The Molecular Mechanism Of Skeletal Muscle PGC-1α On Insulin Resistance Induced By High Fat Diet In Rat

Insulin resistance(IR) is the crucial pathogenesis of type 2 diabetes and the key point of early prevention. High fat diet is a independent risk factor of IR.Peroxisome proliferator-activated receptor γ co-activator 1α(PGC-1α), a nuclear transcription co-activator, may regulate many biology processes, such as mitochondria biogenesis, fatty acid oxidation, and glycometabolism. PGC-1α expression correlate with fatty acid metabolism and insulin sensitivity. In support of this concept, recent studies have reported that raising skeletal muscle triglyceride level by a high-fat diet in humans or feeding high-fat diets in rats, results in decreases in skeletal muscle PGC-1α m RNA. An experiment in vitro showed that overexpression of PGC-1α increased skeletal muscle lipid utilization, reduced the intramuscular lipid content, increase the oxygen consumption, meanwhile improved the insulin signalpathway by increasing the expression of protein kinase beta(AKT) and glucose transport protein 4(GLUT4) m RNA. It has suggested that insulin resistance caused by high fat diet is mediated by PGC-α.Striated activator of Rho signaling(STARS), an actin-binding protein, highly enriched in cardiac, skeletal, and smooth muscle. Recent researches have shown that the expression of STARS in skeletal muscle is related to insulin sensitivity. It has recently been observed that the expression of STARS in skeletal muscle was increased in patients with type 2 diabetes, and there was a negative correlation between STARS and insulin sensitivity. However, another study showed the expression of STARS was increased in skeletal muscle after acute endurance exercise, the expression of PGC-1α and carnitine palmitoyltransferase beta(CPT-1β) were increased, simultaneously. Suppression of STARS reduced the expression of CPT-1β m RNA. It suggested a novel role of STARS in the co-ordination of PGC-1α-induced upregulation of the fat oxidative gene, CPT-1β.It is not clear whether STARS is a downstream target of PGC-1α to regulate the expression of CPT-1β involved in fatty acid oxidation, subsequently impacting on insulin sensitivity. Therefore the primary of the present study was to investigate the molecular mechanism of PGC-1a and its pathway including STARS and CPT-1β, in the development of abnormal fatty acid metabolism and insulin resistance by regulating the expression of PGC-1α. We try to provide useful data to understand the molecualr mechanism of the IR and identify potential novel target for preventing insulin resistance and type 2 diabetes in humans. The present study includes the following two parts: Part I The expression of muscle PGC-1α and STARS in the insulin resistant rats induced by high-fat-dietObjectives: To establish the model of insulin resistant rat induced by high-fat diet, to determine the expression of muscle PGC-1α, STARS and insulin signaling pathway related genes.Methods: Twenty-four male SD rats weighted 125g-145 g were used in the study. After a week of acclimation feeding, rats were divided into two groups: control group(C, n=12) and high-fat group(HF, n=12). Con group were fed with chow diet with the energy contents as follows: 65.5% calories from carbohydrate, 24.2% calories from protein, and 10.3% calories from fat; HF group were fed on a high-fat-diet with the energy contents as follows: 20.1% calories from carbohydrate, 20.1% calories from protein and 59.8% calories from fat. After 6 weeks of diet, an euglycemic-hyperinsulinemic clamp was performed to evaluate insulin sensitivity in two groups. And then killed the rats. Blood samples and quadriceps femoris samples were collected. Serum total cholesterol(TC), Triglycerides(TG), fasting blood glucose(BG) and insulin(INS) was detected. Using reverse transcription polymerase chain reaction(Real-time PCR) to detect the expression of muscle PGC-1α, STARS, and the related genes of insulin signaling pathway.Results:1 Biochemical parameters in two groups:At the end of 6 weeks, BG(6.38±0.23 mmol/L vs 4.05±0.14 mmol/L), INS(19.64±1.50 m U/L vs 11.70±0.86 m U/L), TC(1.17±0.06 mmol/L vs 1.04±0.08 mmol/L) and TG(0.75±0.12 mmol/L vs 0.31±0.02 mmol/L)were significantly higher in high-fat-diet group compared with control group(all P<0.05).2 The comparison of insluin sensitivity between the two groupsGIR was significantly lower in HF group compared with the C group(14.74±0.42 mg·kg-1·min-1 vs. 27.61±0.56 mg·kg-1·min-1)(P<0.05), the difference was statistically significant(P<0.05).3 The gene expression of PGC-1α,STARS in both groupsCompared with the C group, the expression of PGC-1α m RNA in HF group was significantly lower(P<0.05), and STARS m RNA was obviously higher.( P<0.01). Compared with C group, the expression of IRS-1, AKTand GLUT4 m RNA were significantly decreased in HF group(all P<0.01).Conclusions:1 Blood glucose and lipid were increased in rate fed with high fat diet, meanwhile glucose infusion rate was decreased.2 The expression of muscle PGC-1α and genes related to insulin signaling pathway were down-regulated, and STARS was up-regulated in rat fed high fat diet. Part II the molecular mechanism of PGC-1α on insulin resistance induced by palmitic acid in L6 cellsObjectives: To determine the expression of PGC-1α, STARS, CPT-1β and genes related to insulin signaling pathway in L6 cells intervention with palmitic acid, and to explore the molecular mechanism of PGC-1α on insulin resistance induced by high fat.Methods: L6 myoblasts were cultured and differentiated. The skeletal muscle cells were then divided into two groups: control group(Con) and palmitic acid group(PA) intervention with palmitic acid. Control group was randomly divided into five subgroups: control group(Con), empty-vector group(pc DNA3), overexpression group by transfection PGC-1α plasmid(pc DNA3/PGC-1α), negative control si RNA group(NC-si RNA), PGC-1α knockdown group by small interfering RNA(si-PGC-1α); The paltimic acid group(PA) was randomly divided into three subgroups: paltimic acid group(PA), PA group infected with empty-vector group(PA+pc DNA3), PGC-1α overexpression in PA group by transfection PGC-1α plasmid(PA+PGC-1α). Glucose uptake was performed using the glucose oxidase-peroxidase method to evaluate insulin sensitivity. The m RNA expression of PGC-1α, STARS, CPT-1β and genes related to insulin signaling pathway(IRS-1, AKT, GLUT4) were detected by Real-time PCR. The relative protein expression was detected using western-blot. Significance of differences among these groups were determined using Student’s t-test or One-way ANOVA.Results:1 The identification of L6 myoblasts differentiationCompared with undifferentiated group, the gene expression of Desmin and Myogenin in the differentiated group was significantly increased, with statistical significance(P<0.01).2 The expression of PGC-1α, STARS m RNA and genes involved in insulin signaling pathwayCompared with Con group, the medium glucose concentration was increased significantly in PA, with statistical significance(P<0.01). Compared with Con group, The m RNA expression of PGC-1α,IRS-1,AKTand GLUT4 were decreased significantly in PA group(all P<0.05), while the expression of STARS m RNA increased significantly(P<0.05).3 The effect of overexpression PGC-1α on STARS, fatty acid oxidation and insulin signaling pathway in L6 cellsAfter transfected with PGC-1α plasmid, PGC-1α m RNA and protein expression were increased significantly(both P<0.05). Compared with Con group, STARS m RNA and protein were decreased significantly in pc DNA3/PGC-1α(both P<0.05). Similarly, the gene expression of STARS was significantly decreased in PA+pc DNA3/PGC-1α compared with PA group.The expression of CPT-1β gene and p-ACC protein were significantly increased in pc DNA3/PGC-1αcompared with Con(both P<0.05).Compared with PA, the expression of CPT-1β m RNA increased significantly(P<0.05) in PA+pc DNA3/PGC-1α. The expression of IRS-1, AKT and GLUT4 m RNA were significantly increased in pc DNA3/PGC-1α(all P<0.05). Compared with Con group, there was no significant difference in total protein expression of IRS-1 and AKT(P>0.05), nevertheless phosphorylation IRS-1 was significantly decreased, phosphorylation AKT and GLUT4 increased in pc DNA3/PGC-1α group(all P<0.05 or P<0.01). The expression of IRS-1, AKT and GLUT4 m RNA in PA+pc DNA3/PGC-1α were significantly higher than PA(all P <0.05).4 The gene expression after knockdown PGC-1α by small interfering RNACompared with Con, the expression of genes and proteins have no significant difference in NC-si RNA(both P>0.05), PGC-1α m RNA and protein expression decreased significantly in si-PGC-1α group, with statistical difference(both P<0.01), indicating the success of thansfecting. Compared with Con group, the expression of STARS m RNA and protein increased significantly, the expression of CPT-1β m RNA and phosphorylation ACC decreased in si-PGC-1α group(all P<0.05). Compared with Con, IRS-1, AKT and GLUT4 m RNA expression were significantly decreased in si-PGC-1α group, the expression of GLUT4 and phosphorylation AKT were decreased significantly, all with statistical difference(P<0.05 or P<0.01). The expression of phosphorylation IRS-1 significantly increased(P<0.05). There were no significant difference of total protein expression of IRS-1, AKT(all P>0.05).Conclusions: Paltimic acid inhibited the glucose intake rate in L6 muscle cells. It resulted in insulin sensitivity decline with a lower expression of PGC-1α and higher expression of STARS. Upregulation of PGC-1α decreased the expression of STARS, increased the expression of CPT-1β involved in fatty acid oxidization and genes related to insulin signaling pathway. 3. On the contrary, knockdown PGC-1α upregulated STARS expression, inhibited genes expression involved fatty acid oxidization and genes related to insulin signaling. 4. PGC-1α plays a crucial role in insulin resistance induced by high fat diet in rats, which upregulated STARS and downregulated fatty acid oxidation gene.