.1. Changes In Glucose, Metformin And Aicar On The Islet ¦Â-cells (ins-1 Cells), G Protein-coupled Receptor Expression To Adjust 2.kkay Diabetic Mice And High Fat Fed Obese Mouse Skeletal Muscle Insulin Mapk Pathway,

[Background and objective] :The family of G protein-coupled receptors (GPCRs) serves as the target for almost a third of currently marked druges, and provides the predominant mechanism through which extracellular factors transmit signals to the cell. GUP-1R, GIPR and GPR40 are members of GPCRs family abundantly expressed in the isletβcells, and seem to be important in maintaining normalβ-cell functions. Cellular energy status also play a important role in the regulation ofβcell function. Glucose is a major factor for proliferation of pancreatic beta cell in vivo and in vitro. As a key sensor of cellular energy status, the action of 5-AMP-activated protein kinase (AMPK) regulate cellular function in response to energy demand in various cells. A low cellular glucose concentration leads to depletion of ATP and cellular accumulation of AMP, which then leads to AMPK activation. Metformin is an anti-diabetic drug that increases glucose utilization in insulin-sensitive tissues.The effect is in part attributable to stimulation of AMPK.5-amino-4-imidazolecarboxamide riboside(AICAR) is a known activator of AMPK.It is thoutht to be a potential therapy for type 2 diabetes.However,the direct effects of metformin and AICAR on isletβcells have not been fully clarified.It have been demonstrated that glucose down-regulated GIPR gene expression. While the regulations of other GPCRs by glucose have not been elucidated up to now.And the regulations of other GPCRs by metformin and AICAR also have not been elucidated.Thus we investigated in INS-1βcells whether glucose concentrations metformin or AICAR regulate mRNA and protein expression of GPCRs.[Methods]:INS-1 cells were cultured.Different concentration of glucose, merformin and AICAR were incubated with INS-lcells.Real time RT-PCR and western blot are used to analysis the mRNA and protein levels of GLP-1R,GIPR and GPR40.It has been demonstrated that PPARαregulate the expression of GIPR.Thus,we analysis the mRNA and protein levels of PPARαINS-1 cells were incubated in the presence of actinomycin-D which is a transcriptional blocker to analysis the stability of mRNA.[Results]:INS-1 cells were incubated at glucose concentrations from 0 to 30 mmol/1 at 12 hours,and then examined for mRNA and protein expression levels.The expression levels of GLP-IR,GIPR and PPAR a mRNA and protein were down-regulated by elevated glucose in INS-IE beta cells.Moreover, to investigate time-response effects of glucose,INS-1 cells were incubated at 30mM glucose over a time course between 0-24 hours.The regulation of PPARαgene transcription by Glucose(30mM) was strongest and rapidest,with a significant reduction in PPARαmRNA level within 2 hours that reached a maximal decrease of 13%decrease at 6 hours.The mRNA levels of GIPR and GLP-IR were down-regulated significantly at 6 hours, reaching a maximal decrease of 20%and 34%at 24 hours.Perhaps not surprisingly,the time course of glucose-induced GLP-1R,GIPR and PPARαprotein down-regulation lagged behind that of their mRNA levels.INS-1 cells were incubated at AICAR or metformin with different glucose concentrations(10,30mM) for 24 hours.AICAR and metformin stimulated expression of GLP-1R,GIPR and PPAR a gene at 10 and 30 mM glucose concentrations after both 12 and 24 hours.However,glucose,AICAR and metformin had no effects on GPR40 gene expression.The stability of the mRNA encoding the GLP-1R,GIPR,GPR40 and PPARawere not statistically different between at 5mM glucose and 30mM glucose.[Conclusion]:In INS-1E beta cells,glucose down-regulated the expression levels of GLP-1R,GIPR and PPARαmRNA and protein in a dose-dependent manner. Metformin and AICAR have been shown to increase expression of GLP-1R and GIPR.But whether AMPK is a key regulator of glucose-mediated receptor regulation remains to be investigated.In addition,our result indicated that glucose level,metformin and AICAR seemed not to regulate the expression of GPR40. [Background and objective]:Obesity and type 2 diabetes are the most prevalent metabolic disease in the world,and often associated with impaired insulin action or insulin resistance.Insulin resistance is characterized by a decreased ability of insulin to act on peripheral tissues,such as skeletal muscle and adipose tissue,and a failure of insulin to inhibit hepatic glucose output.The defects of insulin signal transduction pathway play a key role in the development of insulin resistance.Insulin signaling along the insulin receptor substrate(IRS)/phosphatidylinositol 3-kinase (PI3K) pathway is considered to be important for the major metabolic actions of insulin,whereas mitogen-activated protein kinase(MAPK) cascades constitute signaling networks by which insulin regulates gene expression.Accumulating evidence suggests that insulin signaling defects along metabolic pathway involving IRS/PI3K are associated with skeletal muscle insulin resistance in obesity and type 2diabetes.In contrast,little is known of the regulation of MAPK cascades in insulin-resistant states,such as obesity and diabetes.Thus,we investigated the effects of insulin on MAPK cascades in skeletal muscle from insulin-sensitive lean (C57/BL6),insulin-resistant obese non- diabetic and diabetic(KKAy) mice.[Research design and methods]:Male C57/BL6 and KKAy mice(12 weeks old) were high-fat feed for 8 weeks to create insulin-resistant obese non- diabetic and diabetic(KKAy) mice.insulin-sensitive lean(C57/BL6) mice were considered as control mice.Mice were injected intraperitoneally with either vehicle or insulin (0.1IU/g).5,10 and 20 minutes after the injection,animal were killed by decapition, and quadriceps muscle was quickly isolated and immediately frozen in liquid nitrogen. Total and phosphorylation of ERKs,JNKs,p38MAPK were assessed by Western Blot.[Results]:Insulin led to a rapid phosphorylation of ERK and JNK signaling in quadriceps muscle of lean mice.JNK1 and JNK2 phosphorylation was increased with maximal effects observed at 5min.ERK1 and ERK2 phosphorylation was increased after 5 min,with maximal effects observed at 20 min.insulin did not induce any increase of P38MAPK phosphorylation.Total JNK1/2,ERK1/2 and P38MAPK in muscle were similar between lean,obese and KKAy mice.Basal phosphorylation of JNK1,ERK1/2 and p38 was increased in skeletal muscle from obese mice,while basal phosphorylation of JNK2 was similar between obese mice and lean mice. Insulin increased JNK 1/2 phosphorylation 2.1-fold;increased ERK 1/2 phosphorylation 1.6-and 2.1-fold from obese mice.Insulin action on JNK1,ERK1/2 phosphorylation was significantly impaired in skeletal muscle,while insulin action on JNK2 remains normal from obese mice.Basal phosphorylation of JNK1/2,ERK1/2 and p38 was increased in skeletal muscle from KKAy mice.Insulin action on JNK2 phosphorylation was impaired in skeletal muscle from KKAy mice.insulin did not induce any increase of JNK1,ERK1/2 phosphorylation.[Conclusion]:Insulin increased ERK1/2,JNK1/2 but not p38 phosphorylation in skeletal muscle of lean mice in a time-dependent manner.Basal phosphorylation of JNK2,ERK1/2 and p38 was increased in skeletal muscle from obese mice,and basal phosphorylation of JNK1/2,ERK1/2 and p38 was increased in skeletal muscle from KKAy mice.Insulin action on JNK1,ERK1/2 phosphorylation was significantly impaired in skeletal muscle from obese mice;Insulin action on JNK1/2,ERK1/2 phosphorylation was significantly impaired in skeletal muscle KKAy diabetic mice.