Insulin was found to provoke rapid increases in diacylglycerol (DAG) content and [3H]glycerol incorporation into DAG and other lipids during incubations of rat hemidiaphragms and soleus muscles. Insulin also rapidly increased phosphatidic acid and total glycerolipid labeling by [3H]glycerol, suggesting that insulin increases DAG production at least partly through stimulation of the de novo pathway. Increased DAG production may activate protein kinase C (PKC) as reported previously in the rat diaphragm. We also observed apparent insulininduced translocation of PKC from cytosol to membrane in the rat soleus muscle. The importance of insulin-induced increases in DAG-PKC signaling in the stimulation of glucose transport in rat diaphragm and soleus muscles was suggested by 1) PKC activators phorbol esters and phospholipase C stimulation of [3H]-2-deoxyglucose (DOG) uptake and 2) PKC inhibitors staurosporine and polymixin B inhibition of insulin effects on [3H]-2-DOG uptake. Although phorbol ester was much less effective than insulin in the diaphragm, phospholipase C provoked increases in [3H]-2-DOG uptake that equaled or exceeded those of insulin. In the soleus muscle, phorbol ester, like phospholipase C, was only slightly but not significantly less effective than insulin. Similar variability in effectiveness of phorbol ester has also been noted previously in rat adipocytes (weak) and BC3HI myocytes (strong), whereas DAG, added exogenously or generated by phospholipase C treatment, stimulates glucose transport to a degree that is quantitatively more comparable to that of insulin in each of the four tissues. Differences in effectiveness of phorbol ester and DAG could not be readily explained by postulating that the latter acts independently of PKC, because DAG provoked the apparent translocation of the enzyme from cytosol to membranes in rat adipocytes, and effects of DAG on [3H]-2-DOG uptake were blocked by inhibitors of PKC in both rat adipocytes and BC3H1 myocytes. Collectively, our findings provide further support for the hypothesis that insulin increases DAG production and PKC activity, and these processes are important in the stimulation of glucose transport in rat skeletal muscle and other tissues.

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