Effects Of Berberine On Cell Proliferation, Differentiation, Apoptosis And Adipocytines Expression In 3T3-L1 Adipocytes

BackgroundInsulin resistance (IR) is considered as a core component in the pathophysiology of the metabolic syndrome (MS) which represents a cluster of abnormalities such as central obesity, hyperglycemia, hypertension, dyslipidemia. It is regard as a principal risk factor for increased cardiovascular disease, the leading cause of morbidity and mortality. The worldwide prevalence of MS is increasing, leading to serious public health concerns. However, the underlying mechanisms of MS are incompletely understood. Many studies show that it is very important and indispensable to enhance insulin sensitivity for prevention and treatment of MS. Currently, thiazolidinediones (TZD), the agonists of peroxisome proliferator-activated receptors gamma (PPARγ) with insulin sensibilization and antidiabetic action, is most widely used and recommended for diabetes, but there are some side-effects caused by TZD, such as increasing body weight and body fat volume, headache, extremity edema. Accordingly, to find new insulin-sensitizing agents without TZD's side-effect becomes the highlight. Berberine (Ber), an alkaloid originally isolated from Huanglian (Copitis chinensis), shows the beneficial pharmacological actions of decreasing blood glucose, regulating lipid metabolic disorders, increasing insulin sensitivity, consequently, it has been extensively used as a perspective drug for prevention and treatment of type 2 diabetes, obesity and MS.ObjectiveIn order to explore the molecular mechanisms of Berberine on treatment of insulin resistance, 3T3-L1 preadipocytes were cultured with high glucose and high insulin as a insulin resistance cellular model. Then we observed the effects of Berberine on cell proliferation, differentiation, apoptosis and some adipocytines expression in 3T3-L1 preadipocytes and adipocytes.MethodsInsulin resistance cellular model determined by ~3H-glucose uptake rates was induced with high concentration of insulin and glucose medium. To observe the effects of various concentrations of Berberine and different incubation time on the proliferation in 3T3-L1 preadipocytes, the curve of cellular counts and OD570 were set up by XTT methods. Effects of Berberine on apoptosis rates in fully differentiated 3T3-L1 adipocytes were detected by Flow cytometry (Annexin V-FITC/PI, double dye). The expressions of peroxisome proliferation activated receptorγ(PPARγ) and CAAT/enhancer binding protein (C/EBPa) mRNA and protein during cell differentiation were determined by real time PCR and Western blotting respectively. Adiponectin contents in supernatant were detected by ELISA; the expression of adiponectin and visfatin mRNA were assayed by real-time PCR; Western blotting were used to determine the expressions of adiponectin receptor 1, adiponectin receptor 2 and visfatin.ResultsThe rate of ~3H-glucose uptake declined 53.65 %,47.69%,61.40% in the three cell groups cultured with in the high glucose medium, high insulin medium and high glucose + high insulin medium respectively (P<0.05 or 0.01 vs. control group).Berberine with various concentrations in the experiment (10～80μM) could affect 3T3-L1 preadipocytes proliferation and it showed a dose-dependent relationship. 3T3-L1 preadipocytes treated with 10μM Berberine had little lipid droplets in the cytoplasm which were less than cells treated with Rosiglitazone (Ros). Berberine significantly decreased the mRNA and protein expression of PPARγand C/EBPa (P <0.05 or 0.01 vs. control group) in the course of 3T3-LI preadipocyte differentiation.Berberine can induce apoptosis in fully differentiated adipocyte in dose-dependent manner (P<0.05 vs. control group).0.1～10μM Berberine could increase visfatin mRNA level dose-dependently, especially 10μM in which visfatin mRNA level was 4.96 fold as much as control group. After intervention of 10μM Berberine for 3 hours, the visfatin mRNA level began to increase and reached a peak after 12 hours intervention (4.57 fold as much as 0 hours intervention group). Berberine with 5, 10, 20μM could increase visfatin protein expression level by 1.13, 2.46, 2.34 folds respectively (P<0.05 or 0.01 vs. control group).After Berberine intervention, adiponectin content in cultural supernatant and adiponectin mRNA expression increased in time-dependent manner, and its mRNA expression reached a pick after intervention for 18 hours (6.20 fold vs. 0 hours intervention group). The protein secretion and mRNA expression of adiponectin in insulin resistance celluar were decreased, but Berberine could upregulate them (P< 0.05 vs. control group).In insulin resistance celluar, the expression of adiponection receptor 1 was decreased (P < 0.05 vs. control group) while adiponection receptor 2 had no significant changes (P>0.05 vs. control group). Berberine as well as Rosiglitazone can increase protein expression of adiponectin receptor 1 (1.64 and 1.92 folds vs. control group respectively). Rosiglitazone increased protein expression of adiponectin receptor 2 significantly (2.58 folds vs. control group) while Berberine had no notable changes (P>0.05 vs. control group).ConclusionsIn vitro, Berberine can inhibit proliferation and differentiation of 3T3-L1 preadipocyte, induce adipocyte apoptosis, decrease mRNA and protein expression of PPARγand C/EBPa, increase mRNA and protein expression of adiponectin and visfatin, up-regulate adiponectin receptor 1 expression. These beneficial effects may be one of its mechanisms for amelioration of insulin resistance.