| Type 2 diabetes mellitus is a kind of heterogeneous syndrome of polygenic origin and involves both defective insulin secretion and peripheral insulin resistance.β-cell dysfunction is a sine qua non for the development of the disease.Some metabolic factors such as hyperglycemia and hyperlipidemia contribute to the progressive deterioration of glucose homeostasis characteristic of this disease.Prolonged exposure of pancreaticβ-cells to free fatty acids(FFAs)impairs insulin secretion function of islets of Langerhans,so called lipotoxicity.On the one hand,elevated FFAs aggravate the apoptosis ofβ-cells in islets.On the other hand,FFAs accumulate in the non-fat tissue and cause cell dysfunction.Professor McGarry,one famous Banting science fund winner even regarded diabetes mellitus as "Diabetes Mellipitus" in order to emphasize the important role of lipotoxicity effect on the pathogenesis of diabetes.So the studies about the effect and mechanism of lipotoxicity and novel anti-lipotoxicity drugs come into light.At present,mechanism of lipotoxicity includes:mitochondrion pathway, peroxisome proliferator-activated receptor(PPAR)pathway,oxidation and stress pathway,et al.Recently,researches focus on the role of PPARα.There exist been still two opposite arguments against PPARαand lipotoxicity.Some studies supported the idea that PPARαactivation was one way of lipotoxicity.Others reported PPARαwas inhibited by the high levels of FFAs and the function defect ofβ-cells induced by fatty acids was improved by PPARαactivation induced by fibrates.The underlying mechanism remains unclear.Fenofibrate,an agonist of PPARα,is a representative drug of fibrates used in the therapy of hyperlipidemia and diabetes mellitus.It was further testified that fenofibrate has more benefits to diabetic patients through potentiating insulin secretion and improving insulin resistance in target tissues such as muscle and liver.Then the most important question arises that what the direct effect of the fenofibrate onβ-cells is?Pancreatic/duodenal homeobox-1(PDX-1),a major transactivator of tightly regulating insulin and glucose transporter-2(GLUT2)at the transcriptional level,is an early and critical event in facilitating development and maintaining the function of pancreaticβ-cells.Its activation,leads to insulin transcription potentiation that subsequently results in insulin synthesis increase.PDX-1 bonds with the A1 and A3/A4 site at the promoter of insulin,which is characterized by containing core TAAT sequence,and directs insulin mRNA translocation from nuclear to cytoplasm, and then activates translation of insulin gene.The extracellular glucose must be transferred into cytoplasm by GLUT2,and then phosphorylated by glucose kinase (GK).ATP is a metabolic production of glucose and enhances the ATP/ADP ratio accompanied by K+-ATP channel shutting,and then membrane depolarization induces secretion of insulin granules.Research has reported that FFAs inhibit the expression of PDX-1,GLUT2 and insulin.But the upstream mechanism is unknown.AMP-activated protein kinase(AMPK)is a key regulator of cellular energy metabolism and is closely associated with insulin secretion ofβ-cells.Stresses such as exercise,hypoxia,or prolonged starvation could activate AMPK via increasing intracellular AMP/ATP ratio.Once activated,it phosphorylates and inactivates acetyl-CoA carboxylase(ACC),resulting in a decrease in malonyl-CoA,thus relieving inhibition of CPT1 and facilitating FFAs to entry into mitochondria forβ-oxidation.Inβ-cells,AMPK activity is inhibited by high concentration of glucose. Then,what is the effect of high FFAs on the expression and activity of AMPK inβ-cells?OBJECTIVES:The present study aims to explore several questions in cultured INS-1β-cells treated with and without palmitate,in the presence or absence of fenofibrate(PPARα agonist)or MK886(PPARαantagonist).1.To observe the effects of chronic palmitate exposure on insulin secretion and the expression of PPARα,PDX-1,insulin and GLUT2 in rat INS-1 cells and to explore the mechanism ofβ-cell lipotoxicity.2.To observe the effects of fenofibrate on insulin secretion and the expression of PPARα,PDX-1,insulin and GLUT2 in INS-1 cells and to explore the mechanism of fenofibrate improvingβ-cell lipotoxicity.3.To explore the relationship between PPARαand PDX-1 in INS-1 cells.4.To observe the effects of chronic exposure of palmitate to INS-1 cells on the expression and activity of AMPKαand to investigate the role of AMPKαinβ-cell lipotoxicity.METHODS:1.The culture and groups of INS-1 cells:The rat insulinoma cell line-INS-1 cells were cultured in RPMI1640 medium containing 10%fetal calf serum,2 mM glutamic acid,50μMβ-mercaptoethanol.Cells were divided into six groups through the whole experiment:C group(control group),P group(with 0.2 mM palmitate),PF group(0.2 mM palmitate and 5×10-6M fenofibrate),F group(with 5×10-6M fenofibrate),PM group(0.2 mM palmitate and 1×10-6M MK886)and M group (1×10-6M MK886).Treatment time:48 hours.2.Cell morphology:INS-1 cells were cultured in different groups for 48 hours and then were observed under invert microscope.3.Cell viability:INS-1 cells were cultured in different groups for 48 hours and then cell viability was detected by MTT experiment.4.The detection and analysis of glucose-induced insulin secretion in INS-1 cells: INS-1 cells were routinely seeded in 24-well-plate for insulin secretion studies and were divided into 6 groups with different treatment medium.After 48 hours treatment, insulin secretion was detected by radioimmunoassay in response to 3 and 20 mM glucose in Krebs-Ringer bicarbonate buffer(KRB),named as basal insulin secretion (BIS)and glucose-stimulated insulin secretion(GSIS),respectively.And insulin secretion index(ISI)was calculated by the ratio of GSIS/BIS. 5.Detection of the expression of PPARα,PDX-1,insulin and GLUT2:The mRNA levels of PPARα,PDX-1,insulin and GLUT2 were detected by real-time PCR.And the protein expression of PDX-1 and GLUT2 was measured by western blot while the PPARαprotein expression was determined by immunoprecipitation. Radioimmunoassay was exerted to measure the intracellular insulin content. Immunofluorescence was used to detect the location and expression of PDX-1 and insulin.Electrophoretic mobility shift assay(EMSA)was performed to measure the binding activity of PPARα.6.Pdx-1#6 cell line's culture:ISIS-1 stable cell lines,Pdx-1#6,allowing inducible expression of wild-type PDX-1 depending on doxycycline,were cultured as described in INS-1 cells supplemented with 100 ng/ml hygromycin and 100 ng/ml G418.In order to induce PDX-1 expression in current experiment,different concentration of doxycycline(0,75,150,500 ng/ml)was added for 24 or 48 hours.Then mRNA levels of PDX-1,PPARα,insulin,GLUT2 were measured by RT-PCR.7.The measure of AMPKαsubunit expression and activity:mRNA levels of alpha 1 subunit and alpha 2 subunit of AMPK were measured using real-time PCR.Protein levels of total AMPKα(T-AMPKα)subunit were detected using western blot. Activities of AMPK were evaluated,by measuring the protein levels of phosphorated AMPKα(P-AMPKα)subunit using western blot.RESULTS:1.Cell morphology:The control cells grew well.Round vesicle may be observed in about 60%palmitate-treated cells,which looked like contraction and round.PF group cells had the less vesicle compared with P group cells.No marked change presented in F group cells over control.2.Cell viability:Compared with control group,cell viability was decreased by 53%(p＜0.05)in P group.While cell viability in PF group was increased by 74%(p＜0.05)compared with P group.There is no obvious change in F group over control.3.Insulin release:Compared with control group,0.2 mM palmitate enhances BIS by about 24%(p＜0.05)and reduces GSIS to 86%(p＜0.05).ISI was decreased by palmitate from 2.34-fold to 1.69-fold(p＜0.05).Compared with P group,BIS was decreased by 27%(p＜0.05)in PF group and GSIS and ISI were increased by 1.14- and 1.56-fold(p＜0.05).ISI was increased by 12%in F group over control. Enhanced BIS was measured by 56%and 34%in PM group and M group(p＜0.05), respectively and the GSIS was reduced by 58%and 69%(p＜0.05).4.The expression of PPARα,PDX-1,insulin and GLUT2:In P group compared with control group,the expression of PPARαwas decreased by 38%in mRNA level and by 58%in protein expression(p＜0.05),respectively.PDX-1 expression was reduced by 46%in mRNA level and by 49%in protein expression(p＜0.05).The expression of insulin and GLUT2,PDX-1 downstream targets,were decreased by 41%and 54%in mRNA levels(p＜0.05),and by 29%and 31%in protein levels(p＜0.05).Immunofluorescence results showed the weaker staining of PDX-1,which located mainly in nucleus,and insulin,which located in cytoplasm.The staining of both showed weaker in P group over control,indicating that palmitate may inhibit the expression of PPARαand PDX-1,in turn to decrease the expression of insulin and GLUT2,which may be a possible mechanism of palmitate impairing insulin secretion.In PF group compared with P group,the expression of PPARαwas increased by 61%in mRNA level and by 157%in protein expression(p＜0.05),respectively. PDX-1 expression was enhanced by 61%in mRNA level and by 78%in protein expression(p＜0.05).The expression of insulin and GLUT2,PDX-1 downstream targets,were decreased by 114%and 61%in mRNA levels,and by 68%and 179%in protein levels(p＜0.05).Immunofluorescence results showed a stronger staining in PDX-1 and insulin.All indicate that fenofibrate,as an agonist of PPARα,may enhance the expression of PPARαand PDX-1,and in turn to enhance the expression of insulin and GLUT2,which may be a possible mechanism of fenofibrate improving insulin secretion in INS-1 cells.In order to explore whether PPARαmay regulate the expression of PDX-1,we employed MK886,a PPARαantagonist.Results showed that PPARαmRNA level was decreased by 66%(p＜0.05)in M group and PDX-1 expression was inhibited by 54%in mRNA levels and 53%in protein levels(p＜0.05).Accordantly,insulin and GLUT2 expression levels were decreased.A weaker stainning was observed in immunofluorescence experiment.All reached a maximum reduction in PM group. This implies that PPARαmay positively regulate the expression of PDX-1 in INS-1 cells under normal or highlipid state,which may be a possible mechanism of palmitate impairing insulin secretion in INS-1 cells.To further test the relationship between PPARαand PDX-1,we employed Pdx-1#6 cells with a characteristic of overexpressed PDX-1 in a doxycycline-inducible manner. With the gradual increase in PDX-1 protein over 24 hours in the range of 0～500 ng/ml doxycycline,we detected the gradual enhancement in gene transcription levels of insulin and GLUT2,but no obvious change in PPARαmRNA level.All suggest that PDX-1 does not mediate PPARα,but positively regulate the gene trancription of insulin and GLUT2.In order to elucidate the action site of PPARαregulation of PDX-1,we performed EMSA and found that palmitate reduced the DNA binding activity of PPARαand an improved effect was shown in PF group.Excitedly,we found a direct binding band of PPARαwith PDX-1 promoter,the change trend of which was consistent with above experiments.5.The expression and activity of AMPK:Compared with control group, palmitate induced a 34%reduction in the protein expression of T-AMPKα(p＜0.05), and a 38%decrease in P-AMPKαprotein expression(p＜0.05).mRNA levels analysis showed that AMPKα1 was reduced by 40%while AMPKα2 was enhanced, which indicated that AMPKα1 subunit is the main functional subunit inβ-cells.Compared with P group,PF group enhanced the expression of T-AMPKαand P-AMPKαby 40%and 52%(p＜0.05).The mRNA expression of AMPKα1 was increased by 26%but no obvious change presented in AMPKα2 subunit.No signnificant change happened,to F group compared with control group.CONCLUSIONS:1.PPARαpositively regulates the PDX-1 expression in physiological and palmitate-induced pathological state,which may be via the direct binding of PPARαwith PDX-1 promoter. 2.Chronic exposure of INS-1 cells to palmitate may impair the insulin secretion by reducing the expression of PPARα,PDX-1 and its downstream targets,insulin and GLUT2.3.Fenofibrate may improve the impaired insulin secretion by palmitate through activating PPARαand upregulating the expression of PDX-1,insulin and GLUT2.4.Chronic exposure of INS-1 cells may reduce the expression and activity of AMPKα,which is mainly from the change of AMPKα1 subunit.This may be a possible mechanism of palmitate impairing insulin secretion.
|
PDF Full Text Request