Akt-dependent phosphorylation of hepatic FoxO1 is compartmentalized on a WD40/ProF scaffold and is selectively inhibited by aPKC in early phases of diet-induced obesity.

Initiating mechanisms that impair gluconeogenic enzymes and spare lipogenic enzymes in diet-induced obesity (DIO) are obscure. Here, we examined insulin signaling to Akt and atypical protein kinase C (aPKC) in liver and muscle and hepatic enzyme expression in mice consuming a moderate high-fat (HF) diet. In HF diet-fed mice, resting/basal and insulin-stimulated Akt and aPKC activities were diminished in muscle, but in liver, these activities were elevated basally and were increased by insulin to normal levels. Despite elevated hepatic Akt activity, FoxO1 phosphorylation, which diminishes gluconeogenesis, was impaired; in contrast, Akt-dependent phosphorylation of glycogenic GSK3ß and lipogenic mTOR was elevated. Diminished Akt-dependent FoxO1 phosphorylation was associated with reduced Akt activity associated with scaffold protein WD40/Propeller/FYVE (WD40/ProF), which reportedly facilitates FoxO1 phosphorylation. In contrast, aPKC activity associated with WD40/ProF was increased. Moreover, inhibition of hepatic aPKC reduced its association with WD40/ProF, restored WD40/ProF-associated Akt activity, restored FoxO1 phosphorylation, and corrected excessive expression of hepatic gluconeogenic and lipogenic enzymes. Additionally, Akt and aPKC activities in muscle improved, as did glucose intolerance, weight gain, hepatosteatosis, and hyperlipidemia. We conclude that Akt-dependent FoxO1 phosphorylation occurs on the WD/Propeller/FYVE scaffold in liver and is selectively inhibited in early DIO by diet-induced increases in activity of cocompartmentalized aPKC.

Insulin signaling and insulin sensitizing in muscle and liver of obese monkeys: peroxisome proliferator-activated receptor gamma agonist improves defective activation of atypical protein kinase C.

Obesity, the metabolic syndrome, and aging share several pathogenic features in both humans and non-human primates, including insulin resistance and inflammation. Since muscle and liver are considered key integrators of metabolism, we sought to determine in biopsies from lean and obese aging rhesus monkeys the nature of defects in insulin activation and, further, the potential for mitigation of such defects by an in vivo insulin sensitizer, rosiglitazone, and a thiazolidinedione activator of the peroxisome proliferator-activated receptor gamma. The peroxisome proliferator-activated receptor gamma agonist reduced hyperinsulinemia, improved insulin sensitivity, lowered plasma triglycerides and free fatty acids, and increased plasma adiponectin. In muscle of obese monkeys, previously shown to exhibit defective insulin signaling, the insulin sensitizer improved insulin activation of atypical protein kinase C (aPKC), the defective direct activation of aPKC by phosphatidylinositol (PI)-3,4,5-(PO4)3, and 5'-AMP-activated protein kinase and increased carnitine palmitoyltransferase-1 mRNA expression, but it did not improve insulin activation of insulin receptor substrate (IRS)-1-dependent PI 3-kinase (IRS-1/PI3K), protein kinase B, or glycogen synthase. We found that, although insulin signaling was impaired in muscle, insulin activation of IRS-1/PI3K, IRS-2/PI3K, protein kinase B, and aPKC was largely intact in liver and that rosiglitazone improved insulin signaling to aPKC in muscle by improving responsiveness to PI-3,4,5-(PO4)3.

The critical role of atypical protein kinase C in activating hepatic SREBP-1c and NFkappaB in obesity.

Obesity is frequently associated with systemic insulin resistance, glucose intolerance, and hyperlipidemia. Impaired insulin action in muscle and paradoxical diet/insulin-dependent overproduction of hepatic lipids are important components of obesity, but their pathogenesis and inter-relationships between muscle and liver are uncertain. We studied two murine obesity models, moderate high-fat-feeding and heterozygous muscle-specific PKC-lambda knockout, in both of which insulin activation of atypical protein kinase C (aPKC) is impaired in muscle, but conserved in liver. In both models, activation of hepatic sterol receptor element binding protein-1c (SREBP-1c) and NFkappaB (nuclear factor-kappa B), major regulators of hepatic lipid synthesis and systemic insulin resistance, was chronically increased in the fed state. In support of a critical mediatory role of aPKC, in both models, inhibition of hepatic aPKC by adenovirally mediated expression of kinase-inactive aPKC markedly diminished diet/insulin-dependent activation of hepatic SREBP-1c and NFkappaB, and concomitantly improved hepatosteatosis, hypertriglyceridemia, hyperinsulinemia, and hyperglycemia. Moreover, in high-fat-fed mice, impaired insulin signaling to IRS-1-dependent phosphatidylinositol 3-kinase, PKB/Akt and aPKC in muscle and hyperinsulinemia were largely reversed. In obesity, conserved hepatic aPKC-dependent activation of SREBP-1c and NFkappaB contributes importantly to the development of hepatic lipogenesis, hyperlipidemia, and systemic insulin resistance. Accordingly, hepatic aPKC is a potential target for treating obesity-associated abnormalities.

Repletion of atypical protein kinase C following RNA interference-mediated depletion restores insulin-stimulated glucose transport.

The role of atypical protein kinase C (aPKC) in insulin-stimulated glucose transport in myocytes and adipocytes is controversial. Whereas studies involving the use of adenovirally mediated expression of kinase-inactive aPKC in L6 myocytes and 3T3/L1 and human adipocytes, and data from knock-out of aPKC in adipocytes derived from mouse embryonic stem cells and subsequently derived adipocytes, suggest that aPKCs are required for insulin-stimulated glucose transport, recent findings in studies of aPKC knockdown by small interfering RNA (RNAi) in 3T3/L1 adipocytes are conflicting. Moreover, there are no reports of aPKC knockdown in myocytes, wherein insulin effects on glucose transport are particularly relevant for understanding whole body glucose disposal. Presently, we exploited the fact that L6 myotubes and 3T3/L1 adipocytes have substantially different (30% nonhomology) major aPKCs, viz. PKC-zeta in L6 myotubes and PKC-lambda in 3T3/L1 adipocytes, that nevertheless can function interchangeably for glucose transport. Accordingly, in L6 myotubes, RNAi-targeting PKC-zeta, but not PKC-lambda, markedly depleted aPKC and concomitantly inhibited insulin-stimulated glucose transport; more importantly, these depleting/inhibitory effects were rescued by adenovirally mediated expression of PKC-lambda. Conversely, in 3T3/L1 adipocytes, RNAi constructs targeting PKC-lambda, but not PKC-zeta, markedly depleted aPKC and concomitantly inhibited insulin-stimulated glucose transport; here again, these depleting/inhibitory effects were rescued by adenovirally mediated expression of PKC-zeta. These findings in knockdown and, more convincingly, rescue studies, strongly support the hypothesis that aPKCs are required for insulin-stimulated glucose transport in myocytes and adipocytes.

Insulin-sensitive protein kinases (atypical protein kinase C and protein kinase B/Akt): actions and defects in obesity and type II diabetes.

Glucose transport into muscle is the initial process in glucose clearance and is uniformly defective in insulin-resistant conditions of obesity, metabolic syndrome, and Type II diabetes mellitus. Insulin regulates glucose transport by activating insulin receptor substrate-1 (IRS-1)-dependent phosphatidylinositol 3-kinase (PI3K) which, via increases in PI-3,4,5-triphosphate (PIP(3)), activates atypical protein kinase C (aPKC) and protein kinase B (PKB/Akt). Here, we review (i) the evidence that both aPKC and PKB are required for insulin-stimulated glucose transport, (ii) abnormalities in muscle aPKC/PKB activation seen in obesity and diabetes, and (iii) mechanisms for impaired aPKC activation in insulin-resistant conditions. In most cases, defective muscle aPKC/PKB activation reflects both impaired activation of IRS-1/PI3K, the upstream activator of aPKC and PKB in muscle and, in the case of aPKC, poor responsiveness to PIP(3), the lipid product of PI3K. Interestingly, insulin-sensitizing agents (e.g., thiazolidinediones, metformin) improve aPKC activation by insulin in vivo and PIP3 in vitro, most likely by activating 5'-adenosine monophosphate-activated protein kinase, which favorably alters intracellular lipid metabolism. Differently from muscle, aPKC activation in the liver is dependent on IRS-2/PI3K rather than IRS-1/PI3K and, surprisingly, the activation of IRS-2/PI3K and aPKC is conserved in high-fat feeding, obesity, and diabetes. This conservation has important implications, as continued activation of hepatic aPKC in hyperinsulinemic states may increase the expression of sterol regulatory element binding protein-1c, which controls genes that increase hepatic lipid synthesis. On the other hand, the defective activation of IRS-1/PI3K and PKB, as seen in diabetic liver, undoubtedly and importantly contributes to increases in hepatic glucose output. Thus, the divergent activation of aPKC and PKB in the liver may explain why some hepatic actions of insulin (e.g., aPKC-dependent lipid synthesis) are increased while other actions (e.g., PKB-dependent glucose metabolism) are diminished. This may explain the paradox that the liver secretes excessive amounts of both very low density lipoprotein triglycerides and glucose in Type II diabetes. Previous reviews from our laboratory that have appeared in the Proceedings have provided essentials on phospholipid-signaling mechanisms used by insulin to activate several protein kinases that seem to be important in mediating the metabolic effects of insulin. During recent years, there have been many new advances in our understanding of how these lipid-dependent protein kinases function during insulin action and why they fail to function in states of insulin resistance. The present review will attempt to summarize what we believe are some of the more important advances.

Requirements for pYXXM motifs in Cbl for binding to the p85 subunit of phosphatidylinositol 3-kinase and Crk, and activation of atypical protein kinase C and glucose transport during insulin action in 3T3/L1 adipocytes.

Cbl is phosphorylated by the insulin receptor and reportedly functions within the flotillin/CAP/Cbl/Crk/C3G/TC10 complex during insulin-stimulated glucose transport in 3T3/L1 adipocytes. Cbl, via pYXXM motifs at tyrosine-371 and tyrosine-731, also activates phosphatidylinositol (PI) 3-kinase, which is required to activate atypical protein kinase C (aPKC) and glucose transport during thiazolidinedione action in 3T3/L1 and human adipocytes [Miura et al. (2003) Biochemistry 42, 14335-14341]. Presently, we have examined the importance of Cbl in activating PI 3-kinase and aPKC during insulin action in 3T3/L1 adipocytes by expressing Y371F and Y731F Cbl mutants, which nullify pYXXM binding of Cbl to SH2 domains of downstream effectors. Interestingly, these mutants inhibited insulin-induced increases in (a) binding of Cbl to both Crk and the p85 subunit of PI 3-kinase, (b) activation of Cbl-dependent PI 3-kinase, (c) activation and translocation of aPKC to the plasma membrane, (d) translocation of Glut4 to the plasma membrane, (e) and glucose transport. Importantly, coexpression of wild-type Cbl reversed the inhibitory effects of Cbl mutants. In contrast to Cbl-dependent PI 3-kinase, Cbl mutants did not significantly inhibit the activation of PI 3-kinase by IRS-1, which is also required during insulin action. Our findings suggest that (a) Cbl uses pYXXM motifs to simultaneously activate PI 3-kinase and Crk/C3G/TC10 pathways and (b) Cbl, along with IRS-1, functions upstream of PI 3-kinase and aPKCs during insulin-stimulated glucose transport in 3T3/L1 adipocytes.

Insulin substrates 1 and 2 are corequired for activation of atypical protein kinase C and Cbl-dependent phosphatidylinositol 3-kinase during insulin action in immortalized brown adipocytes.

Phosphatidylinositol 3-kinase (PI3K)-dependent activation of atypical protein kinase C (aPKC) is required for insulin-stimulated glucose transport. Although insulin receptor substrate-1 (IRS-1) and IRS-2, among other factors, activate PI3K, there is little information on the relative roles of IRS-1and IRS-2 during aPKC activation by insulin action in specific cell types. Presently, we have used immortalized brown adipocytes in which either IRS-1 or IRS-2 has been knocked out by recombinant methods to examine IRS-1 and IRS-2 requirements for activation of aPKC. We have also used these adipocytes to see if IRS-1 and IRS-2 are required for activation of Cbl, which is required for insulin-stimulated glucose transport and has been found to function upstream of both PI3K/aPKC and Crk during thiazolidinedione action in 3T3/L1 adipocytes [Miura et al. (2003) Biochemistry 42, 14335]. In brown adipocytes in which either IRS-1 or IRS-2 was knocked out, insulin-induced increases in aPKC activity and glucose transport were markedly diminished. These effects of insulin on aPKC and glucose transport were fully restored by retroviral-mediated expression of IRS-1 or IRS-2 in their respective knockout cells. Knockout of IRS-1 or IRS-2 also inhibited insulin-induced increases in Cbl binding to the p85 subunit of PI3K, which, along with IRS-1/2, may be required for activation of PI3K, aPKC, and glucose transport during insulin action in 3T3/L1 adipocytes. These findings provide evidence that directly links both IRS-1 and IRS-2 to aPKC activation in immortalized brown adipocytes, and further suggest that IRS-1 and IRS-2 are required for the activation of Cbl/PI3K during insulin action in these cells.

Tissue-specific differences in activation of atypical protein kinase C and protein kinase B in muscle, liver, and adipocytes of insulin receptor substrate-1 knockout mice.

Insulin receptor substrates (IRSs) 1 and 2 are postulated to control the activation of phosphatidylinositol 3-kinase (PI3K)-dependent signaling factors, namely, atypical protein kinase C (aPKC) and protein kinase B (PKB)/Akt, which mediate metabolic effects of insulin. However, it is uncertain whether aPKC and PKB are activated together or differentially in response to IRS-1 and IRS-2 activation in insulin-sensitive tissues. Presently, we examined insulin activation of aPKC and PKB in vastus lateralis muscle, adipocytes, and liver in wild-type and IRS-1 knockout mice, and observed striking tissue-specific differences. In muscle of IRS-1 knockout mice, the activation of both aPKC and PKB was markedly diminished. In marked contrast, only aPKC activation was diminished in adipocytes, and only PKB activation was diminished in liver. These results suggest that IRS-1 is required for: 1) activation of both aPKC and PKB in muscle; 2) aPKC, but not PKB, activation in adipocytes; and 3) PKB, but not aPKC, activation in liver. Presumably, IRS-2 or other PI3K activators account for the normal activation of aPKC in liver and PKB in adipocytes of IRS-1 knockout mice. These complexities in aPKC and PKB activation may be relevant to metabolic abnormalities seen in tissues in which IRS-1 or IRS-2 is specifically or predominantly down-regulated.

Insulin-induced activation of atypical protein kinase C, but not protein kinase B, is maintained in diabetic (ob/ob and Goto-Kakazaki) liver. Contrasting insulin signaling patterns in liver versus muscle define phenotypes of type 2 diabetic and high fat-induced insulin-resistant states.

Insulin resistance in type 2 diabetes is characterized by defects in muscle glucose uptake and hepatic overproduction of both glucose and lipids. These hepatic defects are perplexing because insulin normally suppresses glucose production and increases lipid synthesis in the liver. To understand the mechanisms for these seemingly paradoxical defects, we examined the activation of atypical protein kinase C (aPKC) and protein kinase B (PKB), two key signaling factors that operate downstream of phosphatidylinositol 3-kinase and regulate various insulin-sensitive metabolic processes. Livers and muscles of three insulin-resistant rodent models were studied. In livers of type 2 diabetic non-obese Goto-Kakazaki rats and ob/ob-diabetic mice, the activation of PKB was impaired, whereas activation of aPKC was surprisingly maintained. In livers of non-diabetic high fatfed mice, the activation of both aPKC and PKB was maintained. In contrast to the maintenance of aPKC activation in the liver, insulin activation of aPKC was impaired in muscles of Goto-Kakazaki-diabetic rats and ob/ob-diabetic and non-diabetic high fat-fed mice. These findings suggest that, at least in these rodent models, (a) defects in aPKC activation contribute importantly to skeletal muscle insulin resistance observed in both high fat feeding and type 2 diabetes; (b) insulin signaling defects in muscle are not necessarily accompanied by similar defects in liver; (c) defects in hepatic PKB activation occur in association with, and probably contribute importantly to, the development of overt diabetes; and (d) maintenance of hepatic aPKC activation may explain the continued effectiveness of insulin for stimulating certain metabolic actions in the liver.

Protein kinase C-lambda knockout in embryonic stem cells and adipocytes impairs insulin-stimulated glucose transport.

Atypical protein kinase C (aPKC) isoforms have been suggested to mediate insulin effects on glucose transport in adipocytes and other cells. To more rigorously test this hypothesis, we generated mouse embryonic stem (ES) cells and ES-derived adipocytes in which both aPKC-lambda alleles were knocked out by recombinant methods. Insulin activated PKC-lambda and stimulated glucose transport in wild-type (WT) PKC-lambda(+/+), but not in knockout PKC-lambda(-/-), ES cells. However, insulin-stimulated glucose transport was rescued by expression of WT PKC-lambda in PKC-lambda(-/-) ES cells. Surprisingly, insulin-induced increases in both PKC-lambda activity and glucose transport were dependent on activation of proline-rich tyrosine protein kinase 2, the ERK pathway, and phospholipase D (PLD) but were independent of phosphatidylinositol 3-kinase (PI3K) in PKC-lambda(+/+) ES cells. Interestingly, this dependency was completely reversed after differentiation of ES cells to adipocytes, i.e. insulin effects on PKC-lambda and glucose transport were dependent on PI3K, rather than proline-rich tyrosine protein kinase 2/ERK/PLD. As in ES cells, insulin effects on glucose transport were absent in PKC-lambda(-/-) adipocytes but were rescued by expression of WT PKC-lambda in these adipocytes. Our findings suggest that insulin activates aPKCs and glucose transport in ES cells by a newly recognized PI3K-independent ERK/PLD-dependent pathway and provide a compelling line of evidence suggesting that aPKCs are required for insulin-stimulated glucose transport, regardless of whether aPKCs are activated by PI3K-dependent or PI3K-independent mechanisms.

Defective Activation of Protein Kinase C-z in Muscle by Insulin and Phosphatidylinositol-3,4,5,-(PO(4))(3) in Obesity and Polycystic Ovary Syndrome.

Insulin resistance occurs frequently in metabolic syndrome components, obesity, and the polycystic ovary syndrome, and is partly due to impaired glucose transport into skeletal muscle, but underlying mechanisms are uncertain. Atypical protein kinase C and protein kinase B, operating downstream of phosphatidylinositol 3-kinase, mediate insulin effects on glucose transport, but their importance in these syndromes is poorly understood. Presently, we examined these signaling factors in muscle biopsies obtained during euglycemic/hyperinsulinemic clamp studies. In lean subjects, insulin provoked approximately twofold increases in muscle atypical protein kinase C activity. In obese subjects and obese subjects who had evidence of the polycystic ovary syndrome, insulin-stimulated glucose disposal and atypical protein kinase C activation were diminished, whereas activation of insulin receptor substrate-1-dependent phosphatidylinositol 3-kinase and protein kinase B trended lower, but not significantly. Interestingly, direct activation of atypical protein kinase C by phosphatidylinositol-3,4,5-(PO(4))(3), the lipid product of phosphatidylinositol 3-kinase, was readily apparent in immunoprecipitates prepared from muscles of lean subjects, but to a lesser degree or poorly if at all in subjects who were obese or had the obesity/polycystic ovary syndrome. Our findings suggest that activation of muscle atypical protein kinase C by insulin and phosphatidylinositol-3,4,5-(PO(4))(3) is defective and may contribute to skeletal muscle insulin resistance in women who are obese, or have obesity associated with the polycystic ovary syndrome.

Cbl PYXXM motifs activate the P85 subunit of phosphatidylinositol 3-kinase, Crk, atypical protein kinase C, and glucose transport during thiazolidinedione action in 3T3/L1 and human adipocytes.

The thiazolidinedione (TZD), rosiglitazone, has previously been found to tyrosine-phosphorylate Cbl and activate Cbl-dependent phosphatidylinositol (PI) 3-kinase and atypical protein kinase Cs (aPKCs) while stimulating glucose transport in 3T3/L1 adipocytes. Presently, the role of Cbl in rosiglitazone action was further assessed in both 3T3/L1 and human adipocytes by expressing Y371F and/or Y731F mutant forms of Cbl that nullified the functionality of canonical pYXXM motifs in Cbl. These mutants diminished the interaction of Cbl with the p85 subunit of PI 3-kinase and inhibited subsequent increases in Cbl-dependent PI 3-kinase activity, aPKC activity, and glucose transport. These mutants also inhibited the interaction of Cbl with Crk, which has been implicated in the activation of other PI 3-kinase-independent signaling factors that have been found to be required during activation of glucose transport by insulin and other agonists. We conclude that pYXXM motifs in Cbl serve to activate PI 3-kinase-dependent and possibly PI 3-kinase-independent pathways that are required for TZD-dependent glucose transport in adipocytes.

Increased atypical PKC activity in endurance-trained human skeletal muscle.

Exercise training may modulate protein content and enzyme activities in skeletal muscle. However, it is not known whether atypical protein kinase C (aPKC) is affected by training. Thus, we investigated aPKC, extracellular-regulated protein kinase 1/2 (ERK 1/2), and P38 mitogen-activated protein kinase (P38 MAPK) activities and expression in skeletal muscle from untrained and endurance-trained subjects at rest and after 20min of cycle exercise (80% of VO(2peak)). Activities of aPKC (P<0.05) and ERK 1/2 (P=0.06), but not phosphorylation of P38 MAPK, were higher in trained than in sedentary subjects at rest. Exercise increased the activities of ERK 1/2 (P<0.01) and aPKC (P<0.05) and the phosphorylation (Thr180/Tyr182) of P38 MAPK (P<0.01) similarly in muscle from trained and sedentary subjects. Protein expression of the kinases was similar in trained and sedentary muscle. The increased aPKC activity in exercise-trained subjects could be important in explaining the enhanced insulin action in these individuals.

Activation of protein kinase C-zeta by insulin and phosphatidylinositol-3,4,5-(PO4)3 is defective in muscle in type 2 diabetes and impaired glucose tolerance: amelioration by rosiglitazone and exercise.

Insulin resistance in type 2 diabetes is partly due to impaired glucose transport in skeletal muscle. Atypical protein kinase C (aPKC) and protein kinase B (PKB), operating downstream of phosphatidylinositol (PI) 3-kinase and its lipid product, PI-3,4,5-(PO(4))(3) (PIP(3)), apparently mediate insulin effects on glucose transport. We examined these signaling factors during hyperinsulinemic-euglycemic clamp studies in nondiabetic subjects, subjects with impaired glucose tolerance (IGT), and type 2 diabetic subjects. In nondiabetic control subjects, insulin provoked twofold increases in muscle aPKC activity. In both IGT and diabetes, aPKC activation was markedly (70-80%) diminished, most likely reflecting impaired activation of insulin receptor substrate (IRS)-1-dependent PI 3-kinase and decreased ability of PIP(3) to directly activate aPKCs; additionally, muscle PKC-zeta levels were diminished by 40%. PKB activation was diminished in patients with IGT but not significantly in diabetic patients. The insulin sensitizer rosiglitazone improved insulin-stimulated IRS-1-dependent PI 3-kinase and aPKC activation, as well as glucose disposal rates. Bicycle exercise, which activates aPKCs and stimulates glucose transport independently of PI 3-kinase, activated aPKCs comparably to insulin in nondiabetic subjects and better than insulin in diabetic patients. Defective aPKC activation contributes to skeletal muscle insulin resistance in IGT and type 2 diabetes, rosiglitazone improves insulin-stimulated aPKC activation, and exercise directly activates aPKCs in diabetic muscle.

Defective activation of atypical protein kinase C zeta and lambda by insulin and phosphatidylinositol-3,4,5-(PO4)(3) in skeletal muscle of rats following high-fat feeding and streptozotocin-induced diabetes.

UNLABELLED: Insulin-stimulated glucose transport in skeletal muscle is thought to be effected at least partly through atypical protein kinase C isoforms (aPKCs) operating downstream of phosphatidylinositol (PI) 3-kinase and 3-phosphoinositide-dependent protein kinase-1 (PDK-1). However, relatively little is known about the activation of aPKCs in physiological conditions or insulin-resistant states. Presently, we studied aPKC activation in vastus lateralis muscles of normal chow-fed and high-fat-fed rats and after streptozotocin (STZ)-induced diabetes. In normal chow-fed rats, dose-dependent increases in aPKC activity approached maximal levels after 15-30 min of stimulation by relatively high and lower, presumably more physiological, insulin concentrations, achieved by im insulin or ip glucose administration. Insulin-induced activation of aPKCs was impaired in both high-fat-fed and STZ-diabetic rats, but, surprisingly, IRS-1-dependent and IRS-2-dependent PI 3-kinase activation was not appreciably compromised. Most interestingly, direct in vitro activation of aPKCs by PI-3,4,5-(PO(4))(3), the lipid product of PI 3-kinase, was impaired in both high-fat-fed and STZ-diabetic rats. Defects in activation of aPKCs by insulin and PI-3,4,5-(PO(4))(3) could not be explained by diminished PDK-1-dependent phosphorylation of threonine-410 in the PKC-zeta activation loop, as this phosphorylation was increased even in the absence of insulin treatment in high-fat-fed rats. CONCLUSIONS: 1) muscle aPKCs are activated at relatively low, presumably physiological, as well as higher supraphysiological, insulin concentrations; 2) aPKC activation is defective in muscles of high-fat-fed and STZ-diabetic rats; and 3) defective aPKC activation in these states is at least partly due to impaired responsiveness to PI-3,4,5-(PO(4))(3), apparently at activation steps distal to PDK-1-dependent loop phosphorylation.

Skeletal muscle insulin resistance in obesity-associated type 2 diabetes in monkeys is linked to a defect in insulin activation of protein kinase C-zeta/lambda/iota.

Rhesus monkeys frequently develop obesity and insulin resistance followed by type 2 diabetes when allowed free access to chow. This insulin resistance is partly due to defective glucose transport into skeletal muscle. In this study, we examined signaling factors required for insulin-stimulated glucose transport in muscle biopsies taken during euglycemic-hyperinsulinemic clamps in nondiabetic, obese prediabetic, and diabetic monkeys. Insulin increased activities of insulin receptor substrate (IRS)-1-dependent phosphatidylinositol (PI) 3-kinase and its downstream effectors, atypical protein kinase Cs (aPKCs) (zeta/lambda/iota) and protein kinase B (PKB) in muscles of nondiabetic monkeys. Insulin-induced increases in glucose disposal and aPKC activity diminished progressively in prediabetic and diabetic monkeys. Decreases in aPKC activation appeared to be at least partly due to diminished activation of IRS-1-dependent PI 3-kinase, but direct activation of aPKCs by the PI 3-kinase lipid product PI-3,4,5-(PO(4))(3) was also diminished. In conjunction with aPKCs, PKB activation was diminished in prediabetic muscle but, differently from aPKCs, seemed to partially improve in diabetic muscle. Interestingly, calorie restriction and avoidance of obesity largely prevented development of defects in glucose disposal and aPKC activation. Our findings suggest that defective activation of aPKCs contributes importantly to obesity-dependent development of skeletal muscle insulin resistance in prediabetic and type 2 diabetic monkeys.

Cbl, IRS-1, and IRS-2 mediate effects of rosiglitazone on PI3K, PKC-lambda, and glucose transport in 3T3/L1 adipocytes.

The thiazolidenedione, rosiglitazone, increases basal and/or insulin-stimulated glucose transport in various cell types by diverse but uncertain mechanisms that may involve insulin receptor substrate (IRS)-1-dependent PI3K. Presently, in 3T3/L1 adipocytes, rosiglitazone induced sizable increases in basal glucose transport that were: dependent on PI3K, 3-phosphoinositide-dependent protein kinase-1 (PDK-1), and PKC-lambda; accompanied by increases in tyrosine phosphorylation of Cbl and Cbl-dependent increases in PI3K and PKC-lambda activity; but not accompanied by increases in IRS-1/2-dependent PI3K or protein kinase B activity. Additionally, rosiglitazone increased IRS-1 and IRS-2 levels, thereby enhancing insulin effects on IRS-1- and IRS-2-dependent PI3K and downstream signaling factors PKC-lambda and protein kinase B. Our findings suggest that Cbl participates in mediating effects of rosiglitazone on PI3K, PDK-1, and PKC-lambda and the glucose transport system and that this Cbl-dependent pathway complements the IRS-1 and IRS-2 pathways for activating PI3K, PDK-1, and PKC-lambda during combined actions of rosiglitazone and insulin in 3T3/L1 cells.

Sorbitol activates atypical protein kinase C and GLUT4 glucose transporter translocation/glucose transport through proline-rich tyrosine kinase-2, the extracellular signal-regulated kinase pathway and phospholipase D.

Sorbitol, "osmotic stress", stimulates GLUT4 glucose transporter translocation to the plasma membrane and glucose transport by a phosphatidylinositol (PI) 3-kinase-independent mechanism that reportedly involves non-receptor proline-rich tyrosine kinase-2 (PYK2) but subsequent events are obscure. In the present study, we found that extracellular signal-regulated kinase (ERK) pathway components, growth-factor-receptor-bound-2 protein, son of sevenless (SOS), RAS, RAF and mitogen-activated protein (MAP) kinase/ERK kinase, MEK(-1), operating downstream of PYK2, were required for sorbitol-stimulated GLUT4 translocation/glucose transport in rat adipocytes, L6 myotubes and 3T3/L1 adipocytes. Furthermore, sorbitol activated atypical protein kinase C (aPKC) through a similar mechanism depending on the PYK2/ERK pathway, independent of PI 3-kinase and its downstream effector, 3-phosphoinositide-dependent protein kinase-1 (PDK-1). Like PYK2/ERK pathway components, aPKCs were required for sorbitol-stimulated GLUT4 translocation/glucose transport. Interestingly, sorbitol stimulated increases in phospholipase D (PLD) activity and generation of phosphatidic acid (PA), which directly activated aPKCs. As with aPKCs and glucose transport, sorbitol-stimulated PLD activity was dependent on the ERK pathway. Moreover, PLD-generated PA was required for sorbitol-induced activation of aPKCs and GLUT4 translocation/glucose transport. Our findings suggest that sorbitol sequentially activates PYK2, the ERK pathway and PLD, thereby increasing PA, which activates aPKCs and GLUT4 translocation. This mechanism contrasts with that of insulin, which primarily uses PI 3-kinase, D3-PO(4) polyphosphoinositides and PDK-1 to activate aPKCs.

Knockout of PKC alpha enhances insulin signaling through PI3K.

Insulin stimulates glucose transport and certain other metabolic processes by activating atypical PKC isoforms (lambda, zeta, iota) and protein kinase B (PKB) through increases in D3-polyphosphoinositides derived from the action of PI3K. The role of diacylglycerol-sensitive PKC isoforms is less clear as they have been suggested to be both activated by insulin and yet inhibit insulin signaling to PI3K. Presently, we found that insulin signaling to insulin receptor substrate 1-dependent PI3K, PKB, and PKC lambda, and downstream processes, glucose transport and activation of ERK, were enhanced in skeletal muscles and adipocytes of mice in which the ubiquitous conventional diacylglycerol-sensitive PKC isoform, PKC alpha, was knocked out by homologous recombination. On the other hand, insulin provoked wortmannin-insensitive increases in immunoprecipitable PKC alpha activity in adipocytes and skeletal muscles of wild-type mice and rats. We conclude that 1) PKC alpha is not required for insulin-stimulated glucose transport, and 2) PKC alpha is activated by insulin at least partly independently of PI3K, and largely serves as a physiological feedback inhibitor of insulin signaling to the insulin receptor substrate 1/PI3K/PKB/PKC lambda/zeta/iota complex and dependent metabolic processes.

PKC-zeta mediates insulin effects on glucose transport in cultured preadipocyte-derived human adipocytes.

Insulin-stimulated glucose transport is impaired in the early phases of type 2 diabetes mellitus. Studies in rodent cells suggest that atypical PKC (aPKC) isoforms (zeta, lamda, and iota) and PKB, and their upstream activators, PI3K and 3-phosphoinositide-dependent protein kinase-1 (PDK-1), play important roles in insulin-stimulated glucose transport. However, there is no information on requirements for aPKCs, PKB, or PDK-1 during insulin action in human cell types. Presently, by using preadipocyte-derived adipocytes, we were able to employ adenoviral gene transfer methods to critically examine these requirements in a human cell type. These adipocytes were found to contain PKC-zeta, rather than PKC-lamda/iota, as their major aPKC. Expression of kinase-inactive forms of PDK-1, PKC-zeta, and PKC-lamda (which functions interchangeably with PKC-zeta) as well as chemical inhibitors of PI 3-kinase and PKC-zeta/lamda, wortmannin and the cell-permeable myristoylated PKC-zeta pseudosubstrate, respectively, effectively inhibited insulin-stimulated glucose transport. In contrast, expression of a kinase-inactive, activation-resistant, triple alanine mutant form of PKB-alpha had little or no effect, and expression of wild-type and constitutively active PKC-zeta or PKC-lamda increased glucose transport. Our findings provide convincing evidence that aPKCs and upstream activators, PI 3-kinase and PDK-1, play important roles in insulin-stimulated glucose transport in preadipocyte-derived human adipocytes.