Previous findings in diabetic rodents suggest that insulin activation of atypical protein kinase C (aPKC) is impaired in muscle, but surprisingly conserved in liver, despite impaired hepatic PKB/Akt activation. Moreover, aPKC at least partly regulates two major trans-activators, viz.,hepatic sterol receptor binding protein-1c (SREBP-1c), which controls lipid synthesis, and NFκB, which promotes inflammation and systemic insulin resistance. Presently, in type 2 diabetic Goto-Kakizaki (GK) rats, we examined whether: (a) differences in hepatic aPKC and PKB activation reflect differences in activation of insulin receptor substrate (IRS)-1- and IRS-2-dependent phosphatidylinositol 3-kinase (PI3K); (b) hepatic SREBP-1c and NFκB are excessively activated by aPKC; and (c) metabolic consequences of excessive hepatic aPKC/SREBP-1c/NFκB activation. We found that, in liver as well as in muscle, whereas IRS-1/PI3K activation by insulin was impaired, IRS-2/PI3K was intact. Moreover, selective inhibition of hepatic aPKC by adenoviral-mediated expression of either kinase-inactive aPKC, or shRNA that targets and partially depletes hepatic IRS-2, which controls hepatic aPKC during insulin activation, diminished hepatic SREBP-1c expression and NFκB activities, and concomitantly improved serum lipids and insulin signalling in muscle and liver. Similar improvements in SREBP-1c, NFκB and insulin signaling were seen in ob/ob mice following inhibition of hepatic aPKC. Our findings suggest that, in diabetic rodents: (a) in liver, diminished PKB activation largely reflects impaired IRS-1/PI3K activation, and conserved aPKC activation reflects retained IRS-2/PI3K activity; (b) hepatic aPKC contributes importantly to excessive SREPB-1c and NFκB activities; and (c) excessive hepatic aPKC-dependent activation of SREBP-1c and NFκB contributes importantly to hyperlipidaemia and systemic insulin resistance.

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-in L6 myotubes and PKC-in 3T3/L1 adipocytes, that nevertheless can function interchangeably for glucose transport. Accordingly, in L6 myotubes, RNAi-targeting PKC-, but not PKC-, markedly depleted aPKC and concomitantly inhibited insulin-stimulated glucose transport; more importantly, these depleting/inhibitory effects were rescued by adenovirally mediated expression of PKC-. Conversely, in 3T3/L1 adipocytes, RNAi constructs targeting PKC-, but not PKC-, markedly depleted aPKC and concomitantly inhibited insulin-stimulated glucose transport; here again, these depleting/inhibitory effects were rescued by adenovirally mediated expression of PKC-. 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.Transport is the initial rate-limiting step for cellular uptake and utilization of glucose in skeletal muscle and adipocytes. Insulin is a major controller of glucose transport in adipocytes and skeletal muscle, and defects in insulin-stimulated glucose transport, particularly in skeletal muscle, contribute importantly to the development of insulin resistance in obesity and type 2 diabetes mellitus.Insulin regulates glucose transport in adipocytes and skeletal muscle through the activation of insulin receptor substrate (IRS) 2 -1/2-dependent phosphatidylinositol 3-kinase. Distal effectors of phosphatidylinositol 3-kinase that are postulated to mediate insulininduced increases in glucose transport include protein kinase B (PKB/Akt) and atypical protein kinase C (aPKC) isoforms, and . The initial evidence suggesting requirements for PKB (1-4) and aPKC (5-13) in insulin-stimulated glucose transport was based largely upon inhibitory effects ensuing from expression of kinaseinactive (dominant-negative) forms of these protein kinases in adipocytes and muscle cells. More recently, genetically manipulated adipocytes deficient in either PKB (14) or aPKC (15) have been used to further implicate these protein kinases in insulin-stimulated glucose transport. In these gene knock-out studies, insulin-stimulate...

Aims/hypothesis: Metformin is widely used for treating type 2 diabetes mellitus, but its actions are poorly understood. In addition to diminishing hepatic glucose output, metformin, in muscle, activates 5'-AMP-activated protein kinase (AMPK), which alone increases glucose uptake and glycolysis, diminishes lipid synthesis, and increases oxidation of fatty acids. Moreover, such lipid effects may improve insulin sensitivity and insulinstimulated glucose uptake. Nevertheless, the effects of metformin on insulin-sensitive signalling factors in human muscle have only been partly characterised to date. Interestingly, other substances that activate AMPK, e.g., aminoimidazole-4-carboxamide-1-β-D-riboside (AICAR), simultaneously activate atypical protein kinase C (aPKC), which appears to be required for the glucose transport effects of AICAR and insulin. Methods: Since aPKC activation is defective in type 2 diabetes, we evaluated effects of metformin therapy on aPKC activity in muscles of diabetic subjects during hyperinsulinaemic-euglycaemic clamp studies. Results: After metformin therapy for 1 month, basal aPKC activity increased in muscle, with little or no change in insulin-stimulated aPKC activity. Metformin therapy for 8 to 12 months improved insulinstimulated, as well as basal aPKC activity in muscle. In contrast, IRS-1-dependent phosphatidylinositol (PI) 3-kinase activity and Ser473 phosphorylation of protein kinase B were not altered by metformin therapy, whereas the responsiveness of muscle aPKC to PI-3,4,5-(PO 4 ) 3 , the lipid product of PI 3-kinase, was improved. Conclusions/ interpretation: These findings suggest that the activation of AMPK by metformin is accompanied by increases in aPKC activity and responsiveness in skeletal muscle, which may contribute to the therapeutic effects of metformin.