The Regulation Effect Of Jinlida On Palmitate-induced Insulin Resistance In L6 Cells

Insulin resistance(IR) is the common pathogenesis of metabolic disease including obesity, type 2 diabetes mellitus(T2DM), hyperlipidemia, hypertension, coronary heart disease and so on, which is also initiating factors of the onset and progression of such diseases. IR defined as the decreased the reactivity of the target tissues such as liver, fat, and skeletal muscles to insulin. Skeletal muscle is the vital organ to uptake and utilization of glucose and lipid, which is also the main location of energy metabolism. Accordingly, IR of skeletal muscle can lead to IR in the whole body. Studies have shown that a variety of factors including lipid deposition, oxidative stress and inflammation can lead to the occurrence of IR.5’-AMP activated protein kinase(AMPK) is the key molecule of energy metabolism regulation. It is critical to the oxidation of fatty acids. It expresses in all kinds of metabolism related organs, including skeletal muscle. It can be activated by various stimulation, then acting on the downstream molecule- es, promoting fatty acid oxidation in the mitochondria. Mitochondria is the power plant in cell, where fatty acid was oxidized and release the energy, finally. Various factors can influence on quantity and form of mitochondria. AMPK can interact with PGC1α and NRF1, so as to regulate the biosynthesis of mitochondrial oxidative phosphorylation related molecules and mitochon- drial function.Jinlida is a compound Chinese patent prescriptions made by 17 kinds of chinese traditional medicine including Ginseng, polygonatum kingian- um, rhizoma atractylodis, kuh-seng, Radix Ophiopogonis and so on, which is used to treatment T2 DM. Clincal studies have shown it can protect pancreatic βcell and improve insulin resistance. Experiment on animals demonstrated that Jinlida can remarkably reduce the serum total cholesterol and low density lipoprotein in rat and enhance the insulin sensitivity, but the specific molecular mechanism is still not clear. In the present study,We will observe the effect of Jinlida on insulin resistance and lipid deposition in L6 skeletal muscle cells, and further explore the mechanisms.Objective: Examined the effect of Jinlida on the insulin resistance of L6 cells induced by palmitic acid, and to explore the potential mechanism by assaying the expression of AMPK signal pathway and mitochondrial function related molecules.Method and Materials: Recovery L6 myoblasts, cultured and induced to differentiate into skeletal muscle cells. The skeletal muscle cells were randomly divided into two groups: control(Con) group and palmitic acid(PA) group. To establish the model of insulin resistance, PA group were incubated in the medium that contained 0.4mmol / L palmitate for 24 h. The palmitic acid(PA) group were then randomly divided into five subgroups: palmitic acid(PA) group, Jinlida low(JLD-L), medium(JLD-M), high(JLD-H) dose group and pioglitazone(PIO) group. After 48 h of drug intervention, glucose uptake in each group was performed using the glucose oxidase-peroxidase method to evaluate insulin sensitivity; the concentration of triglycerides(TG) and free fatty acid(FFA)in the cells were measured with commercial enzyme assay kits; the expression of AMPK, ACC,GLUT4, CPT1, NRF1, COXⅣ, ACADM, PPARα, PPARγ and PGC-1α m RNA in each group were detected using Real-Time PCR; the protein expression of AMPK, P-AMPK, ACC, P-ACC were assayed by Western-blot.Results:1.Comparison of insulin sensitivity in L6 cells:The glucose uptake were significantly decreased in PA group compared with Con group(P<0.05), and the glucose uptake were increased significantly after the intervention of Jinlida and Pioglitazone(each P<0.05).2.Comparison of the lipid content of L6 cells:The concentration of TG and FFA in PA group were significantly higher than in Con group(both P<0.05).After the intervention with Jinlida and Pioglitazone, the concentrate- ion of TG and FFA were significantly lower than in the PA group(each P<0.05).3.The gene and protein expression of AMPK signal pathway: There was no significant difference among groups in protein expression of AMPK and ACC(both P>0.05).The protein exprssion of P-AMPK and P-ACC were down-regulated in PA group compared with the Con group(both P<0.05); The expression of P-AMPK and P-ACC were significantly up-regulated by Jinlida and Pioglitazone(all P<0.05). The m RNA and protein exprssion of CPT1 and GLUT4 were down-regulated in PA group compared with the Con group(both P<0.05); and after the intervention of Jinlida and Pioglitazone, the m RNA and protein expression of CPT1 and GLUT4 were up-regulated(each P<0.05).4.The gene and protein expression of mitochondrial function related molecules: The m RNA expression of PGC-1α, NRF1, PPARα, PPARγ, COXⅣ and ACADM were notably decreased(all P<0.05). The m RNA expression of PGC-1α, PPARα, PPARγ and ACADM were significantly increased by Jinlida and Pioglitazone(each P<0.05). The m RNA expression of COXIV was significantly increased in Jinlida medium dose group(P<0.05), however, there were no difference between Jinlida low dose group and high dose group(each P>0.05). The m RNA expression of NRF1 were not affected by Jinlida and Pioglitazone(each P>0.05). The protein expression of PGC-1α was consistant with the gene expression, it has been down-regulated in PA group(P<0.05), and it has been significantly up-regulated after drug intervention(each P<0.05).Conclusions:1.The palmitic acid incubation induced insulin resistance in L6 skeletal muscle cells, and Jinlida intervention improved insulin resistance in cells, it is equivalent to pioglitazone.2.Jinlida reversed the decline of the expression of AMPK signaling pathway and mitochondria-related molecules, and improved the lipid deposition in L6 cells.3.Jinlida improved mitochondrial function, increased of fatty acid oxidation, promote glucose uptake, thereby improving insulin sensitivity by the activation of AMPK in L6 cells.