A Novel Role For Farnesoid X Receptor In The Bile Acid-mediated Intestinal Glucose Homeostasis

Backgroud: Intestinal bile acid deficiency(IBAD)is a serious disorder caused by obstruction of bile duct,decreased synthesis or excessive loss of bile acids(BAs),leading to abnormal content or composition of BAs in the intestine.It is often secondary to obstructive jaundice and external drainage of bile,and results in disturbances in the digestive and absorption,glycolipid metabolism,mucosal barrier and intestinal microbiota,which is seriously harmful to the health of human beings.Farnesoid X receptor(FXR)is an important BA receptor,which is involved in regulating the synthesis,transport and metabolism of BAs and maintaining the homeostasis of cholesterol.As the research going,new function of FXR in regulating glucose metabolism and energy balance has been gradually revealed.Based on this,the new molecule-targeted agents have been gradually developed and applied,and expected as novel drugs for the treatment of IBAD-related dysglycemia.However,the specific role of FXR in the glucose homeostasis and the effect on glucose metabolism in the intestine are still unknown.Therefore,it is of great clinical and scientific significance to clarify the pathogenesis of IBAD-related dysglycemia,elucidate the function of FXR in the regulation of intestinal glucose homeostasis,and to find a new therapeutic target for IBAD-related dysglycemia.Objectives: To clarify the relationship between IBAD and blood glucose,we analyzed the changes of blood glucose in IBAD patients.We selected the jejunal tissues from IBAD mouse model and systematically studied the expression pattern and function of FXR in IBAD patients and mice.We elucidated the role of FXR in the regulation of glucose uptake by small intestinal cells in vitro and glucose transport by intestinal mucosa in vivo experiments,respectively.This study may give new ideas for FXR in regulating glucose metabolism,and provide a rationale for FXR agonists in the treatment of IBAD-related dysglycemia.Methods: 1.To clarify the relationship between IBAD and blood glucose,the fasting glucose in 248 IBAD(mild in 56,moderate in 66,severe in 126)and 58 non-IBAD patients were analyzed.2.The jejunal samples from IBAD and sham mice were selected for microarray analysis,and then the BA receptors and glucose transporters were studied by qRT-PCR,Western Blot or Immunohistochemistry.3.The jejunal tissues from 24 IBAD and 21 non-IBAD patients were collected and analyzed by Western Blot or qRT-PCR to observe the expression of FXR,GLUT2 and SGLT1.Statistics were performed using the Pearson correlation test between fasting blood glucose and m RNA levels of GLUT2 and SGLT1 in IBAD patients.4.The effect of BAs on glucose absorption in the intestinal epithelial cells were analyzed by using the 2-NBDG,a fluorescent tracer.2-NBDG uptake in the small intestinal cells were detected to analyze the effect of FXR on glucose uptake when FXR was silenced or its agonist and antagonist were used.5.After the intestinal epithelial cells were treated with CDCA,glucose or FXR antagonists,the effect of FXR on SGLT1 and GLUT2 were analyzed by Western Blot and Immunofluorescence.6.Based on the microarray analysis,Western Blot was performed to validate the signaling pathway related to BAs and glucose metabolism.7.Based on the literature searching,the intermediate target(S1PR2)between FXR and MEK/ERK pathway was found out.After the treatment of intestinal epithelial cells with S1PR2 si RNA,ligand or antagonist,the Western Blot,qRT-PCR and CO-IP were used to verify the results.8.The effects of FXR on BA-mediated glucose metabolism in the intestinal epithelial cells were detected by the Seahorse Extracellular Flux analyzer.9.The glucose tolerance test was performed to assess the effect of CDCA on glucose levels in mice.10.The effects of CDCA,FXR agonist/antagonist and GLUT2 inhibitor on the transepithelial transport of glucose in intestinal mucosa were detected by Ussing Chamber system.11.The inulin test was performed to detect the effect of FXR agonist on intestinal permeability.12.The small intestine was perfused with FXR agonist and/or glucose,and incubated for indicated time,then the brush border membrane(BBM)were extracted.Western Blot was performed to analyze the effect of FXR on SGLT1 and GLUT2.Results: 1.The ratios of aberrantly elevated serum glucose were substantially increased as the IBAD progressed.The incidence of adverse event was 6.90%(4/58)for non-IBAD,16.07%(9/56)for mild deficiency,22.73%(15/66)for moderate deficiency,27.78%(35/126)for severe deficiency.2.The analysis of microarray and qRT-PCR showed that the m RNA levels of FXR,GLUT2 and SGLT1 were increased in jejunums from IBAD mice compared to sham groups.Western Blot and IHC staining showed that the protein levels of FXR were decreased,which is contrary to its m RNA expression.Further studies indicated that the level of protein ubiquitination was significantly increased after IBAD,implicating a posttranscriptional mechanism by which BAs regulates FXR.3.Western Blot showed that the expression of FXR was decreased in the jejunal tissues from IBAD patients.The qRT-PCR revealed that GLUT2 m RNA levels were increased in the jejunal tissues of IBAD patients,whereas SGLT1 m RNA levels were unchanged compared with the control groups.Pearson correlation analysis showed that the fasting blood glucose levels were positively correlated with GLUT2 m RNA levels in the jejunum(p=0.0001,r=0.5426),while not with SGLT1 m RNA levels(p=-0.8745,r=0.0012).4.2-NBDG uptake assays showed that BA-mediated FXR enhanced glucose uptake in the intestinal epithelial cells.5.Both Western Blot and Immunofluorescence showed that BA-activated FXR promoted glucose uptake in the intestinal epithelial cells by increasing the GLUT2 at the cell membrane.6.Based on microarray analysis and further Western Blot analysis,MEK/ERK signaling pathway has been shown to be involved in FXR-mediated glucose uptake in the intestinal epithelial cells.FXR increased glucose uptake by activating MEK/ERK pathway,which can be inhibited by U0126,an inhibitor of MAP kinases.7.Based on the literature searching and further study,we found that S1PR2,an intermediate molecule between FXR and MEK/ERK pathway,was involved in intestinal glucose uptake.2-NBDG uptake assays showed that glucose uptake in the intestinal epithelial cells was increased by S1P(S1PR2 ligand)and inhibited by JTE-013(S1PR2 antagonist).S1PR2 is a downstream effector of FXR,and FXR regulated ERK1/2 phosphorylation through its interaction with S1PR2.8.Seahorse cell energy metabolism analysis showed BA-activated FXR inhibited cell oxidative phosphorylation and decreased ATP production,while had no effect on glycolysis.9.The glucose tolerance test showed that CDCA,the most potent natural activator of FXR,inhibited the elevated blood glucose induced by glucose in mice,suggesting that FXR might inhibit the transepithelial transport of glucose.10.Ussing chamber assays showed that bile acids inhibited the transepithelial transport of glucose in mice by FXR-mediated GLUT2.11.Inulin test showed that the activation of FXR in the intestine did not affect the intestinal permeability(p > 0.05),indicating that FXR-mediated transmembrane transport of glucose was independent of intestinal permeability.12.The BBM assays revealed that the activation of FXR adjusted the ratio of SGLT1 to GLUT2 abundance in the BBM,which in turn inhibited the transepithelial transport of glucose in the intestine.Conclusion: The ratios of aberrantly elevated serum glucose were substantially increased in IBAD patients and were closely related to severity of disease.IBAD decreased expression of FXR in the intestine,which in turn increased GLUT2 abundance on the BBM,suggesting that GLUT2 may be an important therapeutic target for IBAD-related hyperglycemia.In vitro,BA-activated FXR markedly enhanced glucose uptake by increasing the expression of GLUT2 in the intestinal epithelial cells,which depended on ERK1/2 phosphorylation via S1PR2.Meanwhile,BA-activated FXR inhibited oxidative phosphorylation and ATP production rather than glycolysis in the intestinal epithelial cells.Because glucose transport in the intestinal epithelial cells is a highly energy consuming process that requires the participation of Na+-K+ ATPase,the decrease of ATP production will directly affects the function of Na+-K+ ATPase,leading to the decrease of glucose transport.In vivo,FXR potently inhibited specific glucose flux through the epithelium to the circulation,which in turn reduced the increase in blood glucose levels in mice following oral glucose administration.This trend was supported by the changed ratio of SGLT1 to GLUT2 in the BBM.These findings may account for the pathogenesis of hyperglycaemia in the IBAD patients,uncover a novel mechanism by which BAs sustain glucose homeostasis in the intestine.Meanwhile,oral administration of FXR agonists may be promising for the treatment of IBAD-related dysglycaemia.