Activation of Protein Kinase C (α, β, and ζ) by Insulin in 3T3/L1 Cells

We presently studied (a) insulin effects on protein kinase C (PKC) and (b) effects of transfection-induced, stable expression of PKC isoforms on glucose transport in 3T3/L1 cells. In both fibroblasts and adipocytes, insulin provoked increases in membrane PKC enzyme activity and membrane levels of PKC-α and PKC-β. However, insulin-induced increases in PKC enzyme activity were apparent in both non-down-regulated adipocytes and adipocytes that were down-regulated by overnight treatment with 5 μM phorbol ester, which largely depletes PKC-α, PKC-β, and PKC-ϵ, but not PKC-η. Moreover, insulin provoked increases in the enzyme activity of immunoprecipitable PKC-η. In transfection studies, stable overexpression of wild-type or constitutively active forms of PKC-α, PKC-β1, and PKC-β2 failed to influence basal or insulin-stimulated glucose transport (2-deoxyglucose uptake) in fibroblasts and adipocytes, despite inhibiting insulin effects on glycogen synthesis. In contrast, stable overexpression of wild-type PKC-η increased, and a dominant-negative mutant form of PKC-η decreased, basal and insulin-stimulated glucose transport in fibroblasts and adipocytes. These findings suggested that: (a) insulin activates PKC-η, as well as PKC-α and β; and (b) PKC-η is required for, and may contribute to, insulin effects on glucose transport in 3T3/L1 cells. We presently studied (a) insulin effects on protein kinase C (PKC) and (b) effects of transfection-induced, stable expression of PKC isoforms on glucose transport in 3T3/L1 cells. In both fibroblasts and adipocytes, insulin provoked increases in membrane PKC enzyme activity and membrane levels of PKC-α and PKC-β. However, insulin-induced increases in PKC enzyme activity were apparent in both non-down-regulated adipocytes and adipocytes that were down-regulated by overnight treatment with 5 μM phorbol ester, which largely depletes PKC-α, PKC-β, and PKC-ϵ, but not PKC-η. Moreover, insulin provoked increases in the enzyme activity of immunoprecipitable PKC-η. In transfection studies, stable overexpression of wild-type or constitutively active forms of PKC-α, PKC-β1, and PKC-β2 failed to influence basal or insulin-stimulated glucose transport (2-deoxyglucose uptake) in fibroblasts and adipocytes, despite inhibiting insulin effects on glycogen synthesis. In contrast, stable overexpression of wild-type PKC-η increased, and a dominant-negative mutant form of PKC-η decreased, basal and insulin-stimulated glucose transport in fibroblasts and adipocytes. These findings suggested that: (a) insulin activates PKC-η, as well as PKC-α and β; and (b) PKC-η is required for, and may contribute to, insulin effects on glucose transport in 3T3/L1 cells.