The Study Of Genipin On The Hepatic Insulin Resistance Through Ameliorating Mitochondrial Dysfunction In Vivo And In Vitro

Insulin resistance(IR)is a common pathophysiological feature of obesity,type 2 diabetes mellitus(T2DM)and cardiovascular diseases(CVD).Mitochondria are the major sites for intracellular fatty acid oxidation,ATP synthesis and generation of reactive oxygen species(ROS).Mitochondrial dysfunction is supposed to be one of the major factors leading to insulin resistance.As a prominent target of insulin,the liver plays a key role in maintaining the glucose homeostasis and energy metabolisms.Hepatic insulin resistance was reported to precede the occurrence and development of systemic insulin resistance.It is important to explore the mitochondrial mechanisms of hepatic insulin resistance and develop drugs targeting mitochondrial functions for the prevention and treatment of T2DM and related metabolic diseases.Genipin is an iridoid compounds extracted from Gardenia jasminoides Ellis fruit which has long been used in Asian countries as an herbal medicine with hepatoprotective,choleretic,anti-inflammatory,anti-oxidative,anit-tumor,and hypoglycemic effects.Genipin has recently been reported to promote ATP levels and stimulate insulin secretion in islet β cells by inhibiting mitochondrial uncoupling protein 2(UCP2).However,the potential action mechanisms of genipin on insulin resistance,and especially hepatic insulin resistance remain unknown.In this study,the effects of genipin on insulin resistance and mitochondrial functions were observed in aging rats,palmitate-treated hepatocytes and UCP2 overexpression hepatocytes.The purpose of this study is to explore the mitochondrial mechanisms and the molecular targets of genipin on hepatic insulin resistance servicing for the early prevention and therapy of T2DM.Part Ⅰ The action mechanisms of genipin on age-related insulin resistance in ratsPurposes:Aging is one of the independent risk factors for insulin resistance.Oxidative stress and mitochondrial dysfunction were supposed to be major factors leading to age-related insulin resistance.In this part,the effects of genipin on insulin sensitivity and hepatic mitochondrial functions were observed in 18-month old rats.The purpose is to investigate the potential mitochondrial mechanisms of genipin on hepatic insulin resistance in vivo.Methods:The rats were randomly assigned to receive intraperitoneal injections of either 25 mg/kg genipin or vehicle once daily for 12 days.After administration,at least six rats from each group randomly were used to perform intravenous insulin tolerance tests(ITT).The area under the insulin tolerance curve(AUCITT)and glucose decay constant rate during insulin tolerance test(KITT)were caculated to represent the degree of insulin resistance.The other rats were weighed and sacrificed by decapitation.Blood samples were collected for biochemical measurements.Visceral fat were obtained and weighed,and the ratio of visceral fat/body weight was calculated.Liver tissues were used for histological observation,malondialdehyde(MDA)determination and glycogen analysis,respectively.Hepatic mitochondria were isolated from freshly harvested livers and used for measurements of levels of ROS,mitochondrial membrane potential(MMP)and ATP.Results:(1)Genipin ameliorates age-related insulin resistance:The aging rats showed increased body weight,the ratio of visceral fat/body weight,triglycerides and insulin levels,however,genipin reversed these changes.The AUCITT was increased and KITT was decreased in aging rats representing the insulin resistant state.Genipin improved insulin sensitivity in aging rats by recovering AUCiITT and KITT obviously.(2)Genipin ameliorates hepatic insulin resistance in aging rats:The hepatic glycogen contents were obviously decreased in aging rats.Morphologcial observation suggested that the glycogen granules were reduced,on the contrary,the lipid deposit was increased in aging rats.Genipin intervention significantly reversed these changes,promoted the hepatic insulin sensitivity.(3)Genipin inhibit hepatic oxidative stress and improved hepatic mitochondrial function:The aging rats showed higher levels of hepatic MDA and mitochondrial ROS,lower levels of MMP and ATP,swelling mitochondria compared with the normal control rats.Administration of genipin relieved hepatic oxidative stress and mitochondrial dysfunction in aging rats.(4)Genipin inhibited hepatic mitochondrial UCP2 expression:UCP2 protein expression was significantly increased in liver of aging rats,however,genipin obviously inhibited UCP2 expression.Conclusions:Genipin ameliorates age-related insulin resistance and the attenuation of mitochondrial oxidative stress and UCP2 expression could be involved in.Part Ⅱ The action mechanisms of genipin on palmitate-induced insulin resistance in L02 hepatocytesPurposes:The elevation of plasma free fatty acid(FFA)was considered the decisive factor leading to insulin resistance.Mitochondrial dysfunction plays a key role in FFA-induced insulin resistance.In this part,the effects of genipin on hepatic insulin sensitivity,mitochondrial function and insulin signal transduction were observed in L02 hepatocytes treated with palmitate.The purpose is to explore the effects of genipin on hepatic insulin resistance and the potential mitochondrial mechanisms in vitro.Methods:The effects of palmitate on insulin-stimulated glycogen synthesis,insulin-stimulated glucose consumption,cell viability,and lipid deposition were observed in L02 hepatocytes according to which the palmitate-induced insulin resistant cell model was established.The effect of palmitate on cellular ROS,MMP and ATP level were detected.After that,the cells were cultivated with palmitate at 0.25mM with or without genipin at various concentrations for 24h,and the insulin sensitivity,mitochondrial function and insulin signalling molecules were detected.In addition,the antiox idative effect of genipin was assessed on a luminol-H2O2-Co2+chemiluminescence system.Results:(1)The establishment of palmitate-induced insulin resistant cell model:0.25mM palmitate significantly impaired the insulin-stimulated glycogen synthesis and glucose consumption,and increased lipid deposition for 24h treatment in L02 hepatocytes.(2)Palmitate induced mitochondrial dysfunction:When treatment with 0.25mM palmitate for various duration,L02 cells showed significantly increased ROS level at time point of 8h,16h and 24h;MMP and ATP level were significantly decreased at time point of 16h and 24h.(3)Genipin alleviated insulin resistance in palmitate-treated L02 hepatocytes:Genipin recovered insulin-stimulated glycogen synthesis and glucose consumption dose dependently in palmitate-treated L02 hepatocytes.(4)Genipin improved mitochondrial function in palmitate-treated L02 hepatocytes:Palmitate induced obvious decline of levels of ATP and MMP,and significant increment of cellular ROS production in L02 hepatocytes,however,genipin reversed these changes in a dose dependent manner.(5)Genipin restored insulin signaling in palmitate-treated L02 hepatocytes:SAPK/JNK was activated in response to palmitate treatment in L02 hepatocytes,and insulin-stimulated Akt phosphorylation was obviously reduced after exposure to palmitate.100p,M genipin significantly inhibited SAPK/JNK.phosphorylation and restored Akt phosphorylation.(6)The anti-oxidative role of genipin in vitro:Genipin directly scavenged H2O2 in vitro dose dependently.Conclusions:Genipin ameliorates oxidative stress,mitochondrial dysfuntion and thus insulin resistance,recovered insulin signalling transduction in L02 hepatocytes treated with palmitate.Part Ⅲ The effects of genipin on mitochondrial function and insulin sensitivity in Chang liver cells overexpressing UCP2Purposes:UCP2 belongs to the mitochondrial carrier family and partially uncouple respiration from ATP synthesis by dissipating the proton electrochemical gradient across the mitochondrial inner membrane.It was reported that UCP2 did not express in normal hepatocytes,but was upregulated in the condition of obesity,T2DM and non-alcoholic steatohepatitis and thus may be involved in hepatic insulin resistance.Our study in the first part had revealed that UCP2 involved in the protective role of genipin in age-related insulin resistance.In this part,we will further observe the effects of genipin on mitochondrial function and insulin sensitivity in UCP2 overexpressing Chang liver cells.The purpose is to explore the association of upregulation of UCP2 expression or activity and the protective role of genipin in hepatic insulin resistance.Methods:The Chang liver cell line was transfected with recombinant plasmid containing full-length human UCP2 cDNA(pcDNA3.1-hUCP2)or pcDNA3.1 empty vector.The UCP2 overexpression cells were treated with 50μM genipin,and the levels of insulin stimulated glycogen synthesis,MMP and ATP were detected.Results:(1)The establishment of UCP2 overexpression Chang liver cell line:The results from immunocytochemistry and western blot both showed increased UCP2 expression in cells transfected with UCP2 plasmids than in cells transfected with empty vector and in normal cells.(2)Genipin improved insulin sensitivity in UCP2 overexpression cells:The insulin stimulated glycogen synthesis was obviously decreased in UCP2 overexpression cells,however,genipin significantly recovered glycogen synthesis by 20%in UCP2 overexpression cells.(3)Genipin improved mitochondrial function in UCP2 overexpression cells:Compared with normal control group,the levels of MMP and ATP were obviously reduced in UCP2 overexpression cells;50μp.M genipin intervention promoted levels of MMP and ATP depressed in UCP2 overexpression cells.Conclusions:UCP2 overexpression impaired mitochondrial function and insulin sensitivity,however,genipin reversed these changes probably through inhibiting UCP2.