NAFLD is strongly linked to hepatic insulin resistance, and T2DM and is now the most common chronic liver disease in the United States (14
). We have previously shown that hepatic steatosis results in insulin resistance with the development of fat-induced defects in the insulin signaling pathway. Specifically, the ability of the insulin receptor to tyrosine-phosphorylate its substrates IRS1 and IRS2 is diminished. This proximal defect in the insulin signaling cascade limits the ability of insulin to suppress hepatic glucose production by inhibiting gluconeogenesis and stimulating glycogen synthesis. Moreover, we observed that the development of diet-induced hepatic steatosis was associated with the activation of PKCε (7
). The present set of studies was undertaken to ascertain whether or not PKCε plays a causal role in the development of hepatic insulin resistance.
Using ASOs, we can specifically inhibit a gene of interest in adult animals. In this instance, we inhibited PKCε in adult rats and then assessed changes in glucose metabolism after subjecting them to 3 days of high-fat feeding to induce hepatic steatosis and hepatic insulin resistance. Here we establish that decreasing PKCε expression protects against the development of hepatic insulin resistance and improves adipose insulin action by augmenting the activity of the insulin signaling pathway. Furthermore, these studies show that PKCε may induce insulin resistance by directly interfering with the activation of the insulin receptor kinase by insulin.
PKCε ASO specifically inhibits PKCε, without altering the levels of other PKC isoforms. Specifically, there was no change in the abundance of PKCδ, another novel PKC isoform that has been implicated in the development of hepatic insulin resistance after Intralipid infusion (15
). In addition, none of the treatments altered liver lipid content (i.e., triglyceride and DAG). This is important since DAG is a known activator of classic and novel isoforms of PKC and DAG concentrations have closely paralleled insulin resistance in other models (7
). Thus, in this model system, we specifically inhibit PKCε without altering other PKC isoforms or the concentration of PKC activators.
Neither fasting plasma glucose concentrations nor basal hepatic glucose production rates were affected by PKCε ASO therapy. Despite the lack of differences in these parameters, fasting plasma insulin concentrations were approximately 50% lower in PKCε ASO–treated rats compared with saline- and control ASO–treated rats. The mean insulin concentration in the PKCε rats was similar to that seen in normal rats on a low-fat diet. This observation, in and of itself, suggests that knockdown of PKCε expression improves insulin sensitivity in high-fat fed rats. The results of hyperinsulinemic-euglycemic clamp studies confirm this. Under hyperinsulinemic conditions, suppression of hepatic glucose production was markedly greater in the PKCε group than in the saline and control ASO groups. Moreover, analysis of the insulin signaling pathway demonstrated that insulin signaling was improved in PKCε-treated rats. Whereas the ability of insulin to increase IRS2 tyrosine phosphorylation was blunted in the saline- and control ASO–treated rats, there was a robust increase in IRS2 tyrosine phosphorylation in the PKCε ASO–treated rats. This same pattern of activation was reflected in insulin-stimulated AKT2 activity. Thus, PKCε ASO treatment prevents the defects in insulin signaling that lead to the development of hepatic insulin resistance.
In addition to the improvements in hepatic insulin action, PKCε ASO improved adipose insulin action. Specifically, augmented insulin activation of AKT2 was associated with improved insulin-stimulated glucose uptake. These findings raise the possibility that PKCε may also play a role in adipose insulin action. While PKCε has been shown to be expressed in adipocytes and in 3T3 fibroblasts (17
), its role in the adipocyte is uncertain. PKCε expression has been shown to promote differentiation of 3T3 cells into adipocytes and increase production of IL-6 (19
). However, in the present study, we did not find any significant difference in the concentrations of key adipokines. It is possible that PKCε may directly improve adipose insulin signaling analogously to what we have documented in the liver.
The protective ability of PKCε ASO suggests that PKCε plays a crucial role in the pathogenesis of fat-induced hepatic insulin resistance. Specifically, the finding that decreasing expression of PKCε preserves the ability of the insulin receptor to tyrosine-phosphorylate IRS2 suggests that PKCε interferes with this critical step. Several reports have suggested that insulin resistance may in fact develop from PKC-mediated inhibition of the insulin receptor (20
). Coghlan and colleagues identified several potential PKC phosphorylation sites on the insulin receptor but failed to detect increased phosphorylation of the insulin receptor in muscle biopsies obtained from diabetic subjects (24
). Thus, while PKCs, in general, have been implicated in the pathogenesis of insulin resistance, we delineate the specific role of PKCε in the development of fat-induced hepatic insulin resistance. First, we show here that PKCε and insulin receptor directly associate in vivo, as they each coimmunoprecipitate the other. Second, we demonstrate that incubation of active PKCε with active insulin receptor-β led to a dose-dependent decrease in insulin receptor-β kinase activity, thereby suggesting that PKCε may constrain insulin signaling by hindering the ability of the insulin receptor kinase to tyrosine-phosphorylate its substrates. Finally, we assayed the kinase activity of insulin receptor purified from liver. Insulin activation of insulin receptor kinase activity was diminished in saline- and control ASO–treated high-fat-fed rats as compared with control (low-fat-fed) animals. In comparison, PKCε ASO treatment significantly improved insulin receptor kinase activity. Thus, these data suggest that the diminished tyrosine phosphorylation of IRS2 seen in steatotic livers is a consequence of PKCε-mediated inhibition of the insulin receptor kinase.
Taken together, these data strongly support an important role for PKCε in mediating fat-induced hepatic insulin resistance. On the basis of these results, we hypothesize that fat-induced hepatic insulin resistance arises from DAG-induced activation of PKCε, which directly binds to and inhibits insulin receptor tyrosine kinase activity. A similar mechanism may be present in patients with diabetes; this is supported by Considine et al., who observed activation of PKCε in the livers of obese diabetic patients compared with lean, normoglycemic control subjects (26
). Taken together, these data suggest that PKCε inhibition is a novel therapeutic strategy for treatment of hepatic insulin resistance in patients with NAFLD and T2DM.