Effects of Cell-Permeable Ceramides and Tumor Necrosis Factor-α on Insulin Signaling and Glucose Uptake in 3T3-L1 Adipocytes

Abstract

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 factor-alpha (TNF-alpha) 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-alpha, 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-alpha and ceramides in increasing non-insulin-dependent glucose uptake and decreasing insulin-stimulated uptake in vivo.

Adipocyte and erythrocyte plasma membrane phospholipid composition and hyperinsulinemia: a study in nondiabetic and diabetic obese women

“…Ceramide mediates the downregulation of glucose transporter (GLUT4) mRNA 30 by TNF-a and the decrease in GLUT4 translocation in 3T3-L1 adipocytes. 31,32 Recent data also implicate ceramide in the impaired tyrosine phosphorylation of the IRS-1. 33 Although phosphatidylethanolamine (PE) and phosphatidylcholine (PC) have not been included in the multivariable model (in adipocyte or in erythrocyte), their role cannot be ruled out.…”

Cell-permeable ceramides increase basal glucose incorporation into triacylglycerols but decrease the stimulation by insulin in 3T3-L1 adipocytes

“…20 In other work, TNFa did not decrease the tyrosine phosphorylation of IRS-1, but rather it decreased the concentrations of IRS-1 and GLUT4 in adipocytes. 21,22 This effect was not mediated by signalling through ceramides. 22 In fact, ceramides stimulated rather than inhibited PI 3-K activity associated with IRS-1 and the ceramide-induced inhibition of insulin-stimulated glucose probably resulted from decreased insulin-induced transport of vesicles containing GLUT1 and GLUT4 to the cell surface.…”

“…Other reagents were purchased as follows: electrophoresis reagents, Bio-Rad, Mississauga, Canada; polyvinylidene difluoride membranes, Millipore, Bedford, MA, USA; DMEM and foetal bovine serum, Gibco Cell culture Murine 3T3-L1 fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum and then differentiated as described previously. 22,31 The medium was then changed and the cells were treated with the indicated agents for various times. The final concentrations of C 2 -and C 6 -ceramides were 50 or 100 mM as established previously 22 and they were added to the albumin or serum containing solution in DMSO (final concentration < 0.3%).…”

“…22 In fact, ceramides stimulated rather than inhibited PI 3-K activity associated with IRS-1 and the ceramide-induced inhibition of insulin-stimulated glucose probably resulted from decreased insulin-induced transport of vesicles containing GLUT1 and GLUT4 to the cell surface. 22 Our later studies with Rat2 fibroblasts demonstrate that TNFa, sphingomyelinase and C 2 -ceramide rapidly stimulate tyrosine kinases, production of Ras-GTP and its association with activated PI 3-K. 23,24 Furthermore, incubation of 3T3-L1 adipocytes with TNFa increases the tyrosine phosphorylation of IRS-1 and its binding to PI 3-K. 25 Ceramides also activate Src and PI 3-K in smooth muscle cells. 26 In L6 myocytes, ceramides inhibit insulin-stimulated glucose uptake, but this did not depend upon decreased IRS-1 phosphorylation, or PI 3-K activity.…”

“…28 In contrast to these effects on insulin-stimulated glucose uptake, TNFa and ceramides stimulate insulin-independent glucose uptake in the long-term in 3T3-L1 adipocytes through PI 3-K activation. 22,29,30 The present work was performed to expand this hypothesis and to understand how ceramide-induced changes in insulin sensitivity modify glucose storage as triacylglycerol (TAG). Ceramides rapidly decreased the insulin-stimulated incorporation of glucose into the fatty acid (FA) and glycerol moieties of triacylglycerol.…”