriglyceridaemia, which may arise during periods of increased hepatic lipogenesis. The inhibitory effect of glucagon on cholesterol secretion was much less pronounced than that on the secretion of triacylglycerol. The inhibitory effects of glucagon were reversed by pyruvate on cholesterol secretion differed according to whether glucagon was present or absent. These results suggest that the rate of hepatic VLDL triacylglycerol secretion is not necessarily coupled to the rate of lipogenesis in the liver; nor is there any obligatory coupling between the output of triacylglycerol and cholesterol associated with VLDL.     

Mechanism of insulin resistance in the post receptor events in sheep: 3-O-methylglucose transport in ovine adipocytes

A severe resistance to the stimulatory action of insulin on glucose metabolism has been shown in ruminant adipose tissue or isolated adipocytes as compared to that of rats. To elucidate the mechanism of insulin resistance in ruminants, we measured the stimulatory effect of insulin on 3-O-methylgulose transport and on intracellular glucose metabolism in isolated adipocytes from sheep and rats. At a glucose concentration (0.1 mM) where transport is thought to be rate-limiting for metabolism, lipogenesis from [U-14C]glucose by ovine adipocytes was markedly less than by rat adipocytes in both the basal state and at all insulin concentrations. The responsiveness to insulin assessed by percent increase above basal was reduced to about 15% of that in rat adipocytes, but the insulin sensitivity was similar, because the insulin concentration giving half-maximal stimulation, ED50, did not differ significantly between ovine and rat adipocytes. The maximal insulin-stimulated 3-O-methylglucose transport in ovine adipocytes per cell was less than 20% of that in rat adipocytes, with a significant lowering in basal rates of transport. However, when data was expressed per 3-O-methylglucose equilibrium space no significant differences were found between ovine and rat in the basal transport rates, but a lowered ability of insulin to stimulate glucose transport was still seen in ovine adipocytes. The dose-response curve for glucose transport was slightly shifted to the right in ovine adipocytes compared to rat adipocytes, indicating a small decrease in insulin sensitivity. The decrease in glucose transport was due to 60% reduction in the maximum velocity in the insulin--stimulated state, with no change in the Km.     

Regulation of insulin receptor function by a small molecule insulin receptor activator

In type 2 diabetes mellitus, impaired insulin signaling leads to hyperglycemia and other metabolic abnormalities. TLK19780, a non-peptide small molecule, is a new member of a novel class of anti-diabetic agents that function as activators of the insulin receptor (IR) beta-subunit tyrosine kinase. In HTC-IR cells, 20 microm TLK19780 enhanced maximal insulin-stimulated IR autophosphorylation 2-fold and increased insulin sensitivity 2-3-fold. In contrast, TLK19780 did not potentiate the action of insulin-like growth factor-1, indicating the selectivity of TLK19780 toward the IR. The predominant effect of TLK19780 was to increase the number of IR that underwent autophosphorylation. Kinetic studies indicated that TLK19780 acted very rapidly, with a maximal effect observed 2 min after addition to insulin-stimulated cells. In 3T3-L1 adipocytes, 5 microm TLK19780 enhanced insulin-stimulated glucose transport, increasing both the sensitivity and maximal responsiveness to insulin. These studies indicate that at low micromolar levels small IR activator molecules can enhance insulin action in various cultured cells and suggest that this effect is mediated by increasing the number of IR that are tyrosine-phosphorylated in response to insulin. These studies suggest that these types of molecules could be developed to treat type 2 diabetes and other clinical conditions associated with insulin resistance.