Study The Mechanism Of Calcium Signal- And Short Chain Fatty Acids-regulated Glucose Metabolism

Objective:The skeletal muscle and liver are the main peripheral tissues of glucose metabolism,which play important roles in maintaining glucose homeostasis of the body.Metabolism disorder leads to inflammation and insulin resistance.Chronic interaction between inflammatory and insulin resistance induces type 2 diabetes,which involves traditional insulin target tissues,immune cells and endothelial.Insulin and exercise/muscle contraction are two physiology regulators of blood glucose level.Insulin-stimulated glucose uptake is impaired under insulin resistance condition,but exercise/muscle contraction-regulated glucose uptake is normal.Compared with the insulin signal pathway,the mechanism of glucose uptake promoted by exercise/muscle contraction is not clear yet.In addition,short chain fatty acids(SCFAs)produced by gut microbiota from dietary fiber and resistant starch in food can reduce body weight,improve insulin sensitivity and anti-inflammation.But the roles of SCFAs on insulin target tissues and endothelial cell are not clear.The aim of this study is to investigate the molecular mechanism of calcium signal-regualted glucose metabolism in skeletal muscle,and the effects of SCFAs on glucose metabolism,inflammation in skeletal muscle cells,liver cells and endothelial cells.Methods:Part 1.L6 myoblast stably expressing GLUT4myc(L6-GLUT4myc),L6-GLUT4 myc with over-expressing AS160(L6-GLUT4myc-AS160 WT),L6-GLUT4 myc with AS160 4A on four phosphorylation sites mutated(L6-GLUT4myc-AS160 4A)were used to investigate the mechanisms of calcium signal-regulated glucose metabolism in skeletal muscle.RNA interference was used to down-regulate PKCδ,PKCδ and PKCε,PKCα,PKCθ,Rab10,Rab13 protein level.Cells were treated with calcium ionophore ionomycin for 10 min.GLUT4 myc levels on cell surface,GLUT4 myc endocytosis and GLUT4 myc exocytosis were detected by enzyme-linked immune sorbent assay(ELISA).Protein expressions of PKCδ,PKCα,PKCθ,PKCε,Rab10,Rab13,GLUT4,AS160 and phosphorylations of AS160,TBC1D1,AMPK,CaMKII were detected by Western blotting.Part 2.Investigate the effects of SCFAs on metabolism and inflammation in skeletal muscle cells.Different doses of sodium acetate,sodium propionate,sodium butyrate were applied to L6-GLUT4 myc myoblasts for 24 hours.The GLUT4 myc levels on cell surface were detected by ELISA.GLUT4 expression,phosphorylation of AMPK and ACC were detected by Western blotting.Co-incubate L6-GLUT4 myc myoblasts with palmitic acid(PA)and sodium acetate,sodium propionate,or sodium butyrate together.Then phosphorylations of NF-κB,JNK and protein expression of SOCS3 were detected by Western blotting.Part 3.Investigate the effect of SCFAs on metabolism in AML12 mouse liver cells.AML12 were incubated with different doses of sodium acetate,sodium propionate or sodium butyrate for 24 hours.The phosphorylation of AMPK,ACC and GSK-3β were detected by Western blotting.Part 4.Explore the effect of sodium propionate on inflammation in HUVECs.Human umbilical vein endothelial cells(HUVECs)were co-incubated with sodium propionate and PA together for 24 hours.The phosphorylation of AMPK,NF-κB and JNK were detected by Western blotting.Results:Part 1.siPKCδ had no effect on ionomycin-regualted GLUT4 myc traffic.Both siPKCα and siPKCθ inhibited ionomycin-phosphorylated AS160 and TBC1D1.Ionomycin-increased cell surface GLUT4 myc levels were reduced in L6-GLUT4myc-AS160 4A myoblasts.siRab10 showed no effect on ionomycin-regualted GLUT4 myc traffic.siRab13 inhibited ionomycin-stimulated GLUT4 myc exocytosis.Part 2.Sodium acetate,sodium propionate,and sodium butyrate enhanced GLUT4 traffic and expression,promoted phosphorylations of AMPK and ACC,inhibited JNK phosphorylations induced by PA,sodium propionate and sodium butyrate inhibited NF-κB phosphorylations caused by PA,sodium propionate inhibited the up-regulation of SOCS3 protein expression induced by PA.Part 3.Sodium acetate,sodium propionate and sodium butyrate phosphorylated AMPK,ACC in mouse liver cells,and sodium butyrate but not sodium acetate and sodium propionate phosphorylated GSK-3β.Part 4.Sodium propionate phosphorylated AMPK,inhibited PA-induced phosphorylation of NF-κB and JNK.Conclusion:1.In L6 skeletal muscle cells,PKCδ is not involved in ionomycin-regulated GLUT4 traffic,PKCα and PKCθ mediate ionomycin-stimulated AS160 and TBC1D1 phosphorylation,AS160 mediates ionomycin-regulated GLUT4 traffic,Rab10 is not involved in ionomycin-regulated GLUT4 traffic,Rab13 is involved in ionomycin-stimulated GLUT4 exocytosis.2.In L6 skeletal muscle cells,SCFAs promote GLUT4 traffic in L6 skeletal muscle cells,increase AMPK and ACC phosphorylations,and also increase GLUT4 protein expression in L6 skeletal muscle cells,reduce the expression of inflammation molecules induced by PA.3.SCFAs promote the phosphorylations of AMPK,ACC and GSK-3β in liver cells.4.Propionate significantly decrease expressions of inflammation molecules induced by PA in endothelial cells.