The Effect Of DOCK5 On Glucose Metabolism Regulation And Its Mechanism

PART ONE INTRODUCTION, REPRODUCTION AND ITS OFFSPRING GENOTYPING OF DOCK5-/- MICEObjective To obtain, reproduce and genotype DOCK5 gene knock-out mice, for further investigation on DOCK5 gene function.Methods DOCK5 gene knock-out mice were obtained by contacting with Canada IRCM cytoskeleton and cell migration laboratory. The import of mice was entrusted to professional research institute. The mice situation of reproduction and phenotypic change was observed. Different offspring genotype DOCK5 mice are available according to Mendel’s law, the PCR technique is employed for genotyping offspring mice.Results Six DOCk5+/- mice were introduced, they reproduced smoothly according to Mendel’s law. Proper PCR amplification system is explored for DOCK5 genotyping, and then technique of genotyping is carried out in DOCK5 gene knock-out mice.Conclusion DOCK5 gene knock-out mice were working well in the process of introduction, reproduction and its genotyping. Genotyping PCR method of knock-out mice is successfully built. Enough animal models were available for related investigation in future.PART TWO EFFECT OF DOCK5 ON GLUCOSE METABOLISM IN VIVOObjective To investigate the effect of DOCK5 knock-out on insulin resistance and glucose metabolism in high fat diet induced mice.Methods Male DOCK5+/+mice and DOCK5-/-mice of C57BL/6J background were included in this study. They were randomly divided into four groups: DOCK5+/+ standard diet(SD) fed group(SD-DOCK5+/+ group, n=30), DOCK5+/+ high fat diet(HFD) fed group(HFD-DOCK5+/+ group, n=30), DOCK5-/- SD fed group(SD-DOCK5-/- group, n=30) and DOCK5-/-HFD fed group(HFD-DOCK5-/- group, n=30)。The animal models of environmental and genetic changes were established by HFD induction and gene knock-out. The changes of mice body weight, food intake, respiration quotient(RQ) and energy expenditure(EE) were observed to evaluate the general metabolism of mice model. Glucose metabolism and insulin sensitivity were estimated by blood glucose and insulin level in basal status and in load situation of intra-peritoneal glucose tolerance test(GTT), insulin tolerance test(ITT) and pyruvic acid tolerance test(PTT). Hyperinsulinemic-euglycemic clamp(HEC) study was involved in evaluated glucose metabolism and insulin resistance, as well as tissue glucose uptake(TGU) test.Results The animal model was successfully built with interaction of genetic and environmental changes. Compared to SD-DOCK5+/+ group, all metabolism indexes were found without significant change(P>0.05) in SD-DOCK5-/- group; corresponding HFD induced changes of metabolism indexes were found in HFD-DOCK5+/+ group. Compared to HFD-DOCK5+/+ group, the body weight, liver weight and epididymal fat weight were increased significantly in HFD-DOCK5-/- group mice(P<0.05); increased food intake, decreased RQ and EE were also significant in this group(P<0.01), as well as increased fasting blood glucose, insulin and blood lipid level(P<0.01 or P<0.05). In load situation of GTT, ITT and PTT, blood glucose and insulin level were significantly elevated in HFD-DOCK5-/- group when compared to HFD-DOCK5+/+ group(P<0.01 or P < 0.05). When it comes to HEC steady-status, significant decreased glucose infusion rate(GIR) and glucose disappearance rate(GRd), increased hepatic glucose production(HGP) and decreased suppression rate of HGP were found in HFD-DOCK5-/- group when compared to HFD-DOCK5+/+ group(P<0.01 or P<0.05). TGU rate of liver and epididymal fat tissue in HFD-DOCK5-/- group were significant decreased(P<0.01), comparing to those of HFD-DOCK5+/+ group, but there is no difference in TGU of skeletal muscle between the two groups.Conclusion HFD fed DOCK5-/- mice can result in insulin resistance and dysfunction of glucose metabolism, showing closely relationship with type 2 diabetes mellitus.PART THREE MECHANISM OF DOCK5 IN REGULATING GLUCOSE METABOLISM OF LIVER TARGETObjective To observe the effect of DOCK5 gene knock-out or knock-down on PI3K-Akt signaling pathway and glucose metabolism related key signaling molecules in liver. To investigate the molecular biological mechanism involved in the effect of DOCK5 on insulin resistance and glucose metabolism.Methods QPCR technique was employed to analyze the tissue expression distribution of DOCK5 m RNA in C57BL/6J mice. The expression levels of DOCK5 m RNA and protein in liver, muscle and adipose tissue were detected and compared between SD-DOCK5+/+ group and HFD-DOCK5-/- group. The expression and activity change of IR, IRS1, Akt, GSK3β, PEPCK, G6 pase and PP2 A were detected in liver tissue among four groups(SD-DOCK5+/+ group, HFD-DOCK5+/+ group, SD-DOCK5-/- group and HFD-DOCK5-/- group). Cell model of DOCK5 suppression expression(knock-down) was established by transfection of DOCK5-sh RNA plasmid into Hepa1-6 cell line. By treatment of insulin stimulation and/or Okadaic acid(PP2A inhibitor) intervention, the cell glucose uptake rate(GUR) were detected, as well as expression and activity change of IR, IRS1, Akt, GSK3β, PEPCK and G6 pase. The effects of PP2 A inhibitor on those indexes were analyzed.Result DOCK5 m RNA expression can be detected in multiple tissues in C57BL/6J mice; the expression of DOCK5 m RNA in liver is relatively higher than that in muscle and adipose tissue. The DOCK5 m RNA and protein expression levels of liver in HFD-DOCK5+/+ group were significantly increased when compared to that of liver in SD-DOCK5+/+ group, and there is no difference in DOCK5 expression of muscle and adipose tissue between the two groups. Compared to SD-DOCK5+/+ group, the expression and activity of IR, IRS1, Akt, GSK3β, PEPCK, G6 pase and PP2 A were not changed in liver(P>0.05); corresponding HFD induced changes of these indexes were found in liver of HFD-DOCK5+/+ group(P<0.05). Compared to HFD-DOCK5+/+ group, the phosphorylation of IR and IRS1 revealed no difference; phosphorylation of Akt, GSK3β and PP2 A were significantly decreased; and expression of PEPCK and G6 pase were elevated in liver of HFD-DOCK5-/- group(P<0.05). In Hepa1-6 cell model, DOCK5 suppression expression can decreased the cell GUR stimulated by insulin(P < 0.01); PP2 A inhibitor can neutralize this effect. DOCK5 suppression expression decreased phosphorylation of PP2A(P < 0.01), while no effect on phosphorylation of IR and IRS1. DOCK5 suppression expression decreased insulin stimulated phosphorylation of Akt and GSK3β(P<0.05), and increased the expression of PEPCK and G6pase(P<0.01); those effect can be also neutralized by PP2 A inhibitor.Conclusion DOCK5 may regulate insulin signaling transduction and glucose metabolism through PI3K-Akt pathway, PP2 A may be a key factor involved in effect of DOCK5 on glucose metabolism.