Berberine Hydrochloride Improves Glucose Metabolism By Targeting Hepatic Glucokinase

Objective: To explore the anti-diabetic mechanism of berberine hydrochloride(BBR)in db/db mice,and to determine the possible targets.It will provide an important scientific basis for clinical treatment of type 2 diabetes mellitus.Methods: 1.C57BL/Ks J db/db mice were randomly divided into BBR-treated(BBR)and untreated group(db/db)group.C57BL/6J mice were as control group.BBR(210 mg·kg-1·day-1)was administered to the treated mice,while the untreated mice were given an equal volume of normal saline for 4 weeks.Body weight,liver weight and liver-body ratio were observed during the experiment.Serum glucose,hemoglobin A1c(Hb A1c),insulin,glucagon,total cholesterol(TC),triglycerides(TG),highdensity lipoprotein cholesterol(HDL-c),free fatty acid(FFA)and low-density lipoprotein cholesterol(LDL-c)were detected by biochemical method.Enzyme linked immunosorbent assay(ELISA)and PAS staining to detect and observe hepatic glycogen content.ELISA and hematoxylin-eosin staining to detect and observe hepatic lipids content.2.An integrative metabolomics approach was applied to identified the changed metabolites in serum,feces and liver of db/db mice with or without BBR treatment.Multivariate statistical analysis was used to achieve the biomarker affected by BBR.3.The db/db mice were sacrificed at 0.25,0.5,1,2,4,8,and 12 h after BBR gavage(210 mg·kg-1·day-1).The untreated mice(0 h)were used as controls.Pharmacokinetics of BBR in serum and liver was explored,and the correlations between liver BBR level and liver glucose-6-phosphate(G-6-P)level,glycogen level,glucokinase(GK)activity and GK m RNA expression were investigated.4.AML12 cells were maintained in high-glucose medium in the presence or absence of BBR.Immunofluorescence of GK expression in cells was visualized.AML12 cells were treated with 20 ?M BBR,and glycogen content was chemically evaluated and stained with PAS.Expression of key proteins and key genes in gluconeogenesis were detected by Western blot and RT-PCR.5.Western blot analysis and Immunohistochemistry staining were used to analyze the expression of GK and GK regulatory protein(GKRP)in liver lysates,cytoplasm and nucleus.Western blot analysis was also used to analyze the expression of key protein in glucose metabolism.The m RNA levels of hexokinase I-IV and key m RNA in glucose metabolism were quantified by RT-PCR.Results: 1.Compared with the control group,the levels of blood glucose,Hb A1 c,glucagon,TG,TC,HDL-c,LDL-c and FFA were increased,while the levels of insulin and hepatic glycogen were decreased significantly in db/db mice.BBR could restore these biochemical parameters besides TC and HDL-c in db/db mice.2.Metabolomics analysis showed that BBR reduced the levels of glucose-related metabolites in serum,liver and feces of db/db mice,as well as the level of free fatty acids,which suggested the improvement of hyperglycemia and dyslipidemia.Most importantly,hepatic G-6-P was found to be increased upon BBR intervention.3.Pharmacokinetics-pharmacodynamics assessment revealed enriched BBR distribution in the liver,and liver G-6-P had the same trend as the concentration-time curve of BBR.G-6-P is solely catalyzed by GK,and GK activity and expression showed a positive correlation with liver BBR levels.4.BBR upregulated GK immunofluorescence expression in AML12 cells cultured in high glucose and increased glycogen content simultaneously.Meanwhile,it seems no effect on gluconeogenesis at both transcriptional and translation levels.5.Four weeks after BBR intervention in db/db mice,the expression of GK in liver lysates,cytoplasm and nucleus was significantly increased,and BBR treatment restored HK4 m RNA expression.Although the effect of BBR on liver GKRP at the transcriptional and translational levels was negligible,it could promote the dissiociation of GK from GKRP.BBR had no effect on gluconeogenesis,but could upregulate the expression of GLUT2.Moreover,BBR did not affect the expression of GK in animal models with normal blood glucose.Conclusion: 1.BBR could improve hyperglycemia and hyperlipidemia in db/db mice,and increase hepatic glycogen content.2.BBR could reduce glucose-related profiles in serum,liver and feces of db/db mice,and elevate the level of G-6-P in liver.3.The changes of G-6-P and hepatic glycogen in the liver of db/db mice responded to the changes of BBR concentration,and the activity and expression of GK were positively correlated with the level of BBR in the liver.4.BBR could upregulate the expression of GK in AML12 cells and db/db mice,and had no effect on gluconeogenesis.5.Increased GK release from GKRP was one of the potential mechanisms of BBR in treating diabetes.