Berberine Decreases Intestinal Glucose Absorption And Improves Postprandial Hyperglycemic Control In Diabetic Mice

BackgroundEpidemiological studies have demonstrated high proportion of postprandial hyperglycemia in patients with clinic-diagnosed T2 DM,and over 70% patients with pre-diabetes are manifested as impaired glucose tolerance.Postprandial hyperglycemia is an important injury factor in diabetes pathogenesis and many other diabetic chronic complications,controlling postprandial glycemic comes to be the main strategy to prevent and cure diabetes and its chronic complications.As the major organ functioning as nutrient absorption,intestine’s specific roles in pathogenesis of postprandial hyperglycemia and diabetes have been valued much in recent years.Pathological enhancement of intestinal glucose absorption has been observed in patients suffering from T2 DM,which is closely related to postprandial hyperglycemia.There are two glucose transporters related to intestine glucose absorption,sodium glucose co-transporter 1(SGLT-1),and glucose transporter 2(GLUT-2).Intestinal glucose concentration rises up to over 30 m M aftereating,then,GLUT-2 translocates from intestinal epithelial cell cytoplasm to brush border membrane,and then,GLUT-2-dominated glucose absorption makes the major contribution to postprandial glycemia.When it comes to diabetes,intestinal GLUT-2 would be permanently located at brush border membrane,which leads to the enhancement ability of intestinal glucose absorption and postprandial hyperglycemia in patients with T2 DM.It turns out to be a more urgent matter that rectifying the enhancement ability of intestinal glucose absorption in the process of pathogenesis of T2 DM.Berberine,a small molecule derived from Coptidis rhizome,is used in the treatment of diabetes since it was found to cause hypoglycemia in patients with diarrhea.Berberine’s anti-diabetic effects are mainly manifested as improvement of cardiovascular function and glycolipid metabolism,rectifying risk factors of diabetes and diabetic complications.However,due to the low bioavailability of berberine,and its unclear anti-diabetic mechanism,its clinical application remains controversial.Some studies suggest that berberine can exert local effects in intestinal tract,including inhibiting the function of disaccharide,promoting the release of GLP-1 to exert effects of regulating metabolism,and regulating the component part of intestinal microbiota.Nonetheless,the study on whether berberine can improve glucose absorption in the small intestine and postprandial glycemia in T2 DM is still a blank.In view of the problem mentioned above,our research group has made a brand-new exploration on the intestinal mechanism of the anti-diabetic postprandial hyperglycemic effect of berberine on the basis of previous studies.Aims To investigate whether berberine could rectify postprandial hyperglycemia through affecting intestinal glucose absorption in T2 DM,and figure out underlying mechanisms that how berberine impacts intestinal glucose absorptionMethods Diabetes was induced in male C57BL/6 mice by feeding a high-fat diet and administration of a low dose of streptozotocin.We group mice according to the method of random number table.These diabetic mice were treated with berberine(200 mg / kg / day,BW,gavage)for 6 weeks.Body weight and fasting blood glucose were monitored in theprocess of berberine treatment.OGTT and IPGTT were applied to reflect the glucose tolerance.Serum insulin,GLP-1,GLP-2 and IGF system were detected by ELISA kit.Intestine tissue and IEC-6 cell was used in in vitro studies to investigate berberine’s effects on intestinal glucose transport and cellular glucose uptake.The intestinal and cellular membrane proteins were extracted and western blotting was used to evaluate glucose transporter protein expression levels.Immunofluorescence and immune electron microscopy was applied to observe the berberine’s effects on distribution of GLUT-2 in intestinal epithelial cells.Chemical inhibitors and small interference RNA knocking down were applied to investigate mechanism of berberine.Dunnett-t test was used to evaluate the statistical significance of difference between experimental and control groups,while SNK-q test was used between different experimental groups.Results 1.Compared with the control group,the significant increasement in mice’s body weight and fasting blood glucose levels,and the abnormal glucose tolerance were seen in the DM group.After berberine gavage treatment for 6 weeks,compared with the DM group,the body weight and the fasting blood glucose was significantly decreased in DM+BBR group,and the impaired glucose tolerance was improved,moreover the HOMA-IR was significantly decreased in DM+BBR group.2.Compared with control group,DM group mice showed enhanced ability of intestinal glucose absorption,and decreased glucose level in feces excreted by unit feed intake.After berberine gavage treatment for 6 weeks,compared with DM group,DM+BBR group mice showed decreased ability of intestinal glucose absorption,and increased glucose level in feces excreted by unit feed intake.Additionally,berberine instant gavage decreased post load blood glucose in OGTT in normal animals,but it had no influence on post load blood glucose in IPGTT,and serum insulin level was decreased in berberine instant gavage group after OGTT.These results indicated that berberine decreased postprandial glycemic level by decreasing intestinal glucose absorption,which is the direct pharmacological effect of berberine in intestine tissue.3.Intestine tissue and IEC-6 cells in vitro were applied to study preliminary mechanismof berberine’s effects on intestinal glucose absorption.Compared with control group,ability of intestinal glucose absorption and cellular glucose uptake was decreased in control+BBR group in a dose dependent manner.Furthermore,both GLUT-2 inhibitor phloretin and si RNA knocking down GLUT-2 expression could block the berberine’s effects on intestinal glucose absorption and cellular glucose uptake,which indicates that berberine decreased intestinal glucose absorption and cellular glucose uptake by depressing GLUT-2 function.4.Berberine decreased GLUT-2 localization at brush border membrane of intestinal epithelial cells.Compared with control group,intestinal GLUT-2 expression was increased in DM group,and we confirmed that GLUT-2 localization at brush border membrane of intestinal epithelial cells was increased by membrane protein western blotting,immunofluorescence and immune electron microscopy tests.After berberine gavage treatment for 6 weeks,compared with DM group,both GLUT-2 expression in intestine and GLUT-2 localization at brush border membrane of intestinal epithelial cells were decreased in DM+BBR group.Additionally,high concentration of glucose was applied to culture IEC-6 cells to simulate intestinal high concentration of glucose after food intake,and we found that berberine could also decrease IEC-6 cells GLUT-2 translocation.5.Compared with DM group,intestinal IGF-1 and IGFBP-3 were decreased in DM+BBR group,and intestinal IGF-1R and PLC-β2 expression were also decreased in DM+BBR group,which indicated that berberine gavage treatment for 6 weeks could rectify intestinal abnormal IGF system caused by diabetes.6.Berberine down-regulated phosphorylation of IGF-1R in IEC-6 cells,IGF-1R inhibitors AG1024 and si RNA knocking down IGF-1R expression could block berberine’s effects on cellular glucose uptake and GLUT-2 translocation.Berberine also down-regulated PLC-β2 localization at cell membrane in IEC-6 cells,PLC-β2 inhibitors U73122 could block berberine’s effects on cellular glucose uptake and GLUT-2 translocation.Furthermore,we found that PLC-β2 localization at cell membrane in IEC-6 cells could be blocked by IGF-1R inhibitors AG1024 and si RNAknocking down IGF-1R expression.These results indicated that berberine decreased cellular glucose uptake and GLUT-2 translocation by suppressing IGF-1R-PLC-β2-GLUT-2 signal pathway.7.Berberine up-regulated phosphorylation of AMPK in IEC-6 cells,AMPK inhibitors Compound C and si RNA knocking down AMPK expression could block berberine’s effects on cellular glucose uptake and GLUT-2 translocation.Furthermore,we found that phosphorylation of IGF-1R in IEC-6 cells could be blocked by AMPK inhibitors Compound C and si RNA knocking down AMPK expression.These results indicated that berberine decreased cellular glucose uptake and GLUT-2 translocation through AMPK dependent suppression of IGF-1R.8.Berberine didn’t up-regulate phosphorylation of Akt in IEC-6 cells,PI3 K inhibitors Wortmannin couldn’t block berberine’s effects on cellular glucose uptake and GLUT-2 translocation.These results indicated that PI3K-Akt signaling pathway didn’t pariticipant in berberine’s effects of decreasing cellular glucose uptake and GLUT-2 translocation.Conclusions 1.Berberine decreases intestinal glucose absorption and improve postprandial hyperglycemic control in T2 DM through decreasing GLUT-2 localization at brush border membrane in intestinal epithelial cells.2.Berberine decreases GLUT-2 localization at brush border membrane in intestinal epithelial cells through AMPK dependent suppression of IGF-1R-PLC-β2-GLUT-2 signal pathway.