The Effect Of DHA On Skeletal Muscle Insulin Resistance And Molecular Mechanism Under High-fat Condition

Objectives: With the improvement of people's quality of life and the change of dietary structure,the incidence of diabetes,especially type 2 diabetes(T2DM),has been increasing year by year,bringing a heavy medical burden to families and society.Most people with diabetes are obese or overweight.Excessive intake of fat energy increases the level of free fatty acids in the plasma,leading to decreased efficiency of insulin target tissue uptake and utilization of glucose,resulting in insulin resistance(IR).Insulin resistance is one of the major causes of T2 DM,and it can also induce metabolic diseases such as hypertension and cardiovascular and cerebrovascular diseases.Therefore,improving glucose uptake and improving insulin sensitivity have become important measures for the prevention and treatment of IR.The IR animal and cell model induced by high fat conditions is an important model to study the pathogenesis of IR and its prevention and treatment measures.DHA is an important n-3 polyunsaturated fatty acid(n-3PUFA)mainly found in deep-sea fish oils.Fish oil is not only effective in regulating blood lipids and lowering blood pressure,also plays an active role in the uptake and metabolism of glucose,and in maintaining the body's blood sugar level.Studies have shown that increased n-3 PUFA levels in the diet can reduce the risk of insulin resistance and increase insulin sensitivity in rats,but the mechanism of DHA at the cellular level to improve insulin resistance has not been clearly studied.In this experiment,fish oil was used to replace the fat energy in high-fat diets to feed insulin-resistance rats.Under the premise of ensuring that the energy of high-fat diets was constant,the effects of fish oil diet intervention on insulin sensitivity of IR rats were analyzed.Based on the results of animal experiments,after the successful construction of IR-L6 cell model with suitable concentration of PA,the cells were treated with different concentrations of DHA to further study the effect of DHA on improving insulin resistance and insulin signaling pathway.The results of animal experiments and cell experiments were combined to investigate the role of DHA in the regulation of insulin signaling pathways and possible mechanisms of improving insulin resistance.Methods: Sixty male Sprague-Dawley rats were randomly divided into two groups for insulin resistance: 12 in the control group(NC)and 48 in the high-fat group(HF).48 rats in the HF group with IR were randomly divided into 4 groups: HF,20% FO,50% FO,and 100% FO,12 in each group.The HF group continued to feed high-fat homozygous diets,and 20%FO,50%FO,and 100%FO groups were fish oil substitutes for 20%,50%,and 100% of the fat energy of high-fat diets,respectively.After 4 weeks of feeding,the intake of fish oil was recorded and the fasting blood glucose and insulin sensitivity were measured.Western blot was used to detect the protein expression of IRS1 and GLUT4.The L6 cells were first cultured in DMEM high-glucose medium containing 10% fetal bovine serum to 80% confluence,and then replaced with DMEM high-glucose medium containing 2% fetal bovine serum to grow out myotubes.PA were added at concentrations of 0.25,0.5,0.75,and 1 mmol/L for 24 hours,and appropriate modeling concentrations were selected based on cell activity and glucose uptake.DHA Intervention Experiment 1.0.75 mmol/L PA and DHA mixed media with concentrations of 0.01,0.05,0.1,and 0.2 mmol/L were added to the IR-L6 cell model.Intervention experiment 2,using 0.75mmol/LPA and DHA mixed medium with concentrations of 0.01,0.05,0.1,and 0.2mmol/L,to intervene cells for 24 h.The cell viability and glucose uptake were measured.The m RNA levels of key genes in the insulin signaling pathway were detected by real time q PCR.Western blot was used to detect the expression of key genes in the insulin signaling pathway.Results: 1 Rats(1)Modeling results showed that intake and body weight of rats in the HF group were significantly higher than those in the NC group.The hyperinsulinemic euglycemic clamp test results showed that the glucose perfusion rate in the HF group was significantly lower than that in the NC group(P<0.05).The rats in the HF group developed insulin resistance,indicating that the model was successful.(2)Compared with HF group,FINS gradually decreased with the increase of FO replacement ratio,there was a significant difference in 100%FO group(P<0.05);FPG did not change significantly,and ISI value increased significantly(P<0.05).(3)Protein expression results showed that the protein expression of IRS1 and GLUT4 in the HF group was decreased by 21% and 19%,respectively,compared with the NC group,with statistically significant differences(P<0.05).Compared with HF group,the expression of IRS1 and GLUT4 protein in FO group increased significantly with the increase of fish oil replacement ratio(P<0.05).2 L6 cells(1)After L6 cells differentiated,they formed a muscle-type structure,arranged more regularly,showed a long spindle,and the cells fused to form a distinct myotube.(2)After 24 hours of PA culture,compared with the control group,the cell activity gradually decreased with the increase of PA concentration;the cellular glucose uptake was significantly decreased with the increase of PA concentration(P<0.05),indicating that the IR-L6 cell model was successfully constructed.Western blot results showed that the expression of IRS1 and GLUT4 decreased with the increase of PA concentration.(3)After DHA intervention,the activity of cells was significantly increased(P<0.05),but decreased when the DHA concentration was high;the glucose uptake was significantly increased with the increase of DHA concentration(P<0.05).(4)After DHA intervention,compared with PA group,the expression of IRS1 increased gradually with the increase of DHA concentration,and the expression of PI3 K and m TOR gradually decreased with the increase of DHA concentration.Akt and PGC-1? gene expression slightly decreased at DHA concentrations of 0.01 and 0.05 mmol/L,but increased significantly at 0.1 and 0.2 mmol/L.The expression of GLUT4 increased significantly with the increase of DHA concentration.(5)Compared with PA group,the expression of IRS1,Akt and GLUT4 protein increased with the increase of DHA concentration after DHA intervention,and there was no significant change in the expression of IRS1(Y612)protein,the expression of IRS1(Ser636/639)and PI3 K decreased significantly with the increase of DHA concentration.The expression of m TOR,S6 K and S6K(Thr389)protein decreased significantly with the increase of DHA concentration;the expression of PGC-1? protein was gradually increased and the expression of STARS was significantly decreased(P<0.05).Conclusions: 1 Rats(1)Continuous high-fat diet can increase body weight,decrease insulin sensitivity,and produce insulin resistance.After intervention with fish oil,insulin sensitivity index increases and insulin sensitivity increases.(2)Fish oil high-fat diet can promote the expression of IRS1 and GLUT4 protein,promote glucose uptake and improve insulin resistance.2 L6 cells(1)Insulin resistance of L6 cells can be induced by 0.75mmol/LPA cultured cells for 24 h.(2)DHA intervention can improve cell activity and increase cell glucose uptake;cell activity declines when DHA concentration is too high.(3)DHA activates IRS1/PI3K/Akt/GLUT4 insulin signaling pathway by inhibiting the expression of m TOR and S6 K and improves insulin resistance.(4)DHA can inhibit the expression of STARS by increasing the activity of PGC-1?,thereby increasing the activity of IRS1 and Akt,increasing the insulin sensitivity of IR-L6 cells,and promoting glucose uptake.