The Effects Of High Fat Diet On The Expression Of PGC-1αin Mice And The Mechanisms In Its Transcriptional Regulation

Peroxisome proliferator-activated receptor (PPAR) γ coactivator-1α (PGC-1α) is atranscriptional coactivator of mitochondrial related genes. It was found as a coactivatator ofnuclear recptor PPARγ which regulates the differentiation of adipocyte. PGC-1α is amaster player in dissipating the stored energy through increasing mitochondrial production.It has been shown that PGC-1α regulate adaptive thermogenesis, mitochondrial biogenesis,glucose/fatty-acid metabolism, peripheral circadian clock, fiber-type switching in skeletalmuscle, and heart development. PGC-1α functions in oxidative tissues including the heart,kidney, brown fat, skeletal muscle. In those tissues, the expression of PGC-1α is regulatedby enviromental factors such as cold stimulation, starvation, physical exercise andinfections to fit the energy needs of organism. Aberrant expression of PGC-1a is closelyrelated with metabolic syndrome and Type2dibetes.In order to study the roles of PGC-1a in the pathogenesis of insulin resistance, and themechanism of transcriptional regulation of PGC-1a by enviromental variants, we fedC57BL/6mice with high fat diet to induce the development of insulin resistance. We foundthe expression of PGC-1α was decreased under the high fat diet. The mRNA transcriptionand protein expression of mitochondrial-related genes were significantly down-regulated,accompanied with the mitochondrial function disorder, of mice under high fat diet.However, partialy suppression of insulin signaling by PI3K inhibitor LY294002in themeantime of high fatty diet feeding can ameliorate insulin resistance. Next we constructedthe fusion plasmid containing Luc-reporter with PGC-1α promoter and its mutants. Wefound that IRE and CRE sites of the promoter are essential to its basal transcriptionalactivity. We also found that Oleate acid stimulated the PGC-1α transcription throughp38/CREBP/CRE pathway and insulin inhibited it through Akt/FoxO1/IRE pathway. Ourfinding also showed that p38/CREBP/CRE pathway is not strong enough to stimulate the full transcriptional activity of PGC-1α promoter without activated FoxO1/IRE signaling.Results of above indicated that hyperinsulinemia from excessive nutrient intake may be oneof the major contributors to the decreased expression of PGC-1α, mitochondrial functionfailure, and insulin resistance.Part1Hyperinsulinemia induced by high fat diet resulted inmitochondrial function disorder and insulin resistance inmiceObjective: To investigate the relationship between hyperinsulinemia induced by highfat diet and the insulin resistance resulted from mitochondrial dysfunction. Methods:C57BL/6mice were fed with high fat diet. Inspect the changes of phosphorylation of Aktand the expression of Tfam, NRF1by western blotting. Inspect the transcription ofmitochondrial-related gene by real-time PCR. Inspect the mitochondrial function bymtDNA, GSH/GSSG, TG contents. Inspect the HOMA index by static models. Results:Under high fat diet, the phosphorylation of Akt in liver and muscle are increased; theexpression of Tfam and NRF1are decreased; the transcription of mitochondrial-relatedgene are down regulated; the mitochondrial function is disordered; the insulin resistantindex increased. Conclusion: Hyperinsulinemia induced by high fat diet is contribute to thedecreased expression of PGC-1α,mitochondrial function failure, and insulin resistance.Part2The effects of Oleate acid on the expression of PGC-1α in cultured mouse hepatocyte and muscle cells and itsmechanismObjective: To investigate the effects of Oleate acid on the expression of PGC-1α incultured mouse hepatocyte and muscle cells and its mechanism. Methods: Differentconcentrations of Oleate acid were delivered to cultured1c1c7and c2c12cells. Inspectthe changes of phosphorylation of p38by western blotting. Inspect the expression ofPGC-1α by western blotting or real-time PCR. Inspect the changes of the transcription activity of PGC-1α promoter by luciferase assay. Investigate the signaling pathwaysinvolved in by EMSA and ChIP assay. Results: Oleate acid up-regulated thephosphorylation of p38MAPK in cultured liver and muscle cells; the expression ofPGC-1α is increased; the pathway involved in is p38/CREBP/CRE. SB203580, a specificinhibitor of p38, can block those changes mentioned above. Conclusion: The basaltranscriptional activity of PGC-1α promoter depends on the intact IRE and CRE. Oleateacid stimulated PGC-1αtranscription by the p38/CREBP/CRE pathway.Part3Fine-tuned regulation and its mechanisms of PGC-1αgene transcription by Akt and p38MAPKObjective: To determine whether or not insulin/Akt pathway plays an inhibitory role inPGC-1α gene transcription under the regulation of p38. Methods: Different mutants ofPGC-1α promoter were delivered to cultured c2c12myotubes. MKK6E or dn-p38orADA-FoxO1was co-transfected into the cells. Luciferase assay was performed to inspectthe changes of the transcription activity of PGC-1α. Results: Basal level of p38phosphorylation and Akt-dependent signaling were increased in gastrocnemius of miceunder the high fat diet. p38stimulated PGC-1α gene transcription in C2C12myotubes. Insulin suppression of PGC-1α gene transcription was neutralized by the overexpression ofthe constitutively nuclear form of FoxO1. Conclusion: p38-and Akt-dependentintracellular signaling pathways play balancing roles in the transcription of the PGC-1αgene.