Study On The Mechanism Of Geniposide Regulating Glucose And Lipid Metabolism In Mice

Epidemics of obesity and diabetes have been the global issues that threaten human health and quality of life,which link with other chronic metabolic diseases such as metabolic syndrome,cardiovascular and cerebrovascular diseases,and kidney diseases.Therefore,it is important to understand the pathogenesis of obesity and diabetes and to explore the effective interventions or treatments.As a kind of food and medicine resource,Gardenia jasminoides Ellis is widely used in multitudinous foods and medicines.Geniposide and crocin,extracted from Gardenia jasminoides Ellis,are used to develop healthy foods.And geniposide has hypolipidemic and hypoglycemic effects on glucose homeostasis and lipid metabolism.Due to the complexity of the pathogenesis of obesity and diabetes,however,the mechanism of geniposide regulating glucose and lipid metabolism is still not clear,and the pathways through which geniposide regulates glucose and lipid metabolism in mice are still not incomplete.Clarifying the mechanism of geniposide in regulating glucose and lipid metabolism in mice would provide important ideas for the functional research of active ingredients of natural products,and also provide an important theoretical basis for the research and development of healthy foods.This study aims to clarify the effects of geniposide on glucose and lipid metabolism in different mouse models.The mechanisms were studied with multiple approaches,including in vitro cellular assays,physiological/biochemical indexes,histomorphological observation,transcriptomic sequencing and molecular biology analysis.The main research outcomes are summarized as follows:Firstly,the effects of geniposide on lipid metabolism were further verified in normal chow diet-fed mice,high-fat diet-fed mice and atherosclerosis(Apo E^-/-)mice.After the intervention of geniposide(intraperitoneal injection)at a dose of 50 mg/kg/d for 4 or 13weeks,the results showed that the intervention of geniposide did not affect the food intake and feed conversion ratio,but significantly reduced the body weight gain and Lee's index in all tested mice.The organ tissue weighing found that geniposide significantly reduced the liver/body weight ratio and the white fat(epididymal fat and subcutaneous fat)/body weight ratio,but did not affect the brown fat/body weight ratio in the normal chow diet and high-fat diet model mice.The ELISA analysis showed that the levels of total cholesterol(TC)and triglyceride(TG)were decreased in the serum and liver of the three model mice,and the histomorphometric analysis of liver and blood vessels showed the reduced fat deposition in the liver of model mice and the less atherosclerotic plaques in the aorta of Apo E^-/-mice,which could contribute to improve lipid metabolism.Secondly,the effects of geniposide on the homeostasis of glucose metabolism were further verified in normal chow diet/high-fat diet-fed mice.After the intervention of geniposide(intraperitoneal injection)at a dose of 50 mg/kg/d for 4 weeks,the results showed that the intervention of geniposide significantly reduced the random blood glucose and fasting blood glucose levels in different model mice,while geniposide could enhance the glucose tolerance and the insulin sensitivity of model mices.ELISA analysis found that geniposide could decrease the levels of serum insulin and hepatic glycogen in different model mice.The hepatic histomorphological analysis revealed that geniposide significantly reduced the accumulation of hepatic glycogen and improved glucose metabolism of different model mice.Thirdly,the effects of geniposide were evaluated on bile acid enterohepatic circulation in normal chow diet/high-fat diet-fed mice,to determine its impact on cholesterol metabolism.Transcriptomic sequencing revealed that the intervention of geniposide was able to activate the classical and alternative pathways of hepatic bile acid synthesis and to boost cholesterol catabolism,resulting in a compensatory increase of hepatic 3-Hydroxy-3-methylglutaryl-Co A reductase(HMGCR)at transcriptional level.These results were further confirmed by molecular biological analysis,which showed that geniposide significantly inhibited the negative feedback regulation of hepatic farnesoid X receptor(FXR)-mediated bile acid metabolism and promoted the reverse cholesterol transport.Also,geniposide could inhibit intestinal FXR-mediated bile acid reabsorption,allowing more bile acids to be recycled through the enterohepatic circulation and promoting bile acid excretion in feces and urine.Thus,our study suggested that geniposide improved cholesterol metabolic homeostasis by regulating the metabolic cycle of bile acids.Furthermore,the influence of geniposide in bile acid metabolism was investigated in cell study.The results showed that geniposide(100?M�L^-1)was able to inhibit FXR expression in Hep G2 cells.The inhibition effect further promoted the expressions of HNF-4?and LRH-1and bile acid synthesis,but reduced the efficiency of negative feedback regulation of hepatic bile acid metabolism.The intervention of geniposide(100?M�L^-1)inhibited FXR expression in Caco2 cells,following with the decreased expression of ileal-bile acid-binding protein(I-BABP)and apical sodium-dependent bile acid transporter(ASBT)and the inhibition of intestinal bile acid reabsorption.However,the intervention of GW4064(FXR agonist,2.5?M�L^-1)was able to promote the expression of FXR in Hep G2 cells and Caco2 cells,which could counteract the inhibitory effect of geniposide on FXR,which consequently mitigate the function of geniposide.Therefore,the findings suggested that FXR could be an important target for geniposide to regulate the bile acid metabolism between liver and intestine.In addition,the role of geniposide on the homeostasis of glucose metabolism was investigated in skeletal muscle of high-fat diet-fed mice.The results revealed that the intervention of geniposide significantly improved the PI3K-Akt-GSK3?-mediated insulin signaling pathway and enhanced systemic insulin sensitivity in the liver and skeletal muscle of high-fat diet-fed mice.Geniposide also promoted the oxidative utilization of circulating glucose through Fox O1-PDK4-PDH axis and stimulated glucose transporter 4(Glut4)expression which could promote the uptake of circulating glucose.However,data screening and partial least squares-discriminant analysis revealed that geniposide was able to reduce the level of circulating hepatokine Retinol binding protein 4(RBP4),which is responsible of regulating the information exchange of glucose metabolism between liver and skeletal muscle.Finally,the role of geniposide on hepatokine RBP4 was further investigated by constructing adeno-associated virus vectors,plasmids and si RNAs,using in vivo and in vitro experiments.Sequence alignment revealed that the sequences of signal transduction and activator of transcription(STAT5)and hypoxia inducible factor-1 alpha(HIF1?)had binding sites to the promoter region of RBP4.The following molecular experiments showed that growth hormone receptor(GHR)promoted the synthesis and secretion of hepatic RBP4through JAK2-STAT5 axis,hence elevating the level of circulating RBP4.GHR also enhanced the expression of hepatic HIF1?through PI3K-Akt-m TOR axis,and promoted hepatic transthyretin(TTR)synthesis and secretion,thereby maintaining the homeostasis of circulating RBP4.Experiments on liver-specific GHR overexpression mice and obese patients also showed that hepatic GHR was involved in the process of glucose metabolism and regulated the circulatory homeostasis of hepatokine RBP4.The intervention of geniposide significantly inhibited the expression of hepatic GHR,which could decrease the level of circulating RBP4 in high-fat diet-fed mice.