Chronic activation of protein kinase C in soleus muscles and other tissues of insulin-resistant type II diabetic Goto-Kakizaki (GK), obese/aged, and obese/Zucker rats. A mechanism for inhibiting glycogen synthesis.

We examined the possibility that protein kinase C (PKC) is chronically activated and may contribute to impaired glycogen synthesis and insulin resistance in soleus muscles of hyperinsulinemic type II diabetic Goto-Kakizaki (GK) rats. Relative to nondiabetic controls, PKC enzyme activity and levels of immunoreactive PKC-alpha, beta, epsilon, and delta were increased in membrane fractions and decreased cytosolic fractions of GK soleus muscles. In addition, PKC-theta levels were decreased in both membrane and cytosol fractios, whereas PKC-zeta levels were not changed in either fraction in GK soleus muscles. These increases in membrane PKC (alpha, beta, epsilon, and delta) could not be accounted for by alterations in PKC mRNA or total PKC levels but were associated with increases in membrane diacylglycerol (DAG) and therefore appeared to reflect translocative activation of PKC. In evaluation of potential causes for persistent PKC activation, membrane PKC levels were decreased in soleus muscles of hyperglycemic streptozotocin (STZ)-induced diabetic rats; thus, a role for simple hyperglycemia as a cause of PKC activation in GK rats was not evident in the STZ model. In support of the possibility that hyperinsulinemia contributed to PKC activation in GK soleus muscles, we found that DAG levels were increased, and PKC was translocated, in soleus muscles of both (1) normoglycemic hyperinsulinemic obese/aged rats and (2) mildly hyperglycemic hyperinsulinemic obese/Zucker rats. In keeping with the possibility that PKC activation may contribute to impaired glycogen synthase activation in GK muscles, phorbol esters inhibited, and a PKC inhibitor, RO 31-8220, increased insulin effects on glycogen synthesis in soleus muscles incubated in vitro. Our findings suggested that: (1) hyperinsulinemia, as observed in type II diabetic GK rats and certain genetic and nongenetic forms of obesity in rats, is associated with persistent translocation and activation of PKC in soleus muscles, and (2) this persistent PKC activation may contribute to impaired glycogen synthesis and insulin resistance.

Insulin translocates PKC-epsilon and phorbol esters induce and persistently translocate PKC-beta 2 in BC3H-1 myocytes.

Initial studies suggested that insulin increases diacylglycerol and activates protein kinase C (PKC) in BC3H-1 myocytes. In these earlier studies, insulin was found to translocate PKC-beta, but the presence of PKC-epsilon was not appreciated. More recently, the presence of PKC-epsilon was documented, but PKC-beta was not detected, and it was questioned whether insulin activates PKC in BC3H-1 myocytes [Stumpo, D.J., Haupt, D.M. and Blackshear, P.J. (1994) J. Biol. Chem. 269:21184-21190]. We questioned whether insulin translocates PKC-epsilon in BC3H-1 myocytes, and re-evaluated the question of whether myocytes truly contain a PKC-beta isoform whose existence can be verified by its response to phorbol ester treatment. We found that PKC-epsilon was acutely translocated by insulin and phorbol esters from the cytosol to the membrane fraction in BC3H-1 myocytes; in addition, PKC-epsilon, like PKC-alpha, was depleted by chronic phorbol ester treatment. We also found that BC3H-1 myocytes containing a 76,000 Mr PKC-beta isoform that is acutely translocated and subsequently depleted by phorbol esters. Moreover, chronic phorbol ester treatment induced an 84,000 Mr PKC-beta 2 isoform that appeared to be persistently translocated and activated, as suggested by studies of myristoylated arginic-rich C kinase substrate (MARCKS) phosphorylation. We conclude that: (1) insulin acutely translocates PKC-epsilon, as well as PKC-beta, in BC3H-1 myocytes; and (2) PKC-beta is not truly downregulated by phorbol esters in BC3H-1 myocytes.