Relationship Between Hypoglycemic, Hypolipidemic Effects Of Berberine And PPARs/P-TEFb Signal Transduction Pathway

Type 2 diabetes mellitus is a metabolic disorder due to insulin resistance and insulin-secretion deficiency accompanying hyperlipidemia. Peroxisome proliferators-activated receptors (PPARs) are ligand dependent transcription factors that regulate expression of target genes related to lipid and glucose metabolism. The PPARs play critical roles in the regulation of the adipocyte differentiation and lipid metabolism. Cyclin-dependent kinase 9 (CDK9) and cyclin T1 are the two components of the positive transcription elongation factor b (P-TEFb). P-TEFb is required not only as a basic transcription elongation factor, but it is also recruited by some transcription factors to activate transcriptional elongation from specific promoters. P-TEFb involves in several specific differentiation processes of cells. Both PPARγagonists (rosiglitazone) and PPARαagonists (fenofibrate) have actions with distinct benefits for type 2 diabetes. Currently available pharmacological antidiabetic agents such as rosiglitazone, however, have various adverse effects and high rates of secondary failure. Experimental and clinical trials showed that berberine was a potential hypoglycemic drug to treat type 2 diabetic patients with dyslipidemia, but the mechanism still needs to investigate.ObjectiveTo investigate the effects of berberine on blood glucose, blood lipid, glucolipid metabolism in liver and skeletal muscle, and PPARs and P-TEFb mRNA and protein expression in adipose tissue of type 2 diabetic rats. To select optimal sequence of small interference RNA (siRNA) of CDK9 mRNA and the best interference concentration and action time of optimal siRNA, then to determine PPARα, PPARδ, PPARγ, CDK9, cyclin T1 mRNA and protein expression in 3T3-L1 cells and effect of berberine on them under this condition. To probe into beneficial effect of berberine on diabetic rats, relationship between hypoglycemic, hypolipidemic effects of berberine and PPARs/P-TEFb expression, and to illuminate the mechanism of antidiabetic effect of berberine.Methods1. To induce type 2 diabetic rats: Fasted rats were injected 35 mg/kg streptozotocin to induce diabetic rats according to literature and control rats were injected with the same volume citrate-phosphate buffer. After 2 weeks, the diabetic rats with fasting blood glucose level of above 16.7 mmol/L were given a high-carbohydrate/high-fat diet instead of a standard diet to induce hyperlipidemia and control rats were still given the standard diet.2. Experimental group and drug treatment: The rats were divided into 7 groups, 10 animals in each group: age-matched control rats and diabetic rats without any drug treatment; diabetic rats treated with berberine at a dose of 75, 150 or 300 mg/kg every day, respectively; diabetic rats treated with fenofibrate at a dose of 100 mg/kg or rosiglitazone at a dose of 4 mg/kg every day, both served as positive control. The standard diet or the high-carbohydrate/high-fat diet was given only after drug, mixed with the standard diet, was completely ingested by the rats for 16 weeks. Animal weight was measured every 2 weeks throughout the experiment and the drug dose was accordingly adjusted. Fasting blood glucose levels were also detected at the end of week 20, 24, 28 (during treatment) and 32 (before sacrificed).3. Measurement of blood index: Blood samples were collected from the heart after fasted rats were anaesthetized with an overdose of sodium pentobarbital at the end of week 32. Half of each group rats were perfused with 4% paraformaldehyde. Liver, pancreas and adipose tissue were excised and weighted, and fragments of the tissues were postfixed in 4% paraformaldehyde. Tissues of the other half animals were rapidly excised and weighed after blood collection. Part of tissues were cut into slices and frozen in liquid nitrogen for other studies. Another part of tissues was postfixed in 4% paraformaldehyde overnight for paraffin embedding. Total cholesterol (TC), triglyceride (TG), low density lipoprotein-cholesterol (LDL-C), high density lipoprotein-cholesterol (HDL-C), apolipoprotein (Apo) A?, ApoB, free fatty acid (FFA), serum insulin, hemoglobin A1c (HbA1c), total lipidase, tumor necrosis factor alpha (TNF-α) and adiponectin levels in blood were measured by commercial kit.4. Investigation on histopathology, ultrastructure and insulin expression in pancreas: Hematoxylin-eosin (HE) staining and transmission electron microscope were used to observe pancreatic histopathology and ultrastructure ofβcells, respectively. Insulin expression in pancreas was measured by immunohistochemistry. Superoxide dismutase (SOD) activity and malonaldehyde (MDA) level in pancreas were measured by commercial kit.5. Measurement of glucolipid metabolism in liver and skeletal muscle: Oil red O staining and periodic acid-schiff (PAS) staining were used to observe lipid and glycogen distribution in liver, respectively. Glycogen, FFA and TG contents in liver and skeletal muscle were detected by commercial kit.6. Determination of PPARs, P-TEFb mRNA and protein expression in adipose tissue: PPARα, PPARδ, PPARγ, CDK9, cyclin T1 mRNA and protein were determined by real time PCR and western blotting. TNF-α, adiponectin, FFA, total lipidase, SOD and MDA in adipose tissue were measured by commercial kit. Hematoxylin-eosin staining was used to observe the size and number of adipocyte.7. Proliferation, differentiation and lipid accumulation of 3T3-L1 cells: MTT assay and oil red O staining were used to detect proliferation and differentiation of 3T3-L1 cells, respectively. As a differentiation marker of adipocyte, aP2 protein was measured by western blotting.8. Determination of PPARs and P-TEFb expression in 3T3-L1 adipocytes: Effects of berberine, fenofibrate and rosiglitazone on PPARα, PPARδ, PPARγ, CDK9, and cyclin T1 mRNA and protein expression, TNF-αand adiponectin contents were measured in 3T3-L1 adipocytes.9. Selection of optimal RNA interference (RNAi) condition: Fluorescently-labeled FAM-siRNA was used to optimize transfection condition; optimal siRNA sequence of CDK9, and the best interference concentration and action time of optimal siRNA were determined by real time PCR. CDK9 protein expression was analyzed at different time after optimal siRNA transfection.10. Determination of PPARs and P-TEFb expression in 3T3-L1 adipocytes by RNAi: PPARα, PPARδ, PPARγ, CDK9, cyclin T1 mRNA and protein expression, TNF-αand adiponectin contents were measured in 3T3-L1 adipocytes by RNAi. Effects of berberine, fenofibrate and rosiglitazone on them were observed. Results1. Fasting blood glucose, HbA1c, TG, TC, LDL-C, ApoB, FFA and serum insulin levels in diabetic rats were all significantly higher than that of the control ones, while HDL-C and ApoAI levels were significantly lower. These results indicated that type 2 diabetic rats with hyperlipidemia were successfully induced.2. Berberine markedly decreased blood glucose, HbA1c, TG, TC, LDL-C, ApoB and FFA contents of type 2 diabetic rats, while increased HDL-C and ApoAI levels. Berberine increased the declined glycogen content in liver and skeletal muscle of diabetic rats, while decreased the augmented FFA and TG levels in diabetic tissues.3. Berberine significantly decreased serum insulin content and insulin expression in pancreas of diabetic rats, increased insulin sensitivity index. Berberine augmented pancreas to body weight ratio, pancreatic islets area andβcells number. Berberine increased secretory granules, and improved the swollen mitochondrial and endoplasmic reticulum ofβcells in diabetic rats. Berberine increased SOD activity and decreased MDA content.4. Berberine significantly declined FFA, MDA and TNF-αcontents in serum and adipose tissue of diabetic rats, while increased adiponectin level and total lipidase, SOD activities.5. Berberine markedly up-regulated PPARα, PPARδ, PPARγ, CDK9, cyclin T1 mRNA and protein expression in adipose tissue of diabetic rats. Berberine decreased adipose tissue to body weight ratio and adipocyte size, and increased adipocyte number.6. Berberine promoted 3T3-L1 cells proliferation at low concentration, but inhibited proliferation at high concentration. Berberine enhanced 3T3-L1 cells differentiation and degraded lipid accumulation in dose-dependent manner. Berberine up-regulated PPARα, PPARδ, PPARγ, CDK9, cyclin T1 mRNA and protein expression in 3T3-L1 adipocytes. Berberine inhibited TNF-αsecretion and increased adiponectin secretion.7. siRNAa is the optimal siRNA sequence of CDK9, the best interference concentration and action time were 50 nmol/L and 48 h. CDK9 protein expression was significantly attenuated 48 h after transfection and restored to normal level 96 h after transfection.8. PPARα, PPARδ, PPARγ, CDK9, cyclin T1 mRNA and protein expression were all inhibited by RNAi in 3T3-L1 adipocytes, while TNF-αsecretion was augmented and adiponectin secretion was suppressed. Berberine turnovered the decreased PPARα, PPARδ, PPARγ, CDK9, cyclin T1 expression. Berberine inhibited TNF-αsecretion and increased adiponectin secretion.ConclusionsDiabetic rats were developed by injection low dose streptozotocin due to parts ofβcells injury, and then hyperlipidemia was induced with the high-carbohydrate/high-fat diet. The diabetic rats showed insulin resistance and glucolipid metabolic disorder similar to those found in diabetic patients. It was confidently used to screen drugs with hypoglycemic and hypolipidemic effects. Berberine had significant hypoglycemic and hypolipidemic effects on diabetic rats, improved glucolipid metabolism in liver and skeletal muscle. Berberine promotedβcells regeneration and increased insulin content inβcells to preserveβcells function. Berberine decreased serum insulin due to improved metabolism and recoveredβcells function. Berberine restored the decreased PPARα, PPARδ, PPARγ, CDK9, and cyclin T1 mRNA and protein expression in adipose tissue of diabetic rats to the control levels, which attributed to improved glucolipid metabolism. Berberine enhanced 3T3-L1 cells differentiation and decreased lipid accumulation, which may due to up-regulated PPARα, PPARδ, PPARγ, CDK9, and cyclin T1 expression. When CDK9 mRNA expression was inhibited by RNAi, PPARα, PPARδ, PPARγ, CDK9, and cyclin T1 expression were downregulated, which was turnovered and promoted by berberine. Berberine improved glucolipid metabolism both in blood and tissues of diabetic rats, promoted 3T3-L1 cells differentiation and inhibited lipid accumulation via modulating metabolic related PPARs expression and differentiation related P-TEFb expression, so regulation of PPARs/P-TEFb signal transduction pathway is one of the mechanisms of hypoglycemic, hypolipidemic effects of berberine.