The Moleculor Mechanism Study Of Glutamine Improving The Insulin Signaling Pathway And Affecting Glucose Uptake In L6 Cell

Objective: Diabetes mellitus(DM),which is a group of metabolic disorders,is characterized by chronic hyperglycemia resulting from defects in insulin secretion or action,and the incidence of diabetes mellitus is rising rapidly.With the extension of diabetes mellitus,long term hyperglycemia can affect the struture and function of various organs of the body,resulting in diabetic complications,such as diabetic retinopathy,diabetic nephropathy,diabetic cardiovascular disease,and diabetic foot ulcer and so on.The skeletal muscle is the major target of insulin to mediate glucose homeostasis.But when insulin resistance occurs in the body,the ability of skeletal muscle to take and utilize glucose can decrease significantly.The chronic diabetes mellitus induces skeletal muscle damage and atrophy via diabetic neuropathy and the more direct effects of high glucose and low insulin on muscle cell metabolism.Finally,the reduction of skeletal muscle mass and strength come out.Therefore,it is crucial to study how to improve insulin resistance and increase skeletal muscle glucose uptake and utilize,thereby reducing loss and atrophy of skeletal muscle.The insulin,as the important hormone to reduce blood glucose,plays the primary glucose-lowering function via the insulin signaling pathway.The insulin signaling pathway is an enzymatic cascade amplification involving multiple enzymes,which regulates the absorption and metabolism of the glucose and glycogen synthesis.The phosphorylation of insulin receptor(ISR)and protein kinase B(AKT),the activation of phosphatidylinositol-3-kinase(PI3K)plays an important role in insulin’s metabolic function.IRS is activated by insulin and then combines with the regulatory subunit p85 of PI3 K.The phosphatidylinositol diphosphate(PIP2)and phosphatidylinositol triphosphate(PIP3)are from the actived PI3 K,which activate the downstream signaling molecular phosphatidylinositol-dependent kinase(PDK1)and AKT.The protein kinase C zeta(PKCζ)which is the effector of PDK1 in downstream signaling network also is activated.The phosphorylation of AKT and PKCζ increased the glucose transport 4(GLUT4)transmembrane to transport glucose,to accelerate glycometabolism.The GLUT4 plays an explicit role in regulating glucose homeostasis through translocation and activation in skeletal muscle,triggered by insulin dependent PI3K/AKT/PDK1/ PKCζ signaling pathway.Glycogen synthase kinase-3(GSK-3),as a key regulatory factor,also plays a key role in the insulin signaling pathway.After its activity is inhibited,it can seriously affect the insulin signal transduction,glucose metabolism and glycogen synthesis.It is reported that glutamine has the potential to increase insulin sensitivity,to affect glucose transport,glycogen synthesis,as well as lipid degradation.Studies have shown that Gln does have a clinically useful function of lowering blood glucose.Therefore,this study explored the effect of glutamine combined with insulin on skeletal muscle cell L6 on glucose uptake,and its effect on the insulin signaling pathway PI3 K / PDK1 / AKT / PKCζ / GLUT4 in the gene and protein level via the RT-PCR(polymerase chain reaction)and Western Blotting method.And the immunofluorescence method was used to verify the effect of glutamine on GLUT4 protein expression and translocation.This study also used different concentrations of Gln to intervene L6 cells cultured in in high-glucose,and then to detect changes in glucose uptake of L6 cells.Western Blotting was used to detect PI3 K,AKT,and GLUT4 expression in the insulin receptor signaling pathway after intervention with different concentrations of Gln.Method: 1.Before stimulation with the Gln and insulin,L6 cells in the logarithmic phase were incubated under hunger(DMEM without FBS and Gln)for 12 h.Then the cells were cultured in highglucose concentration,serum-free DMEM(High glucose medium glucose concentration is 33 mmol/L).The cells were divided into six groups: the L6 cells treated with highglucose DMEM without Gln and insulin(the control group);the L6 cells intervened by insulin in highglucose DMEM without Gln(the Insulin intervention group);the L6 cells intervened by Gln in highglucose DMEM without insulin(the Gln intervention group);the L6 cells intervened by Gln and GPNA in highglucose DMEM without insulin(the Gln + GPNA intervention group);the L6 cells intervened by Gln and insulin in highglucose DMEM(the Gln + Insulin intervention group);the L6 cells intervened by Gln,insulin and GPNA in highglucose DMEM(the Gln + Insulin + GPNA intervention group).The L6 cells in each group were subjected to different intervention above for 24 h.2.Extract the L6 cell supernatant in different groups.And then Glucose oxidase method was used to detect the glucose uptake of L6 cells in difference groups.The glucose uptake rate can reflect the effect of Gln and insulin on the glucose metabolism.3.Extract the total RNA of L6 cells and detect the the mRNA expression levels of PI3 K,AKT,PDK1,PKCζ and GLUT4 by RT-PCR.The results can reflect the effect of Gln and insulin on moleculars in the insulin signaling pathway in gene level.4.The total cellular protein in difference groups was extracted,and then the protein of PI3 K,AKT,P-AKT,PDK1,PKCζ,P-KCζ,and GLUT4,GSK and P-GSK in the signal transduction pathway were measured by Western Blotting method.The results can show the effect of Gln and insulin on moleculars in the insulin signaling pathway in protein level.5.The membrane protein of L6 cells was extracted and the protein expression of GLUT4 in cell membrane also was analyzed by Western Blotting method,which detected the changes of GLUT4 translocation to the cell membrane.6.The immunofluorescence method was used to observe the total GLUT4 protein expression and GLUT4 protein translocation to the cell membrane.7.After different concentrations of Gln were used to intervene L6 cells for 24 h,the changes of glucose uptake of L6 cells were detected by glucose oxidase method.Western Blotting was used to detect the effect of different concentration of Gln on PI3K/AKT/GLUT4 insulin receptor signaling pathway.Results: Compared with the control group without insulin and glutamine intervention,the results from glucose oxidase assay showed that the glucose uptake rate in the Insulin intervention group and the Gln intervention group increased by 2.21-fold and 1.67-fold,respectively.Whereas the glucose uptake rate in Gln+Insulin group increased by 4.81-fold.The results from RT-PCR and Western Blotting showed Gln up-regulated the expression of PI3 K,PDK1 and GLUT4 gene and protein of L6 cells and promoted the phosphorylation of AKT,PKCζ and GSK in the insulin signaling pathway.The translocation of GLUT4 to the cell membrane activated by the insulin signaling pathway and increases glucose uptake and utilization of L6 cells.The results of immunofluorescence indicated that the intervention of glutamine and insulin increased the brightness of green fluorescence on L6 cells and the intensity of green fluorescence localized on the edge of L6 cell membrane.The results from immunofluorescence are consistant with the results of the Western Blotting and inflected that Gln and insulin promoted the total expression of GLUT4 protein and the translocation of GLUT4 to the cell membrane.The combined effect of glutamine and insulin on hypoglycemic effect was more significant,no matter in glucose uptake and utilization of L6 cells.The effect of glutamine and insulin on the expression of various signaling factors in insulin signaling pathway was more obvious.However,L-Gamma-Glutamyl-p-nitroanilide Monohydrat(GPNA)inhibitors can reduce the hypoglycemic effect of glutamine combined insulin by down-regulating the expression of signal factors in the insulin signaling pathway.Compared with L6 cells cultured by low glucose,high glucose stimulation decreased L6 cell glucose uptake,down-regulated PI3 K and GLUT4 expression and decreased AKT phosphorylation.However,intervention of high concentration Gln can improve the glucose uptake of L6 cells.When Gln concentration is increased by 5 times,L6 cells uptake glucose is the largest,PI3K/AKT/GLUT4 activation is the most obvious;When Gln concentration increases 10 times,L6 cells of glucose absorption is weakened and the activation of PI3K/AKT/GLUT4 is reduced.Conclusion: The study results demonstrated that Gln combined with insulin remarkably up-regulated PI3 K and PDK1 in insulin signaling pathway,while increased AKT and PKCζ phosphorylation rather than AKT and PKCζ expression.In addition,the study certified that Gln enhanced the impact of insulin on the GLUT4 and its translocation from the cytosol to the cell periphery.These results of glucose uptake and GSK phosphorylation also further confirmed that the hypoglycemic effect of Gln accompanied with insulin was more significant.Furthermore,it was found that the GPNA,as a competitive inhibitor of Gln uptake,could reverse the hypoglycemic effect of Gln.These study findings suggested that Gln enhanced the hypoglycemic role of insulin through the PI3K/AKT/GLUT4 signaling pathway and glycogen synthesis pathway.With the concentration of Gln increased in a certain range,the absorption and utilization of glucose in L6 cells also increased.Over a certain concentration range,the effect of glutamine decreased.