Effects Of Salvianolic Acid A And Coptisine On Glucose Metabolism And The Mechanisms Of Their Actions

Diabetes is a chronic metabolic disease with increasing mobidity and mortality. It happens when the pancreatic islets can not produce enough insulin or when the body resists the physiological effects of the insulin. Insulin resistance is the major feature of the type 2 diabeties.Coptsine and berberine are the major constitutes of the traditional Chinese herb, Coptis deltoidea. Berberine has hypoglycemic effect and anti-insulin resistance effect. Coptisine maybe also have the same effect. In this project, we investigated the effect of coptisine on anti-diabetes and anti-insulin resistance.Salvianolic acid A (SalA) is the major water-soluble active constitute of the traditional Chinese herb, salvia miltiorrhiza. Previous studies have indicated the beneficial effects of SalA on preventing oxidative stress, platelet aggregation, ischemia, and hepatocirrhosis Our pilot studies also showed that SalA has effects on anti-diabetic, anti-diabetic vascular complications. Based on these knowledge, we predicted that SalA maybe have the effect on improving insulin resistance. In this project, we investigated the effect of SalA on diabetes and insulin resistance with the aim to find a new drug for the treatment of diabetes and improving insulin resistance.Part I Effect of coptisine on glucose metabolism in vivoNormal ICR mice and alloxan-induced diabetic mice were used to investigate the effect of coptisine on the glucose metabolism in vivo. The results showed that coptisine-10 mg/kg decreased the fasting blood glucose significantly and non-fasting blood glucose in normal ICR mice. Low dose coptisine had a stronger effect than high dose. In addition, we found that coptisine decreased fasting and non-fasting blood glucose, reduced the 24-h water and food intake, improved the glucose tolerance and coptisine-150 mg/kg decreased the body weight of alloxan-induced mice. The results suggested that coptisine has anti-diabetic effect.PartⅡEffect of coptisine on glucose metabolism in vitro and its mechanismsHuman hepatoma cells (HepG2) and rat skeletal muscle myoblasts(L6) were used to investigate the effect of coptisine on the glucose metabolism in vitro. The results showed that:(l)Coptsine increased the glucose consumption of HepG2 cells and L6 myoblasts.(2)Coptisine increased the glucose uptake of L6 myoblasts. Coptisine had a better effect than insulin but lower than berberine in vitro.Studies on anti-diabetic mechanisms of coptisine showed that:(1)Coptisine significantly increased ATP level in HepG2 cells, and berberine had no significant effect. (2)Coptisine and berberine significantly decreased mitochondrial membrane potential in HepG2 cells after incubation with the drugs. (3)After incubation with hepatic mitochondria for 10 min at 25℃, coptisine decreased velocity of state 3, velocity of state 4, respiration control rate index, oxidative phosphorylation rate, and promoted ADP/O in NADH mitochondrial respiratory chain. Coptisine also inhibited mitochondrial NADH respiratory function in skeletal muscle. Coptisine inhibited hepatic mitochondrial respiratory function by inhibiting the mitochondrial complex I. (4) After incubation with hepatic mitochondria for 10 min, coptisine promoted the hepatic mitochondrial F0F1-ATPase activity. Berberine had no effect on the F0F1-ATPase activity. (5) After incubation with hepatic mitochondria for 10 min, coptisine increased the hepatic mitochondrial ATP level and promoted ATP production. Berberine had no effect on ATP productionIn summary, coptisine decreased the glucose level via promoting mitochondrial F0F1-ATPase activity, increasing ATP production and inhibiting mitochondrial complex I.Part III Effect of Sal A and coptisine on improving insulin resistance in vivo and in vitroWe selected KKay mice, a model of type 2 diabetes with insulin resistance, to investigate the effect of SalA, coptisine and berberine on improving insulin resistance in vivo. The results showed that:(1)SalA, coptisine and berberine improved insulin tolerance and glucose tolerance. SalA had a stronger effect than coptisine and berberine. (2) SalA, coptisine and berberine decreased the fasting blood glucose level, non-fasting blood glucose level, fructosamine level in serum, HblAc content. SalA was stronger than both coptisine and berberine. (3)SalA decreased the LDL, TG level in serum, and had no effect on HDL, CHO in serum. Coptisine and berberine didnot change LDL, TQ HDL and CHO level in serum. (4) SalA, coptisine and berberine decreased 24-h food and water intake. SalA had a stronger effect than both coptisine and berberine. (5) SalA, coptisine and berberine decreased body weight.HepG2 human hepatoma cells and L6 rat skeletal muscle myoblasts were insulin sensitive. HepG2 cells and L6 myoblasts were incubated with different dosages of drugs plus 100 nM insulin. The results of insulin synergic experiments showed that:SalA coptisine and berberine increased insulin synergic action significantly. SalA was weaker than both coptisine and berberine.In summary, SalA has a stronger effect than coptisine and berberine in vivo on improving insulin resistance of KKay mice, but has a weaker effect in vitro on the insulin synergic action.Part IV Research of the mechanisms of SalA anti-insulin resistanceWe isolated hepatic and skeletal muscle mitochondria from normal ICR mice. Effect of SalA, coptisine and berberine on mitochondrial enzymatic activities were detected. The results showed that:(1)SalA promoted mitochondrial complex I activity. While coptisine and berberine had no significant effect. (2)SalA and coptisine significantly increased SDH activity but Berberine had no effect. (3) SalA and coptisine increased F0F1ATPase activity, whereas Berberine had no significant effect. L6 myoblasts incubated with 150μM mpalmitic acid for 18 h, at the same time, with different dosages of Sal A, coptisine and berberine. ATP and ROS content were detected. The results showed that (1) SalA, coptisine and berberine increased glucose uptake significantly. (2)SalA and coptisine increased ATP level in L6 myoblasts, whilst berberine had no effect. (3)SalA, coptisine and berberine decreased ROS production. Western blot results showed that:(1) SalA activated PI3K through promoting GLUT4 translocation. (2) SalA, coptisine and berberine activated AMPK. (3)SalA increased the expression of PGC-lαand Sirt3. In summary, SalA improved insulin resistance in L6 myoblasts via increasing mitochondrial function and regulating AMPK-PGC-lα-Sirt3 axis.Part V Effect of pinocembrin on brain mitochondrial respiratory functionThere is growing evidences show that pinocembrin has a superior neuroproective effect. In the present study, we evaluated the effect of pinocembrin on mitochondrial respiratory function in global brain ischemia/reperfusion (4-vessel occlusion,4-VO)rats. The results showed that pinocembrin improved the respiratory activity of 4-VO brain mitochondria, through increasing ADP/O, state 3 respiration state, resporation control rate index, oxidative phosphorylation rate. We verified the effect of pinocembrin on brain mitochondria in vitro further. We found that pinocembrin increased ADP/O, state 3 respiratory state, respiration control rate index, oxidative phosphorylation rate in NADH/FADH2 dependent respiratory chain and decreased state 4 level in NADH dependent respiratory chain. Pinocembrin improved ATP content in brain mitochondria in vitro and in SH-SY5Y cells.