The Role of Sirtuin 1 and Ceramide in t10c12 Conjugated Linoleic Acid Induced Delipidation in 3T3-L1 Adipocytes.

摘要

Project 1: Trans-10, cis-12 conjugated linoleic acid (t10c12 CLA) reduces triglyceride (TG) levels in adipocytes through multiple pathways, with AMP-activated protein kinase (AMPK) generally facilitating, and peroxisome proliferator-activated receptor gamma (PPARgamma) generally opposing these reductions. Sirtuin 1 (SIRT1), a histone/protein deacetylase that affects energy homeostasis, often functions coordinately with AMPK, and is capable of binding to PPARgamma, thereby inhibiting its activity. This study investigated the role of SIRT1 in the response of 3T3-L1 adipocytes to t10c12 CLA by testing the following hypotheses: 1) SIRT1 is functionally required for robust TG reduction; and 2) SIRT1, AMPK, and PPARgamma cross regulate each other. Inhibition of SIRT1 amounts or activity attenuated the amount of TG loss, while SIRT1 activator SRT1720 increased the TG loss. SRT1720 increased AMPK activity while sirtuin-specific inhibitors decreased AMPK activity. Reciprocally, an AMPK inhibitor reduced SIRT1 activity. Treatment with t10 c12 CLA increased PPARgamma phosphorylation in an AMPK-dependent manner and increased the amount of PPARgamma bound to SIRT1. Reciprocally, a PPARgamma agonist attenuated AMPK and SIRT1 activity levels. These results indicated SIRT1 increased TG loss and that cross regulation between SIRT1, AMPK, and PPARgamma occurred in 3T3-L1 adipocytes treated with t10c12 CLA.;Project 2: In the current study profiling of 261 metabolites was conducted to gain new insights into the biological pathways responding to t10c12 CLA in 3T3-L1 adipocytes. Sphinganine, sphingosine, and ceramide levels were observed to be highly elevated in t10c12 CLA treated adipocytes. Exogenous chemicals that increase endogenous ceramide levels decreased lipid levels in adipocytes, and activated AMPK as well as nuclear factor kappa-B (NF-kappaB), both of which are normally activated in CLA treated adipocytes. Concurrent inhibition of de novo ceramide biosynthesis and entry from existing sphingoglipid pools attenuated the lipid lowering and apoptotic effects normally associated with responses to t10c CLA. Our results indicate ceramides are an important component of the lipid lowering response in t10c CLA treated adipocytes.