Characterizing the effects of saturated fatty acids on insulin signaling and ceramide and diacylglycerol accumulation in 3T3-L1 adipocytes and C2C12 myotubes☆
Section snippets
Reagents
Fetal bovine serum was from Atlas Biologicals (Fort Collins, CO) and silica gel 60 thin layer chromatography (TLC) plates were from Merck (Darmstadt, Germany). The FFAs, ceramide standards, and FFA-free bovine serum albumin (BSA) were obtained from Sigma Chemicals (St. Louis, MO). Low glucose Dulbecco’s modified Eagle’s medium (DMEM) was from Life Technologies (Rockville, MD). Antibodies utilized included the following: rabbit polyclonal anti-phospho-Akt (Ser-473), anti-phospho-GSK3β
Results and discussion
To elucidate the molecular mechanisms underlying the inhibitory effects of lipid oversupply on insulin action, we set out to identify an insulin-responsive cell line in which insulin signaling or action was sensitive to exogenous FFAs. We began by studying two well-established models for studies of insulin signaling: C2C12 myotubes and 3T3-L1 adipocytes. Insulin dramatically stimulates early signaling events, including the phosphorylation and activation of the anabolic enzymes Akt/PKB and
Acknowledgements
The authors thank Yusuf Hannun (Medical University of South Carolina) and Al Merrill (Georgia Institute of Technology) for helpful discussions, and Suzanne Stratford and Trina Knotts (Colorado State University) for critically reviewing the manuscript prior to submission.
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This work was supported by National Institutes of Health Grants (R01-DK58784), a Career Development Award from the American Diabetes Association, and a Basil O’Connor Starter Scholar’s Award from the March of Dimes.