We used adenoviral gene transfer methods to evaluate the role of atypical protein kinase Cs (PKCs) during insulin stimulation of glucose transport in L6 myotubes. Expression of wild-type PKC-potentiated maximal and half-maximal effects of insulin on 2-deoxyglucose uptake, but did not alter basal uptake. Expression of constitutively active PKC-enhanced basal 2-deoxyglucose uptake to virtually the same extent as that observed during insulin treatment. In contrast, expression of kinase-defective

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... C-completely blocked insulin-stimulated, but not basal, 2-deoxyglucose uptake. Similar to alterations in glucose transport, constitutively active PKC-mim-icked, and kinase-defective PKC-completely inhibited, insulin effects on GLUT4 glucose transporter translocation to the plasma membrane. Expression of kinase-defective PKC-, in addition to inhibition of atypical PKC enzyme activity, was attended by paradoxical increases in GLUT4 and GLUT1 glucose transporter levels and insulinstimulated protein kinase B enzyme activity. Our findings suggest that in L6 myotubes, 1) atypical PKCs are required and sufficient for insulin-stimulated GLUT4 translocation and glucose transport; and 2) activation of protein kinase B in the absence of activation of atypical PKCs is insufficient for insulin-induced activation of glucose trans-