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Effect of fatty acids on insulin sensitivity in skeletal muscle cells culture: protein phosphorylation, gene expression and PPAR activity

Grant number: 05/00807-5
Support type:Scholarships in Brazil - Post-Doctorate
Effective date (Start): October 01, 2005
Effective date (End): May 31, 2007
Field of knowledge:Biological Sciences - Physiology - Physiology of Organs and Systems
Principal Investigator:Rui Curi
Grantee:Sandro Massao Hirabara
Home Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Insulin resistance occurs in several conditions such as obesity and diabetes mellitus type 2. In these conditions, it is also observed increase in plasma free fatty acid (FFA) levels. The involvement of these metabolites in the development of the insulin resistance has been postulated. However, the mechanism involved is not well established. We demonstrated that FFA alter glucose metabolism in skeletal muscle cells (SMC) through the Randle cycle and the insulin signaling pathway. This project aims to determine if the effects of different FFA on insulin sensibility and insulin signaling pathway are related with changes in gene expression, mitochondrial metabolism and activity of peroxisome proliferator-activated receptors (PPARs) in SMC. Two cell types will be utilized: a) culture of C2C12 SMC and b) primary culture of rat SMC. The following FFA will be studied: caprylic (C8:0), palmitic (C16:0), stearic (C18:0), oleic (C18:1), linoleic (C18:2), eicosapentaenoic (C20:5) and docosahexaenoic (C22:6) acid. The SMC will be cultivated for up to 48h in the presence of 100 microM of different FFA. After this period, the following parameters will be evaluated: 1) glucose metabolism (2-deoxy-D-[2,6-3H]glucose uptake and [14C]glycogen synthesis); 2) insulin signalling (phosphorylation of insulin receptor [IR], insulin receptor substrate-1 and -2 [IRS-1 and -2], protein kinase B [PKB/Akt], glycogen synthase kinase-3 [GSK-3], mitogen-activated protein kinases [MAP kinases] p44 and p42, phosphotyrosine phosphatase-1B [PTP-1B], phosphatase containing SH2 [SHP-2], leukocyte antigen-related [LAR] and protein kinases C [PKC]); 3) expression of genes involved in glucose and FFA metabolism (fatty acid transporter [FAT/CD36], fatty acid binding protein [FABP], glucose transporter-4 [GLUT-4], acyl-CoA synthase [ACS], pyruvate dehydrogenase kinase-4 [PDK-4], monocarboxylate transporter-1 [MCT-1], carnitine palmitoyl transferase-1 [CPT-1] and uncoupling proteins-2 and -3 [UCP-2 and -3]); 4) mitochondrial metabolism (production of NAD(P)H and ATP and mitochondrial coupling), and 5) activity and expression of PPARs. To evaluate the glucose metabolism, the SMC will be incubated for one hour in bicarbonate Krebs-Ringer buffer containing 5.6 mM glucose, 0.3 microCi/mL D-[U-14C]glucose and 0.2 microCi/mL 2-deoxy-D-[2,6-3H]glucose, in the absence or presence of insulin. After this period, the SMC will be frozen in liquid nitrogen and used for determination of 2-deoxy-D-[2,6-3H]glucose uptake and [14C]glycogen synthesis. The effect of the FFA on insulin signalling pathway will be evaluated by western blotting, using specific antibodies anti-IR, anti-IRS-1 and -2, anti-phosphotyrosine, anti-phospho-MAP kinases p44 and p42, anti-phospho-Akt, anti-phospho-GSK-3, anti-PTP-1B, anti-SHP-2, anti-LAR, and anti-phospho-PKC. The gene expression will be determined by real-time RT-PCR. The production of NAD(P)H will be evaluated by fluorometry and ATP production by spectrophotometry. Mitochondrial coupling will be analised by using rhodamine 123 as fluorescent probe. In this project will be possible to determine if the modulation of insulin sensibility and signaling pathway by the different FFA is related to changes in protein phosphorylation, gene expression, mitochondrial activity and PPAR activities in SMC. (AU)