In type 2 diabetes, pancreatic beta cells fail to secrete sufficient insulin to overcome peripheral insulin resistance. Intracellular lipid accumulation contributes to beta cell failure through poorly defined mechanisms. Here we report a role for the lipid-regulated protein kinase C isoform PKCepsilon in beta cell dysfunction. Deletion of PKCepsilon augmented insulin secretion and prevented glucose intolerance in fat-fed mice. Importantly, a PKCepsilon-inhibitory peptide improved insulin availability and glucose tolerance in db/db mice with preexisting diabetes. Functional ablation of PKCepsilon selectively enhanced insulin release ex vivo from diabetic or lipid-pretreated islets and optimized the glucose-regulated lipid partitioning that amplifies the secretory response. Independently, PKCepsilon deletion also augmented insulin availability by reducing both whole-body insulin clearance and insulin uptake by hepatocytes. Our findings implicate PKCepsilon in the etiology of beta cell dysfunction and highlight that enhancement of insulin availability, through separate effects on liver and beta cells, provides a rationale for inhibiting PKCepsilon to treat type 2 diabetes.

Aims/hypothesis This study aimed to determine whether protein kinase C (PKC) δ plays a role in the glucose intolerance caused by a high-fat diet, and whether it could compensate for loss of PKCε in the generation of insulin resistance in skeletal muscle. Methods Prkcd−/−

The protein kinase Akt mediates several metabolic and mitogenic effects of insulin, whereas activation of protein kinase C (PKC) isoforms has been implicated in the inhibition of insulin action. We have previously shown that both PKC and PKC⑀ are activated in skeletal muscle of insulin-resistant high fat-fed rats, and to identify potential substrates for these kinases, we incubated recombinant PKC isoforms with rat muscle fractions in vitro. PKC specifically phosphorylated a 48-kDa protein that was subsequently identified by mass spectrometry as Ndrg2. Ndrg2 is highly related to N-Myc downstream-regulated protein 1, which has been linked to stress responses, cell proliferation, and differentiation, although Ndrg2 itself is not repressed by N-Myc. Ndrg2 contains several potential phosphorylation sites, including three Akt consensus sequences. Ndrg2 phosphorylation was enhanced in [ 32 P]orthophosphate-labeled C2C12 muscle cells co-overexpressing either PKC or Akt. Phosphorylation of Ndrg2 was examined further using a phospho (Ser/Thr) Akt substrate antibody. Insulin increased Ndrg2 phosphorylation in C2C12 cells in a wortmannin-and palmitate-inhibitable manner, whereas rapamycin, PD98059, and bisindoylmaleimide I had no effect, supporting a direct role for Akt. Mutation of Ndrg2 indicated that Thr-348 is the major phosphorylation site detected by the antibody and that Akt stimulates phosphorylation of this site, whereas PKC phosphorylates Ser-332. PKC overexpression, however, diminished the effect of insulin on Thr-348 phosphorylation without reducing Akt activation, suggesting that this is mediated through phosphorylation of Ndrg2 at Ser-332. Our data identify Ndrg2 as a novel insulin-dependent phosphoprotein and suggest that PKC may inhibit insulin action in part by reducing its phosphorylation by Akt.The acute activation of the lipid-dependent Ser/Thr protein kinase PKC 1 has been well characterized. Three groups of PKC have been established: the cPKCs ␣, ␤, and ␥, which are activated by calcium and DAG; the nPKCs, e.g. ␦, ⑀, and , which require DAG; and the aPKCs, and /, which are independent of both calcium and DAG (1). The essential role of specific binding partners such as the RACKS and A-kinase anchoring proteins in the spatial co-ordination of PKC isoforms is also becoming apparent (2). Similarly, a number of substrates, which are directly phosphorylated by PKCs in particular cell types and which mediate PKC effects on cellular regulation, is emerging.Specific PKC isoforms have been implicated in the inhibition of insulin action. Insulin resistance of target tissues such as skeletal muscle is a major contributing factor to the development of type 2 diabetes, and activation of nPKCs, including PKC, have been correlated with insulin resistance in a number of studies, especially in association with increased lipid availability (3, 4). The mechanisms involved, however, including the targets for PKC-mediated phosphorylation, remain unclear. PKCs could interfere at one or more steps in insulin signal transduct...

Exposure to prolonged hypoxia can result in pulmonary vascular remodeling and pulmonary hypertension. Hypoxia induces pulmonary vascular smooth muscle cell (PVSMC) proliferation and vascular remodeling by affecting cell adhesion and migration and secretion of extracellular matrix proteins. We previously showed that acute hypoxia decreases cGMP-dependent protein kinase (PKG) activity in PVSMC and that PKG plays a role in maintaining the differentiated contractile phenotype in normoxia. In this study, we investigated the effect of hypoxia on PVSMC adhesion and migration and the role of PKG in these functions. Ovine fetal pulmonary artery SMC were incubated in normoxia (Po(2) approximately 100 Torr) or hypoxia (Po(2) approximately 30-40 Torr) or treated with the PKG inhibitor DT-3 for 24 h in normoxia. To further study the role of PKG in the modulation of adhesion and migration, PVSMC were transiently transfected with a full-length PKG1alpha [PKG-green fluorescent protein (GFP)] or a dominant-negative construct (G1alphaR-GFP). Cell adhesion to extracellular matrix proteins was determined, and integrin-mediated adhesion was assessed by alpha/beta-integrin-mediated cell adhesion array. Exposure to hypoxia (24 h) and pharmacological inhibition of PKG1 by DT-3 significantly promoted adhesion mediated by alpha(4)-, beta(1)-, and alpha(5)beta(1)-integrins to fibronectin, laminin, and tenacin and also resulted in increased cell migration. Likewise, inhibition of PKG by expression of a dominant-negative PKG1alpha construct increased cell adhesion and migration, comparable to that induced by hypoxia. Dynamic actin reorganization associated with integrin-mediated cell adhesion is partly regulated by the actin-binding protein cofilin, the (Ser3) phosphorylation of which inhibits its actin-severing activity. We found that increased PKG expression and activity is associated with decreased cofilin (Ser3) phosphorylation, implying a role for PKG in the modulation of cofilin activity and actin dynamics. Together, these findings identify cGMP/PKG1 signaling as central to the functional differences between PVSMC exposed to normoxia versus hypoxia.

The fetal lung develops in a low oxygen environment with programmed growth of the pulmonary vasculature. To investigate the regulation of proliferation of pulmonary vascular smooth muscle cells (PVSMCs), we studied the effect of hypoxia on near-term ovine fetal PVSMC adhesion, migration, and proliferation. Both arterial (PA) and venous (PV) SMCs were incubated in normoxia (pO2 ≈100 torr) or hypoxia (pO2 ≈30-40 torr) for 24, 48, and 72 h. Cell adhesion to various matrix proteins (collagen I, collagen IV, fibronectin, laminin) was determined by measuring absorbance of a dye staining cell membranes, cell migration was assessed by a wound healing model, and cell proliferation was measured by a cell counting kit. We also measured cGMP-dependent protein kinase (PKG) protein expression by Western blotting. As previously reported, exposure to hypoxia produced a decrease in PKG protein expression in both PA and PV SMC. Hypoxic exposure resulted in a decrease in cell adhesion, an increase in cell migration, and an increase in cell proliferation in both PA and PV SMCs. Inhibition of PKG kinase activity by preincubation with Rp-8-Br-PET-cGMPS (a cyclic guanosine 3Ј,5Ј-cyclic monophosphate analogue) in normoxia decreased cell adhesion in both cell types. We conclude that in hypoxia, inhibition of PKG protein expression and kinase activity promotes PVSMC growth and proliferation by reducing cell adhesion.