Incubation of 3T3-L1 adipocytes with C2- and C6- ceramides (N-acetyl- and N-hexanoylsphingosines) but not dihydro-C2-ceramide increased 2-deoxyglucose uptake in the absence of insulin. This effect was inhibited by PD 98059, LY 294002, and rapamycin, which block the activation of mitogen-activated protein kinase, phosphatidylinositol (PI) 3-kinase, and ribosomal S6 kinase, respectively. Long-term increases in PI 3-kinase activity associated with insulin receptor substrate 1 (IRS-1) increased GLUT1 and GLUT4 concentrations in plasma membranes. This together with increased GLUT1 (but not GLUT4) synthesis explains the increase in non-insulin-dependent glucose uptake. C2-ceramide inhibited insulin-stimulated glucose uptake after 2 h by decreasing insulin-induced translocation of GLUT1 and GLUT4 to plasma membranes. This occurred when there was no increase in basal glucose uptake or decrease in activation of IRS-1 or PI 3-kinase. Incubation for 24 h with tumor necrosis factorα (TNF-α) but not C2-ceramide decreased the concentration and insulin-induced tyrosine phosphorylation of IRS-1 in this experimental system. Cell-permeable ceramides mimic some effects of TNF-α, especially in stimulating basal glucose uptake. We identified a site for inhibiting insulin-stimulated glucose uptake that is downstream of PI 3-kinase. Our work provides further mechanisms for the effects of TNF-α and ceramides in increasing non-insulin-dependent glucose uptake and decreasing insulin-stimulated uptake in vivo.

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