Effect Of Pyrrolidine Dithiocarbamate On Hepatic Glucose Metabolism And Its Protection On Hepatic In T2DM Rat

The liver is an important organ in regulating blood glucose, liver glucose metabolism play a key role in maintaining homeostasis of glucose (glucose homeostasis). Liver regulates blood glucose mainly through glycogen synthesis and gluconeogenesis.When body intakes high carbohydrate diet, insulin secretion is increased,then insulin promotes liver glycogen synthesis and reduces hepatic gluconeogenesis. As a result, it decreases hepatic glucose output and prevent postprandial hyperglycemia. In fasting or starvating situation, insulin secretion is decreased and glucagon secretion is increased, then liver glycogenolysis and gluconeogenesis is increased. As a result,hepatic glucose output increase and prevent hypoglycemia. Absolute insulin deficiency or dysfunction can result in glycogen synthesis and gluconeogenesis dysfunction. The main reason of hyperglycemia in diabetes is hepatic glucose metabolism disorders.Pyrrolidine dithiocarbamate (PDTC) is a dithiocarbamates, which is a thiol-containing compound that can chelate various metal ions and has the function of anti-oxidant. It can specificly suppress nuclear factor-κB (NF-κB) activity, and efficiency inhibit the oxidative damage that induced by the activation of NF-κB. A number of studies have shown that PDTC can reduce the level of blood glucose in diabetic rats, but the mechanism is still unknown.Recent studies have shown that the biological effect of PDTC involved in several signal transduction pathways. Research has shown that PDTC can increase levels of Akt phosphorylation throught PI3K/Akt signaling pathway. In the current study, type 2 diabetic rat model were set up by small dose of streptozotocin (STZ) and high-fat diet. Interventing with pyrrolidine dithiocarbamate (50mg/kg/d, IP) continuously a week,Observation the effects of PDTC on blood glucose and lipid and its protection on hepatic,and the effect of PDTC on hepatic glycogen synthesis throuht affect Akt/FoxO1 signaling pathway,and the effect of PDTC on hepatic gluconeogenesis throuht affect Akt/GSK/GS signaling pathway. The experiment contained four parts as below:Part one: Effects of pyrrolidine dithiocarbamate on glucose and lipid.Objective: The aim of this part study was to observe the effects of pyrrolidine dithiocarbamate on glucose and lipid in diabetic rats.Methods: Male 8-weeks-old Wistar rats weighing about 180-210g were used. A total of 49 healthy male Wistar rats were divided randomly into 2 groups: normal control (NC) group (n=12) received a regular diet and high-fat (HF) diet group (n=37) received high-fat diet. All the rats were weighed every week. After 8 weeks being fed with the high-fat diet animals, insulin resistance was confirmed by oral glucose tolerance test, then STZ was administered via intraperitoneal injection at 27mg/kg to establish diabetes model. NC group was administered the same volume citric acid buffer solution, and each group rats were feds with original forage. After 72 hours diabetes mellitus rats were confirmed by the random blood glucose over 16.7mmol/L. Then the diabetic rats were further divided randomly into 3 groups: diabetic mellitus group (DM), PDTC-treated group (DM+P), insulin-treated group (DM+INS). Rats in the PDTC-treated group were administered PDTC (50 mg/kg) once via intraperitoneally injection into rat daily. At the same time, rats in the normal-diet group,DM group and insulin-treated group were injected with equivalent volume of saline in the same way. The rats in insulin-treated group were injected insulin (1 U/kg) once at the time 1h before they were sacrificed, and each group rats were fed with original forage.After treated with PDTC for one week, the rats were fasted at 20:00 PM in the day before experiment, blood was taken from the angular vein at 8:00 AM of experiment day, and the blood plasma was separated and stored at -20℃for detecting fasting plasma insulin, blood glucose and lipids. Then all the rats were sacrificed, the liver tissues were taken and saved in different ways for PCR, western blot. 2 Measurement of blood glucose and insulinFasting blood glucose was measured by glucose oxidase method, and plasma insulin was measured by ELISA. Homeostasis model assessment of insulin resistance (HOMA-IR) and insulin sensitivity index (ISI) were calculated to evaluate the resistance and sensitivity of insulin.3 Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT)OGTT:The blood glucose and insulin level of 0 min, 30 min, 60 min, 90 min, 120 min were evaluated via intragastric administration glucose at 2g/kg body weight.ITT: The blood glucose level of 0 min, 15 min, 30 min, 60 min, 90 min were evaluated via intraperitoneal injection at 1U/kg insulin.4 Measurement of blood lipidsTriglyceride (TG), total cholesterol (TC), high density lipoprotein- cholesterol (HDL-C), lower density lipoprotein-cholesterol (LDL-C) were tested by automatic biochemistry analyzer. Free fatty acid was measured by Cu²⁺ chromatometry.Results:1 Comparison the weight of each groupThe rats were divided randomly into NC group and HF group, there was no statistically significant difference in weight at the beginning of the experiment. After 4 weeks, the weight of rats in HF group(306.00±15.81 g) was significantly higher than that in NC group(252.90±27.80 g) (P<0.01). After 8 weeks, the weight of rats in HF group (361.92±19.22 g) was significantly higher than that in NC group (313.17±19.95 g) (P<0.01).2 The blood glucose, insulin, HOMA-IR and ISI.After 8 weeks, there was no significantly difference in fasting blood glucose, between HF and NC group; The insulin level of HF group (18.01±2.63μIU/ml) was higher than that of NC group (10.67±0.83μIU/ml) (P<0.05); The HOMA-IR of HF group (1.22±0.17) was higher than that of NC group (0.68±0.09) (P<0.05). The ISI of HF group (-4.43±0.17) was lower than that of NC group (-3.79±0.09) (P<0.05). 3 Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT)After 8 weeks, the blood glucose level of HF group in each time point from OGTT was higher than that of NC group, in which there were significantly difference on 90 min and 120 min (P<0.05); The insulin level of HFgroup in each time point was higher than that of NC group (P<0.05). ITT: The blood glucose of HF group in every time was higher than that of NC group, and at the time of 15min, 60min and 90min has statistically significant different (P<0.05).4 The blood glucose in each groupAt 72h after STZ injection, the fasting blood glucose level of DM group (26.16±3.38 mmol/L) was significantly higher than that of NC group (4.41±0.56 mmol/L) (P<0.01). After treatment with PDTC, the fasting blood glucose of DM+P group (11.55±2.89 mmol/L) was lower than that of DM group (26.55±2.90mmol/L) (P<0.01).5 The blood lipids in each groupAt the end of the experiment, the TC (8.79±1.83 mmol/L), TG (1.12±0.31 mmol/L), LDL (1.72±0.28 mmol/L), FFA (1.82±0.14 mEq/L) in DM group were obviously increased when comparing with those in NC group (TC2.18±0.11 mmol/L), TG (0.66±0.07mmol/L), LDL (1.10±0.07 mmol/L), FFA (1.27±0.13 mEq/L) (P<0.05). The triglyceride was obviously lower than that in DM+P group (0.70±0.10 mmol/L) (P<0.05), there was no significant difference in others blood lipid.Conclusion:1 The type 2 diabetic rat model could be successfully established by long-term high-fat diet feeding with a small dose of intraperitoneal injection of STZ.2 The pyrrolidine dithiocarbamate can reduce blood glucose levels, but can't reduce blood lipids.Part two:Effect of pyrrolidine dithiocarbamate on hepatic glycogen synthesis in diabetic ratsObjective: To determine the effect of pyrrolidine dithiocarbamate on hepatic glycogen synthesis and its mechanism in diabetic rats. Methods:The rat liver tissue was dissolved by alkaline (KOH) solution, and the liver glycogen content was determined by anthrone colorimetric. The rat liver tissue was grinded in liquid nitrogen. Protein was extracted with RIPA and the levels of glycogen synthase kinase-3β(GSK-3β) and protein kinase B (PKB/Akt) phosphorylation in rat liver was examined by Western blot analysis.Results:1 The effect of pyrrolidine dithiocarbamate on hepatic glycogen synthesisGlycogen content was significantly decreased in DM group (5.92±0.99 mg/g) than that in NC group (11.59±1.96 mg/g) (P<0.01). Glycogen content was significantly increased in both DM+P group (8.77±1.00 mg/g) and in DM+INS group (10.25±1.76 mg/g) than that in DM group (P<0.01).2 The effect of pyrrolidine dithiocarbamate on the Akt phosphorylation in diabetic rats'liverThe levels of Akt phosphorylation was significantly decreased in DM group (0.43±0.05) than that in NC group (0.94±0.07) (P<0.01). The Akt phosphorylation was significantly increased in both DM+P group (1.23±0.10) and DM+INS group (1.43±0.11) than that in DM group (P<0.01).3 The effect of pyrrolidine dithiocarbamate on the GSK-3βphosphorylation in diabetic rats'liverThe levels of GSK-3βphosphorylation were significantly decreased in DM group (0.40±0.04) than that in NC group (0.64±0.07) (P<0.01). After drug intervention, the GSK-3βphosphorylation was significantly increased both in DM+P group (0.78±0.05) and in DM+INS group (0.84±0.03) than that in DM group (P<0.01).Conclusion: PDTC can significantly increase the liver glycogen content and decrease blood glucose in diabetic rats, which was associated with significant increase the levels of GSK-3βand Akt phosphorylation.Part three: The effect of pyrrolidine dithiocarbamate on hepatic gluconeogenesis and its mechanism in diabetic ratObjective: To determine the effect of pyrrolidine dithiocarbamate on hepatic gluconeogenesis and its mechanism in diabetic rats.Methods: The rat liver tissue was grinded in liquid nitrogen, protein and RNA was extracted. The levels of Akt and Forkhead box protein O1 (FoxO1) phosphorylation in rat liver were examined by western blot analysis. The mRNA expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-Phosphatase (G6Pase) were detected by Real time RT-PCR. Liver tissue was embedded with OCT glue and section was made in frozen conditions. The sections were stained with immunofluorescence and FoxO1 subcellular localization in the cytoplasm and nucleus were observed by confocal.Results:1 The effect of pyrrolidine dithiocarbamate on the FoxO1 phosp horylation in diabetic rats'liverThe level of FoxO1 phosphorylation was significantly decreased in DM group (0.06±0.01) than that in NC group (0.77±0.06) (P<0.01). After drug intervention, the FoxO1 phosphorylation was significantly increased both in DM+P group (0.64±0.07) and DM+INS group (0.71±0.07) than that in DM group (P<0.01).2 The effect of pyrrolidine dithiocarbamate on the FoxO1 subcellular localization in diabetic rats'liver cellThe level of FoxO1 was significantly decreased in cytoplasm in DM group than that in NC group. After drug intervention, the FoxO1 was significantly increased in cytoplasm both in DM+P group and DM+INS group than that in DM group.3 The effect of pyrrolidine dithiocarbamate on the mRNA levels of PEPCK and G6Pase in diabetic rats'liverThe mRNA levels of PEPCK (1.77±0.32) and G6Pase (3.20±0.76) were significantly increased in DM group than those of PEPCK (1.04±0.19) and G6Pase (1.02±0.08) in NC group (P<0.01). After drug intervention, both the mRNA levels of PEPCK (0.87±0.19) and G6Pase (0.82±0.18) in DM+P group and the PEPCK (0.51±0.13) and G6Pase (0.62±0.11) in DM+INS group were significantly decreased than that in DM group (P<0.01).Conclusion: PDTC can increase the levels of FoxO1 phosphorylation through Akt/FoxO1 signaling pathway and lead to nucleoplasm translocation, and PDTC can decrease blood glucose through decreasing the expression of PEPCK and G6Pase.Part four: The protection of pyrrolidine dithiocarbamate on liver in diabetic rats.Objective: To determine the protection of pyrrolidine dithiocarbamate on liver and its mechanism in diabetic rats.Methods: The rat liver tissue was grinded in liquid nitrogen and RNA was extracted. The expression of inductible Nitric Oxide Synthase (iNOS) mRNA was detected using RT-PCR. The liver tissue was fixed 24 hours in 10% formalin solution and tissue section was made by routine method. The liver tissue section was stained with HE and immunohistochemistry to observe expression of inducible nitric oxide synthase (iNOS) and protein nitration (NT) production and liver injury.Results:1 The morphological changes of liver tissuesComparing with the normal control group, the liver was significantly steatosis, liver cells are damaged in diabetic model group. PDTC significantly reduced liver cells steatosis and damage compared with diabetic model group.2 The relative expression of iNOS mRNAThe expression of iNOS mRNA were significantly higher in DM group (1.29±0.12) than that in NC group (0.19±0.03) (P<0.01), and the expression of iNOS mRNA was significantly decreased in DM+P group (0.22±0.04) than that in the DM group (P<0.01).3 Immunohistochemistry resultsThe expression of iNOS and production of NT in DM group were significantly higher than those in NC group, and the expression of iNOS and production of NT in DM+P group were significantly lower than those in DM group. Conclusion: Hyperglycemia can induce oxidative stress, in the same time iNOS expression and NT was significantly increased. All of these play an important role in liver cell damage. The pyrrolidine dithiocarbamate can decrease the liver cell damage of diabetic rats through suppressing the expression of iNOS and NT production.