In vitro and in vivo activation of the insulin receptor kinase in control and denervated skeletal muscle

Skeletal muscle rapidly develops severe insulin resistance following denervation, although insulin binding is unimpaired. Insulin-stimulated receptor tyrosyl kinase activity was studied in intact and 24-h denervated rat hind limb muscles using three preparations: (a) solubilized insulin receptors incubated +/- insulin with gamma-[32P]ATP and histone H2b; (b) soleus muscles prelabeled in vitro with [32P]phosphate with subsequent insulin-stimulated phosphorylation of the receptor in situ; (c) assessment of in vivo activation of muscle receptor tyrosyl kinase by insulin. The latter was achieved by solubilizing muscle insulin receptors in the presence of phosphoprotein phosphatase and kinase inhibitors and measuring receptor-catalyzed histone H2b phosphorylation in the presence of limiting (5 microM) gamma-[32P]ATP. Receptors isolated 5 and 30 min after intravenous insulin injection catalyzed 32P incorporation into histone H2b twice as fast as those from saline-treated controls; insulin stimulated histone H2b labeling exclusively on tyrosine. In vivo activation was demonstrated using solubilized and insulin-agarose-bound receptors. Autophosphorylation of the beta-subunit and receptor tyrosyl kinase activity toward histone H2b was stimulated by insulin in denervated muscles as in controls, although the biological response to insulin, in vitro and in vivo, was markedly impaired after denervation, suggesting a postreceptor defect. The method developed to assess insulin-stimulated receptor activation in vivo seems useful in characterizing mechanisms of insulin resistance.     

Insulin receptor binding and tyrosine kinase activity in liver and skeletal muscle from fasted rats

Insulin binding and tyrosine kinase activity of the insulin receptor have been measured in the liver and muscles of rats fed or submitted to a 72-h-fasting. In both tissues, insulin binding increased in fasting rats. In liver, the ability of insulin to simulate receptor tyrosine kinase activity greatly unpaired during fasting, but remained unchanged in muscle. The change during fasting of the insulin-stimulated tyrosine kinase activity of the insulin receptor is specific to certain tissue.     

Regulation of the insulin receptor kinase by hyperinsulinism

A murine fibroblast cell line transfected with human insulin receptor cDNA, NIH 3T3 HIR3.5, was observed to display insulin-induced down-regulation of insulin-binding activity in a time- and concentration-dependent manner. Maximal inhibition of insulin-binding activity (54%) occurred within 16 h of exposure to 100 nM insulin in vivo, where in vivo refers to intact cells in tissue culture. The decrease in cellular insulin-binding activity was the consequence of a decrease in the number of cell-associated insulin receptors as determined by Scatchard analysis of insulin binding, 125I-insulin affinity cross-linking, and Western blotting of the insulin receptor beta subunit. Acute insulin treatment in vivo (1-60 min) resulted in the activation of the insulin receptor protein tyrosine kinase as determined by in vitro phosphorylation of glutamic acid:tyrosine (4:1), where in vitro refers to broken cell preparations. This acute in vivo insulin activation of the insulin receptor tyrosine kinase resulted in a greater stimulation (1.4-1.9-fold) of tyrosine kinase activity in the glutamic acid:tyrosine (4:1) assay than the maximal stimulation produced by insulin treatment in vitro. In contrast, long term (24 h) insulin treatment in vivo resulted in a 50-70% decrease in intrinsic protein tyrosine kinase activity of the insulin receptors compared with that of acutely activated (1 min) insulin receptors. Under these conditions, the insulin receptor protein kinase activity remained insulin independent in the in vitro substrate kinase assay. Surprisingly, the insulin-independent activated (1 min in vivo insulin-treated) and uncoupled (24 h in vivo insulin-treated) insulin receptors displayed similar stoichiometries of 32P incorporation into the beta subunit by in vitro autophosphorylation when compared with the control insulin receptors, ranging from 1.5 to 1.8 mol of phosphate incorporated/mol of insulin receptor. Phosphoamino acid analysis demonstrated that the phosphoserine/phosphothreonine content of in vivo 32P-labeled insulin receptors increased markedly within a 1-h exposure to insulin in vivo, whereas insulin-induced receptor desensitization was not apparent until 10-24 h after exposure to insulin. These data suggest that insulin treatment in vivo results initially in the activation of the insulin receptor kinase followed by a subsequent uncoupling of protein kinase activity. This insulin-induced desensitization of the insulin receptor kinase does not correlate with the extent of beta subunit serine/threonine phosphorylation.     

Dephosphorylation increases insulin-stimulated receptor kinase activity in skeletal muscle of obese Zucker rats

Serine/threonine phosphorylation of insulin receptor has been implicated in the development of insulin resistance. To investigate whether dephosphorylation of serine/threonine residues of the insulin receptor may restore the decreased insulin-stimulated receptor tyrosine kinase activity in skeletal muscle of obese Zucker rats, insulin receptor tyrosine kinase activity was measured before and after alkaline phosphatase treatment. Compared to lean controls, insulin-stimulated glucose transport was depressed by 61% (p < 0.05) in obese Zucker rats. The insulin receptor and insulin receptor substrate-1 contents were decreased by 14% (p < 0.05) and 16% (p < 0.05), respectively, in skeletal muscle of obese Zucker rats. In vivo insulin-induced tyrosine phosphorylation of insulin receptor and insulin receptor substrate-1 was depressed by 82% (p < 0.05) and 86% (p < 0.05), respectively. In the meantime, in vitro insulin-stimulated receptor tyrosine kinase activity in obese rats was decreased by 39% (p < 0.05). Dephosphorylation of the insulin receptor by prior alkaline phosphatase treatment increased insulin-stimulated receptor tyrosine kinase activity in both lean and obese Zucker rats, but the increase was three times greater in obese Zucker rats (p < 0.05). These findings suggest that excessive serine/threonine phosphorylation of the insulin receptor in obese Zucker rats may be a cause for insulin resistance in skeletal muscle.     

Chronic selective glycogen synthase kinase-3 inhibition enhances glucose disposal and muscle insulin action in prediabetic obese Zucker rats

Increasing evidence supports a negative role of glycogen synthase kinase-3 (GSK-3) in regulation of skeletal muscle glucose transport. We assessed the effects of chronic treatment of insulin-resistant, prediabetic obese Zucker (fa/fa) rats with a highly selective GSK-3 inhibitor (CT118637) on glucose tolerance, whole body insulin sensitivity, plasma lipids, skeletal muscle insulin signaling, and in vitro skeletal muscle glucose transport activity. Obese Zucker rats were treated with either vehicle or CT118637 (30 mg/kg body wt) twice per day for 10 days. Fasting plasma insulin and free fatty acid levels were reduced by 14 and 23% (P < 0.05), respectively, in GSK-3 inhibitor-treated animals compared with vehicle-treated controls. The glucose response during an oral glucose tolerance test was reduced by 18% (P < 0.05), and whole body insulin sensitivity was increased by 28% (P < 0.05). In vivo insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation (50%) and IRS-1-associated phosphatidylinositol-3' kinase (79%) relative to fasting plasma insulin levels were significantly elevated (P < 0.05) in plantaris muscles of GSK-3 inhibitor-treated animals. Whereas basal glucose transport in isolated soleus and epitrochlearis muscles was unaffected by chronic GSK-3 treatments, insulin stimulation of glucose transport above basal was significantly enhanced (32-60%, P < 0.05). In summary, chronic treatment of insulin-resistant, prediabetic obese Zucker rats with a specific GSK-3 inhibitor enhances oral glucose tolerance and whole body insulin sensitivity and is associated with an amelioration of dyslipidemia and an improvement in IRS-1-dependent insulin signaling in skeletal muscle. These results provide further evidence that selective targeting of GSK-3 in muscle may be an effective intervention for the treatment of obesity-associated insulin resistance.     

Internalization and activation of the rat liver insulin receptor kinase in vivo

The preparation of clearly delineated plasmalemma (PM) and endosomal subcellular fractions from rat liver has allowed us to compare insulin receptor (IR) kinase activity at the cell surface and in hepatic endosomes (ENs) as a function of dose and time after injected insulin. Tyrosine kinase activity in PM and ENs was measured, after solubilization and partial purification by wheat germ agglutinin chromatography (lectin-purified), using poly(Glu:Tyr) as substrate. Following the injection of a subsaturating dose of insulin (1.5 micrograms/100 g body weight), lectin-purified receptor showed peak activation at 30 s in PM and at 2 min in ENs. As observed previously (Khan, M. N., Savoie, S., Bergeron, J. J. M., and Posner, B. I. (1986) J. Biol. Chem. 261, 8462-8472) autophosphorylation activity was also augmented following insulin injection. In a pattern virtually identical to that of exogenous kinase activity, autophosphorylation attained peak activity at 30 s in PM and at 2 min in ENs. The time course of IR autophosphorylation in intact membranes was very similar to that observed for lectin purified receptors and was seen with an injected insulin dose as low as 150 ng/100 g body weight. Phosphatase treatment of the solubilized endosomal receptor abolished its enhanced activity. Hence, insulin treatment led to in vivo receptor phosphorylation which was reflected in the enhancement of both tyrosine kinase and autophosphorylation activities. Significant differences in the phosphorylation activities of PM and ENs were observed. Phosphoamino acid analyses revealed that the activated IR of intact PM was autophosphorylated in vitro, at both serine (55%) and tyrosine (45%) residues; whereas the activated IR of intact ENs was phosphorylated in vitro exclusively on tyrosine autophosphorylation specific activity for the activated IR of ENs was 3- to 4-fold that of the IR of PM. This was observed for the lectin purified IRs as well as for IRs of intact cell fractions. The reduced level of IR autophosphorylation in PM was not due to occlusion of tyrosine acceptor sites by prior in vivo phosphorylation. The rapidity with which activated IR accumulates in ENs as well as the sensitivity of endosomal IR kinase to activation by injected insulin are consistent with the endosomal apparatus serving a physiologically significant site for the regulation of transmembrane signaling.     

Activation of insulin signal transduction pathway and anti-diabetic activity of small molecule insulin receptor activators

We recently described the identification of a non-peptidyl fungal metabolite (l-783,281, compound 1), which induced activation of human insulin receptor (IR) tyrosine kinase and mediated insulin-like effects in cells, as well as decreased blood glucose levels in murine models of Type 2 diabetes (Zhang, B., Salituro, G., Szalkowski, D., Li, Z., Zhang, Y., Royo, I., Vilella, D., Diez, M. T. , Pelaez, F., Ruby, C., Kendall, R. L., Mao, X., Griffin, P., Calaycay, J., Zierath, J. R., Heck, J. V., Smith, R. G. & Moller, D. E. (1999) Science 284, 974-977). Here we report the characterization of an active analog (compound 2) with enhanced IR kinase activation potency and selectivity over related receptors (insulin-like growth factor I receptor, epidermal growth factor receptor, and platelet-derived growth factor receptor). The IR activators stimulated tyrosine kinase activity of partially purified native IR and recombinant IR tyrosine kinase domain. Administration of the IR activators to mice was associated with increased IR tyrosine kinase activity in liver. In vivo oral treatment with compound 2 resulted in significant glucose lowering in several rodent models of diabetes. In db/db mice, oral administration of compound 2 elicited significant correction of hyperglycemia. In a streptozotocin-induced diabetic mouse model, compound 2 potentiated the glucose-lowering effect of insulin. In normal rats, compound 2 improved oral glucose tolerance with significant reduction in insulin release following glucose challenge. A structurally related inactive analog (compound 3) was not effective on insulin receptor activation or glucose lowering in db/db mice. Thus, small molecule IR activators exert insulin mimetic and sensitizing effects in cells and in animal models of diabetes. These results have implications for the future development of new therapies for diabetes mellitus.     

Compartmentalization and in vivo insulin-induced translocation of the insulin-signaling inhibitor Grb14 in rat liver

The molecular adaptor Grb14 binds in vitro to the activated insulin receptor (IR) and inhibits IR signaling. In this study, we have used rat liver subcellular fractionation to analyze in vivo insulin effects on Grb14 compartmentalization and IR phosphorylation and activity. In control rats, Grb14 was recovered mainly in microsomal and cytosolic fractions, but was also detectable at low levels in plasma membrane and Golgi/endosome fractions. Insulin injection led to a rapid and dose-dependent increase in Grb14 content, first in the plasma membrane fraction, and then in the Golgi/endosome fraction, which paralleled the increase in IR beta-subunit tyrosine phosphorylation. Upon sustained in vivo IR tyrosine phosphorylation induced by high-affinity insulin analogs, in vitro IR dephosphorylation by endogenous phosphatases, and in vivo phosphorylation of the IR induced by injection of bisperoxo(1,10 phenanthroline)oxovanadate, a phosphotyrosine phosphatase inhibitor, we observed a striking correlation between IR phosphorylation state and Grb14 content in both the plasma membrane and Golgi/endosome fractions. In addition, coimmunoprecipitation experiments provided evidence that Grb14 was associated with phosphorylated IR beta-subunit in these fractions. Altogether, these data support a model whereby insulin stimulates the recruitment of endogenous Grb14 to the activated IR at the plasma membrane, and induces internalization of the Grb14-IR complex in endosomes. Removal of Grb14 from fractions of insulin-treated rats by KCl treatment led to an increase of in vivo insulin-stimulated IR tyrosine kinase activity, indicating that endogenous Grb14 exerts a negative feedback control on IR catalytic activity. This study thus demonstrates that Grb14 is a physiological regulator of liver insulin signaling.     

Effect of nutritional status on insulin sensitivity in vivo and tissue enzyme activities in the rat.

The hyperinsulinaemic-glucose-clamp technique, in combination with measurement of glucose turnover in conscious unrestrained rats, was used to assess the effects of nutritional status on insulin sensitivity in vivo and glucose metabolism. Liver, heart and quadriceps skeletal-muscle glycogen content and activities of pyruvate dehydrogenase (PDH) and glycogen synthase were measured both basally and at the end of a 2.5 h glucose clamp (insulin 85 munits/h) in rats 6, 24 and 48 h after food withdrawal. Clamp glucose requirement and glucose turnover were unchanged by fasting. Activation of glycogen synthase and glycogen deposition in liver and skeletal muscle during the clamps were also not impaired in rats after a prolonged fast. By contrast with skeletal muscle, activation of cardiac-muscle glycogen synthase and glycogen deposition during the clamps were markedly impaired by 24 h of fasting and were undetectable at 48 h. Skeletal-muscle PDH activity fell with more prolonged fasting (6 h, 15.3 +/- 3.4%; 24 h, 4.7 +/- 0.7%; 48 h, 4.3 +/- 0.6% active; P less than 0.005), but at 24 and 48 h was stimulated by the clamp to values unchanged by the duration of fasting. Stimulation of cardiac PDH activity by the clamp was, however, impaired in rats fasted for 24 or 48 h. Basal hepatic PDH did not change significantly with fasting (6 h, 5.3 +/- 1.1%; 24 h, 4.6 +/- 0.7%; 48 h, 3.9 +/- 0.5%), and, although it could be partly restored at 24 h, very little stimulation occurred at 48 h. Hepatic pyruvate kinase and acetyl-CoA carboxylase activity were both stimulated by the clamps, and this was not impaired with more prolonged fasting. During the glucose clamps, blood concentrations of lactate, pyruvate and alanine were increased to a greater extent in rats fasted for 24 and 48 h than in rats studied 6 h after food withdrawal. The findings suggest that, although sensitivity to insulin of whole-body glucose disposal is unchanged with fasting, there may be qualitative differences in the metabolism of glucose.     

The Male Obese Wistar Diabetic Fatty Rat Is a New Model of Extreme Insulin Resistance

The male obese Wistar Diabetic Fatty (WDF) rat is a genetic model of obesity and non-insulin dependent diabetes (NIDDM). The obese Zucker rat shares the same gene for obesity on a different genetic background but is not diabetic. This study evaluated the degree of insulin resistance in both obese strains by examining the binding and post binding effects of muscle insulin receptors in obese, rats exhibiting hyperinsulinemia and/or hyperglycemia. Insulin receptor binding and affinity and tyrosine kinase activity were measured in skeletal muscle from male WDF fa/fa (obese) and Fa/? (lean) and Zucker fa/fa (obese) and Fa/Fa (homozygous lean) rats. Rats were fed a high sucrose (68% of total Kcal) or Purina stock diet for 14 weeks. At 27 weeks of age, adipose depots were removed for adipose cellularity analysis and the biceps femoris muscle was removed for measurement of insulin binding and insulin-stimulated receptor kinase activity. Plasma glucose (13.9 vs. 8.4 mM) and insulin levels (14,754 vs. 7440 pmoI/L) were significantly higher in WDF obese than in Zucker obese rats. Insulin receptor number and affinity and TK activity were unaffected by diet. Insulin receptor number was significantly reduced in obese WDF rats (2.778 ± 0.617 pmol/mg protein), compared to obese Zucker rats (4.441 ± 0.913 pmol/mg potein). Both obese strains exhibited down regulation of the insulin receptor compared to their lean controls. Maximal tyrosine kinase (TK) activity was significantly reduced in obese WDF rats (505 ± 82 fmol/min/mg protein) compared to obese Zucker rats (1907 ± 610 fmol/min/mg protein). Only obese WDF rats displayed a decrease in TK activity per receptor. These observations establish the obese WDF rat as an excellent model for exploring mechanisms of extreme insulin resistance, particularly post-receptor tyrosine kinase-associated defects, in non-insulin dependent diabetes.     

Oxidative stress--mediated alterations in glucose dynamics in a genetic animal model of type II diabetes

Insulin resistance, characterized by an inexorable decline in skeletal muscle glucose utilization and/or an excessive hepatic glucose production, constitutes a major pathogenic importance in a cluster of clinical disorders including diabetes mellitus, hypertension, dyslipidemia, central obesity and coronary artery disease. A novel concept suggests that heightened state of oxidative stress during diabetes contributes, at least in part, to the development of insulin resistance. Several key predictions of this premise were subjected to experimental testing using Goto-Kakizaki (GK) rats as a genetic animal model for non-obese type II diabetes. Euglycemic-hyperinsulinemic clamp studies with an insulin infusion index of 5 mU/kg bw/min were used to measure endogenous glucose production (EGP), glucose infusion rate (GIR), glucose disposal rate (GDR) and skeletal muscle glucose utilization index (GUI). Moreover, the status of oxidative stress as reflected by the urinary levels of isoprostane and protein carbonyl formation were also assessed as a function of diabetes. Post-absorptive basal EGP and circulating levels of insulin, glucose and free fatty acid (FFA) were elevated in GK rats, compared to their corresponding control values. In contrast, steady state GIR and GDR of the hyperglycemic/hyperinsulinemic animals were reduced, concomitantly with impaired insulin's ability to suppress EGP. Insulin stimulated [3H]-2-deoxyglucose (2-DG) uptake (a measure of glucose transport activity) by various types of skeletal muscle fibers both in vivo and in vitro (isolated muscle, cultured myoblasts) was diminished in diabetic GK rats. This diabetes-related suppression of skeletal muscle glucose utilization was associated with a decrease in insulin's ability to promote the phosphorylation of tyrosine residues of insulin receptor substrate-1 (IRS-1). Similarly, the translocation of GLUT-4 from intracellular compartment to plasma membrane in response to insulin was also reduced in these animals. Oxidative stress-based markers (e.g. urinary isoprostane, carbonyl-bound proteins) were elevated as a function of diabetes. Nullification of the heightened state of oxidative stress in the GK rats with alpha-lipoic acid resulted in a partial amelioration of the diabetes-related impairment of the in vivo and in vitro insulin actions. Collectively, the above data suggest that 1) insulin resistance in GK rats occurs at the hepatic and skeletal muscle levels, 2) muscle cell glucose transport exhibited a blunted response to insulin and it is associated with a major defect in key molecules of both GLUT-4 trafficking and insulin signaling pathways, 3) skeletal muscle insulin resistance in GK rats appears to be of genetic origin and not merely related to a paracrine or autocrine effect, since this phenomenon is also observed in cultured myoblasts over several passages and finally heightened state of oxidative stress may mediate the development of insulin resistance during diabetes.     

Insulin stimulates the tyrosine phosphorylation of caveolin

The specialized plasma membrane structures termed caveolae and the caveolar-coat protein caveolin are highly expressed in insulin- sensitive cells such as adipocytes and muscle. Stimulation of 3T3-L1 adipocytes with insulin significantly increased the tyrosine phosphorylation of caveolin and a 29-kD caveolin-associated protein in caveolin-enriched Triton-insoluble complexes. Maximal phosphorylation occurred within 5 min, and the levels of phosphorylation remained elevated for at least 30 min. The insulin-dose responses for the tyrosine phosphorylation of caveolin and the 29-kD caveolin-associated protein paralleled those for the phosphorylation of the insulin receptor. The stimulation of caveolin tyrosine phosphorylation was specific for insulin and was not observed with PDGF or EGF, although PDGF stimulated the tyrosine phosphorylation of the 29-kD caveolin- associated protein. Increased tyrosine phosphorylation of caveolin, its associated 29-kD protein, and a 60-kD protein was observed in an in vitro kinase assay after incubation of the caveolin-enriched Triton- insoluble complexes with Mg-ATP, suggesting the presence of an intrinsic tyrosine kinase in these complexes. These fractions contain only trace amounts of the activated insulin receptor. In addition, these complexes contain a 60-kD kinase detected in an in situ gel kinase assay and an approximately 60 kD protein that cross-reacts with an antibody against the Src-family kinase p59Fyn. Thus, the insulin- dependent tyrosine phosphorylation of caveolin represents a novel, insulin-specific signal transduction pathway that may involve activation of a tyrosine kinase downstream of the insulin receptor.     

Insulin stimulation of phosphatidylinositol 3-kinase activity and association with insulin receptor substrate 1 in liver and muscle of the intact rat.

Growth factors stimulate the enzyme phosphatidylinositol (PI) 3-kinase in cells in culture. Insulin rapidly stimulates tyrosine phosphorylation of its endogenous substrate, insulin receptor substrate 1 (IRS-1), and in vitro IRS-1 associates with PI 3-kinase, thus activating the enzyme. We have examined whether insulin is capable of stimulating the PI 3-kinase pathway in two physiological target tissues for the actions of insulin in vivo, liver and skeletal muscle. After intraportal injection of insulin into anesthetized rats, there was a 2-fold stimulation of total hepatic PI 3-kinase activity in liver and muscle extracts and a 10- to 20-fold increase in PI 3-kinase activity immunoprecipitated with anti-IRS-1 antibodies. Stimulation of PI 3-kinase was accompanied by an association between this enzyme and IRS-1 as detected by immunoprecipitation of liver and muscle extracts with anti-IRS-1 antibodies and Western blotting with antibodies to the 85-kDa subunit of PI 3-kinase. Immunoprecipitation with anti-p85 antibodies and phosphotyrosine immunoblotting revealed no tyrosine phosphorylation of PI 3-kinase, but demonstrated co-precipitation of tyrosine-phosphorylated IRS-1, as well as another phosphotyrosine protein of approximately 135-140 kDa. Thus, IRS-1 phosphorylation plays a significant role in the activation of PI 3-kinase in vivo by insulin.     

Topiramate is an insulin-sensitizing compound in vivo with direct effects on adipocytes in female ZDF rats

We have studied the in vivo and in vitro effects of Topiramate (TPM) in female Zucker diabetic fatty (ZDF) rats. After weight matching, drug treatment had a marked effect to lower fasting glucose levels of relatively normoglycemic animals as well as during an oral glucose tolerance test. The glucose clamp studies revealed a approximately 30% increased glucose disposal, increased hepatic glucose output (HGO) suppression from approximately 30 to 60%, and an increased free fatty acid suppression from 40 to 75%. Therefore, TPM treatment led to enhanced insulin sensitivity at the level of tissue glucose disposal (increased ISGDR), liver (increased inhibition of HGO), and adipose tissue (enhanced suppression of lipolysis). When soleus muscle strips of control or TPM-treated ZDF rats were studied ex vivo, insulin-stimulated glucose transport was not enhanced in the drug-treated animals. In contrast, when isolated adipocytes were studied ex vivo, a marked increase (+55%) in insulin-stimulated glucose transport was observed. In vitro treatment of muscle strips and rat adipocytes showed no effect on glucose transport in muscle with a 40% increase in insulin-stimulated adipocyte glucose transport. In conclusion, 1) TPM treatment leads to a decrease in plasma glucose and increased in vivo insulin sensitivity; 2) insulin sensitization was observed in adipocytes, but not muscle, when tissues were studied ex vivo or in vitro; and 3) TPM directly enhances insulin action in insulin-resistant adipose cells in vitro. Thus the in vivo effects of TPM treatment appear to be exerted through adipose tissue.     

Phorbol ester-induced serine phosphorylation of the insulin receptor decreases its tyrosine kinase activity

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the function of the insulin receptor was examined in intact hepatoma cells (Fao) and in solubilized extracts purified by wheat germ agglutinin chromatography. Incubation of ortho[32P]phosphate-labeled Fao cells with TPA increased the phosphorylation of the insulin receptor 2-fold after 30 min. Analysis of tryptic phosphopeptides from the beta-subunit of the receptor by reverse-phase high performance liquid chromatography and determination of their phosphoamino acid composition suggested that TPA predominantly stimulated phosphorylation of serine residues in a single tryptic peptide. Incubation of the Fao cells with insulin (100 nM) for 1 min stimulated 4-fold the phosphorylation of the beta-subunit of the insulin receptor. Prior treatment of the cells with TPA inhibited the insulin-stimulated tyrosine phosphorylation by 50%. The receptors extracted with Triton X-100 from TPA-treated Fao cells and purified on immobilized wheat germ agglutinin retained the alteration in kinase activity and exhibited a 50% decrease in insulin-stimulated tyrosine autophosphorylation and phosphotransferase activity toward exogenous substrates. This was due primarily to a decrease in the Vmax for these reactions. TPA treatment also decreased the Km of the insulin receptor for ATP. Incubation of the insulin receptor purified from TPA-treated cells with alkaline phosphatase decreased the phosphate content of the beta-subunit to the control level and reversed the inhibition, suggesting that the serine phosphorylation of the beta-subunit was responsible for the decreased tyrosine kinase activity. Our results support the notion that the insulin receptor is a substrate for protein kinase C in the Fao cell and that the increase in serine phosphorylation of the beta-subunit of the receptor produced by TPA treatment inhibited tyrosine kinase activity in vivo and in vitro. These data suggest that protein kinase C may regulate the function of the insulin receptor.     

Lipocortins 1 and 2 as substrates for the insulin receptor kinase in rat liver

Lipocortins 1 and 2 are major substrates for the epidermal growth factor receptor and the pp60v-src tyrosine kinases in transformed cells. In the present study, we have characterized the phosphorylation of lipocortins 1 and 2 by the insulin receptor tyrosine kinase in vitro and in vivo. In vitro, the solubilized insulin receptor, partially purified from rat liver, catalyzed phosphorylation of human recombinant lipocortin 1 and purified bovine lipocortin 2. Phosphorylation of lipocortin 1 was increased 15-fold upon stimulation with 10(-7) M insulin. The apparent Km of the reaction was 3.3 microM and was not affected by insulin stimulation. Insulin stimulated phosphate incorporation into lipocortin 2 by 20-fold (apparent Km greater than 20 microM). Both lipocortins were phosphorylated exclusively on tyrosine residues as judged by phosphoamino acid analysis. Based upon peptide mapping, lipocortin 1 was phosphorylated on Tyr-21, a site phosphorylated by other tyrosine kinases. Polyclonal anti-phosphotyrosine antibodies recognized the tyrosine-phosphorylated lipocortin 2, but not lipocortin 1 in its phosphorylated form. In hepatocytes from normal and dexamethasone-treated rats, lipocortin 1 content was less than 50 ng/10(6) cells. Insulin-induced phosphorylation of lipocortin 1 was detected in intact hepatocytes from corticosteroid-treated animals but not in cells from normal rats. No phosphorylation of lipocortin 2 was found, although its content was approximately 100 ng/10(6) cells from normal animals and increased to approximately 1 microgram/10(6) cells following treatment of rats with dexamethasone for 4 days. Thus, although lipocortins 1 and 2 are in vitro substrates of the insulin receptor kinase, only lipocortin 1 is phosphorylated in an insulin-dependent manner in intact hepatocytes, and this is only observed after dexamethasone treatment of the rats.     

Leptin increases hepatic insulin sensitivity and protein tyrosine phosphatase 1B expression

Leptin has been shown to improve insulin sensitivity and glucose metabolism in obese diabetic ob/ob mice, yet the mechanisms remain poorly defined. We found that 2 d of leptin treatment improved fasting but not postprandial glucose homeostasis, suggesting enhanced hepatic insulin sensitivity. Consistent with this hypothesis, leptin improved in vivo insulin receptor (IR) activation in liver, but not in skeletal muscle or fat. To explore the cellular mechanism by which leptin up-regulates hepatic IR activation, we examined the expression of the protein tyrosine phosphatase PTP1B, recently implicated as an important negative regulator of insulin signaling. Unexpectedly, liver PTP1B protein abundance was increased by leptin to levels similar to lean controls, whereas levels in muscle and fat remained unchanged. The ability of leptin to augment liver IR activation and PTP1B expression was also observed in vitro in human hepatoma cells (HepG2). However, overexpression of PTP1B in HepG2 cells led to diminished insulin-induced IR phosphorylation, supporting the role of PTP1B as a negative regulator of IR activation in hepatocytes. Collectively, our results suggest that leptin acutely improves hepatic insulin sensitivity in vivo with concomitant increases in PTP1B expression possibly serving to counterregulate insulin action and to maintain insulin signaling in proper balance.     

Integrative metabolic regulation of peripheral tissue fatty acid oxidation by the SRC kinase family member Fyn

Mice null for Fyn (a member of the Src family of nonreceptor tyrosine kinases) display a reduced percentage of adipose mass associated with decreased adipocyte cell size. In parallel, there is a substantial reduction in fasting plasma glucose, insulin, triglycerides, and free fatty acids concomitant with decreased intrahepatocellular and intramyocellular lipid accumulation. Importantly, the Fyn null mice exhibit improved glucose tolerance resulting from increased peripheral tissue (adipose and skeletal muscle) insulin sensitivity with a very small effect in the liver. Moreover, whole-body, adipose, and skeletal muscle fatty acid uptake and oxidation are increased along with AMP kinase activation and acetyl-CoA carboxylase inhibition. Together, these data demonstrate crosstalk between Src-family kinase activity and fatty acid oxidation and show that the loss of Fyn markedly improves peripheral tissue insulin sensitivity by relieving a selective negative modulation of AMP kinase activity in adipose tissue and skeletal muscle.     

Improved glucose homeostasis and enhanced insulin signalling in Grb14-deficient mice

Gene targeting was used to characterize the physiological role of growth factor receptor-bound (Grb)14, an adapter-type signalling protein that associates with the insulin receptor (IR). Adult male Grb14(-/-) mice displayed improved glucose tolerance, lower circulating insulin levels, and increased incorporation of glucose into glycogen in the liver and skeletal muscle. In ex vivo studies, insulin-induced 2-deoxyglucose uptake was enhanced in soleus muscle, but not in epididymal adipose tissue. These metabolic effects correlated with tissue-specific alterations in insulin signalling. In the liver, despite lower IR autophosphorylation, enhanced insulin-induced tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and activation of protein kinase B (PKB) was observed. In skeletal muscle, IR tyrosine phosphorylation was normal, but signalling via IRS-1 and PKB was increased. Finally, no effect of Grb14 ablation was observed on insulin signalling in white adipose tissue. These findings demonstrate that Grb14 functions in vivo as a tissue-specific modulator of insulin action, most likely via repression of IR-mediated IRS-1 tyrosine phosphorylation, and highlight this protein as a potential target for therapeutic intervention.     

Insulin-dependent phosphorylation of the insulin receptor-protein kinase and activation of glucose transport in 3T3-L1 adipocytes

Insulin stimulates hexose transport and phosphorylation of the insulin receptor in monolayer cultures of intact 3T3-L1 adipocytes. To assess the phosphorylation state of the receptor in situ, cells were equilibrated with [32P]orthophosphate and then disrupted under denaturing conditions which preserved the phosphorylation state of the receptor established in the cell. The insulin receptor, isolated by lectin adsorption and two-dimensional nonreducing/reducing polyacrylamide gel electrophoresis, occurred as a single oligomeric species with an apparent alpha 2 beta 2 subunit composition. This oligomeric structure was not altered by treating cells with insulin. Only the beta-subunit of the receptor was phosphorylated; [32P]phosphoserine and [32P] phosphotyrosine were both identified in the beta-subunit from cells in the unstimulated state, but only [32P] phosphotyrosine increased in cells stimulated with insulin. Neither insulin-like growth factors I nor II stimulated insulin receptor beta-subunit phosphorylation, although both activated hexose transport. Upon the addition of insulin, [32P]orthophosphate incorporated into the beta-subunit increased 4.5-fold (7-fold with respect to [32P]tyrosine) and was complete within 1 min (t1/2 = 8 s). Following the removal of insulin from the monolayers, [32P]beta-subunit fell to the basal level (t1/2 = 2.5 min); there was no lag phase before either transition. The tyrosine protein kinase activity, measured in vitro with a model substrate, was higher with immunoaffinity-purified insulin receptor from insulin-stimulated cells than from cells in the basal state. Hexose transport rate, measured using 3-O-[methyl-14C]glucose, was half-maximally stimulated at 2 nM insulin. A 1-min latency period followed insulin addition, after which a 7-fold increase in the steady-state rate of hexose uptake was achieved within 5 min. Upon the removal of insulin, hexose transport continued at the stimulated steady-state rate for 2.5 min and then declined to the basal rate with a half-time of 8 min. These kinetic experiments in situ and protein kinase activity measurements in vitro support the hypothesis that beta-subunit phosphorylation is an intermediate step linking insulin binding to the increased glucose transport rate.