The Effect Of Quercetin On Glucose-6-phosphatase Gene Expression And Activity

Objective:Glucose-6-phosphatase (G-6-Pase) is an important enzyme in glucose metabolism. As a rate-limiting enzyme of the last reaction of glyconeogenesis and glycogenolysis, the change of G-6-Pase in gene expression and activity will affect the output of endogenic glucose directly. Infact, insulin resistance in liver contributes to the excessive hepatic output of glucose, which is highly correlated with hyperglycemia in type 2 diabetes. So, G-6-Pase is a potent therapeutic molecular target of diabetes. The purpose of this experiment is to study the effect of quercetin on G-6-Pase gene expression and activity and provide some theoretic and exprimental basement for developing the new type medicine for DM. Methods:The hepatocytes were isolated from adult Wistar rat by perfusion of the liver with collagenase . The isolated cells were cultured in 1640 with 10% FBS. We select anti-rat CK-18 as primary antibody to identify these cells by immunohistochemistry. After cell attachment ,the hepatocytes were incubated with 25mM glucose and different concentration of quercetin for 16 hours, then 1) total RNA was extracted and levels of mRNA expression of P36 and P46 were measured by RT-PCR;2) the enzymatic activity was detected by glucose dehydrogenase-coupled reaction. Result:1. The yield and the percentage viability of the isolated cell by perfusion of the liver with collagenase are good. The percentage viability of the isolated cell is about 85%.2. The result of immunohistochemistry is positive(with negative control).3. After cultured with 25mM glucose for 16 huors, both mRNA of P36 and P46 and activity of G-6-Pase of rat hepatocyte increased significantly (p<0.05).4. Afrer incubated in medium cantaining different concentration of quercetinand glucose , the mRNA of two summits of G-6-Pase decreased (P<0.05), but it wasn't decreased depending on the concentration of quercetin.5. Quercetin can decrease the activity of G-6-Pase (p<0.05), which is concordant with the result of gene level.Conclusion:It has a good yield and percentage viability of the isolated cell by perfusion of the liver with collagenase. Both the mRNA level of the main component of G-6-Pase P36 and p46 increased in different extent after cultured with glucose, which accord with our design. Different concentration of quercetin can inhibit the mRNA of P36 and P46, but it isn't decreased depend on the concentration of quercetin. Quercetin can also inhibit the activity of G-6-pase (p<0.05),which is concordant with the result of gene level.