n-caproylsphingosine and Insulin-Resistance

The present studies tested the hypothesis that some effects of tumor necrosis factor-alpha (TNF-alpha) are mediated by activation of sphingomyelinases and the production of ceramides. Differentiated 3T3-L1 adipocytes were incubated with short-chain ceramide analogs, (C2- and C6-ceramides: N-acetyl- and N-hexanoyl-sphingosines, respectively), and this treatment increased 2-deoxyglucose uptake in the absence of insulin progressively from 2-24 h. This effect was inhibited by blocking the activations of mitogen-activated protein kinase, phosphatidylinositol 3-kinase (PI 3-kinase), and ribosomal S6 kinase which mediated an increase in GLUT1 concentrations. Long-term increases in PI 3-kinase activity associated with insulin receptor substrate-1 (IRS-1) increased the proportion of GLUT1 and GLUT4 in plasma membranes. These events explain the increases in noninsulin-dependent glucose uptake and incorporation of this glucose into the fatty acid and glycerol moieties of triacylglycerol. The mechanisms by which TNF-alpha and ceramides increase PI 3-kinase activity were investigated further by using rat2 fibroblasts. Incubation for 20 min with TNF-alpha, bacterial sphingomyelinase, or C2-ceramides increased PI 3-kinase activity by about fivefold, and this effect depended upon a stimulation of tyrosine kinase activity and an increase in Ras-GTP. This demonstrates the existence of a novel signaling pathway for TNF-alpha that could contribute to the effects of this cytokine in stimulating basal glucose uptake. By contrast, treating the 3T3-L1 adipocytes for 2-24 h with C2-ceramide diminished insulin-stimulated glucose uptake by decreasing the insulin-induced translocation of GLUT1 and GLUT4 to plasma membranes. This inhibition was observed when there was no increase in basal glucose uptake, and it occurred downstream of PI 3-kinase. Our work provides further mechanisms whereby TNF-alpha and ceramides produce insulin resistance and decrease the effectiveness of insulin in stimulating glucose disposal from the blood. Conversely, TNF-alpha and ceramides increase the ability of adipocytes to take up glucose and store triacylglycerol in the absence of insulin.

Topics: 3T3 Cells; Adipocytes; Animals; Biological Transport; Ceramides; Deoxyglucose; Glucose Transporter Type 1; Glucose Transporter Type 4; Insulin; Insulin Receptor Substrate Proteins; Insulin Resistance; Kinetics; Mice; Monosaccharide Transport Proteins; Muscle Proteins; Phosphatidylinositol 3-Kinases; Phosphoproteins; Sphingosine; Tumor Necrosis Factor-alpha

We have previously demonstrated that chronic exposure of 3T3-L1 adipocytes to tumour necrosis factor-alpha (TNF) resulted in a marked decrease (approximately 90%) in cellular GLUT4 (insulin-responsive glucose transporter) mRNA content as a result of a decreased transcription rate of the GLUT4 gene (approximately 75%) and a reduced half-life of its mRNA (9 to 4.5 h). Investigation of the signalling mechanism responsible for this regulation demonstrated that in the 3T3-L1 adipocytes, sphingomyelin levels decreased to 50% of control levels within 40 min of exposure to TNF, consistent with activation of a sphingomyelinase. In the same manner as with TNF, treatment of the adipocytes with 1-3 microM C6-ceramide, a membrane-permeable analogue of ceramide, decreased GLUT4 mRNA content by approximately 60%. Subsequent investigations revealed that transcription of the GLUT4 gene was reduced by approximately 65% in response to C6-ceramide, demonstrating that the decrease in mRNA content is mediated by a reduction in the transcription of the genc. No effect on GLUT4 mRNA stability was observed after exposure of the adipocytes to C6-ceramide. These observations are interesting in light of our previous data demonstrating that TNF affects both GLUT4 transcription and mRNA stability in the 3T3-L1 adipocytes. In conclusion, the effect of ceramide on GLUT4 gene expression is at the level of transcription, suggesting that another pathway controls mRNA stability. These data establish that ceramide-initiated signal transduction pathways exist within the adipocyte, and provide a potential mechanism for control of GLUT4 gene expression.

Topics: 3T3 Cells; 5,8,11,14-Eicosatetraynoic Acid; Adipose Tissue; Animals; Arachidonic Acid; Ceramides; Choline; Down-Regulation; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Gene Expression Regulation; Glucose Transporter Type 4; Insulin Resistance; Isoenzymes; Mice; Monosaccharide Transport Proteins; Muscle Proteins; NF-kappa B; Phosphorylation; Protein Kinase C; Protein Kinase C-delta; RNA, Messenger; Sphingomyelin Phosphodiesterase; Sphingomyelins; Transcription, Genetic; Tumor Necrosis Factor-alpha