| Obesity is one of the main features of insulin resistance,disorder of metabolism of carbohydrate and lipid are closely related with insulin resistance.A key feature of obesity is excessive lipid accumulation in adipose tissue and ectopic localizations.Liraglutide,a human glucagon-like peptide-1(GLP-1)analogue,its effects on improving blood glucose,obese and insulin resistance has been recognized.The mechanism of improving blood glucose is known clearly.Liraglutide treatment improved obese and insulin resistant parallel with lipids improvement,but the mechanism of this is unclear.Recent studies have shown that adenylyl cyclase 3(AC3)play a critical role in regulation of body weight.AC3 gene knock-out mice become obese when aging mainly due to increased fat mass and larger adipocytes and accompany with the raise of blood lipid.Our preliminary study confirmed that liraglutide can increase the expression of AC3 in the mouse liver,the expression of AC3 m RNA is inversely associated with body weight and homa insulin resistance index(HOMA-IR).We speculate that GLP-1 play a role of improving insulin resistance by regulation the expression of AC3 and then affect the lipid metabolism signal pathways.Therefore,this study investigate the effect of liraglutide on the lipolysis pathway of mice and hepatic cell and the relationship between AC3 and insulin resistance.This study will help to clarify the idea regarding molecular mechanism of GLP-1 and the relationship between AC3 and insulin resistance and focus on clinical remission of insulin resistance.Part One Effects of Liraglutide on AC3 and the Relationship between AC3 and Insulin Resistance in MiceObjectives: To observe the effects of liraglutide on AC3,body weight and the metabolism of carbohydrate and lipid.To investigate the relationship between liraglutide,AC3 and insulin resistance in mice.Methods: The experiments were performed using 4-week-old C57BL/6J mice(Purchased from the Medical Laboratory Animal Centre of Guangzhou Province).The mice were housed in individual cages in a temperature-controlled room with a 12-h light/dark cycle.Mice had free access to standard mouse chow and water.After one week,24 C57BL/6J mice were divided randomly into 2groups.The normal control group(N,n=12)was fed a standard chow diet,and the HFD group was fed an obesogenic diet(34.9% fat and 26.2% protein)for 12weeks(O,n=12).Body weight(BW)and blood glucose were measured weekly.Mice with fasting blood glucose(FBG)levels >13.9 mmol/L(250 mg/dl for three consecutive days were considered to be diabetic.Mice with BW that exceeded normal weight by at least 20% were considered obese.After the obese mouse model was successfully established,all mice were subdivided into the following groups(n=6 per group): N+saline(N+S),N+liraglutide(N+L),O+saline(O+S),and O+liraglutide(O+L).The N+L and O+L groups were administered subcutaneous injections of the GLP-1 analogue liraglutide at a dose of 0.1 mg/kg/12 h.The N+S and O+S groups were treated with subcutaneous injections of the same volume of normal saline.After 8 weeks,the mice were fasted overnight and anaesthetized with sodium pentobarbital(30mg/kg,i.p.).Blood was obtained via the angular vein.Serum was separated by centrifugation at 4°C and stored at-20°C until assayed.The livers were immediately dissected,frozen in liquid nitrogen and stored at-80°C until analysed.Hepatic AC3 m RNA expression levels were measured by real-time quantitative polymerase chain reaction(RT-q PCR).Hepatic AC3 protein expression levels were measured by Western bloting.Serum levels of AC3,triglycerides(TGs),glycerol,free fatty acide(FFA)and insulin were determined via enzymatic methods using commercial kits.Results:1.Before liraglutide treatment,the BW of mice in the O group exhibited significantly higher than the mice in the N group(p <0.01).The BW of mice in the O group higher than in the N group at least 20%.FBG levels were elevated in the O group relative to those in the N groupp(<0.01).2.BW was significantly higher in obese mice than in normal control mice(p<0.01).Liraglutide treatment can significantly decreased BW(p <0.01).No significant differences in FBG were observed between normal control mice and obese mice or between liraglutide-treated mice and saline-treated mice.Serum insulin level was significantly higher in obese mice than in normal control mice(p <0.01).Liraglutide treatment can significantly decreased serum insulin level(p <0.01).HOMA-IR scores was significantly higher in obese mice than in normal control mice(p <0.01).Liraglutide treatment can significantly decreased HOMA-IR scores(p <0.01).The serum levels of TGs,glycerol and FFA were significantly higher in obese mice than in normal control mice(p <0.01).The liraglutide treatment significantly decreased the serum levels of TGs and FFA and significantly increased serum glycerol levels(p <0.01).The effects of liraglutide on BW,insulin,HOMA-I R,TGs,glycerol and FFA levels in obese mice were significantly higher than that in normal contral mice(p <0.05).3.Serum AC3 level was significantly lower in obese mice than in normal control mice(p <0.01).Liraglutide treatment significantly increased serum AC3 level in normal control and obese mice(p <0.01).The serum AC3 levels in obese mice increased significantly higher than that in normal contral mice(p <0.05).4.The hepatic levels of AC3 m RNA was significantly lower in obese mice than in normal control mice(p <0.01).Liraglutide treatment can significantly increased the hepatic levels of AC3 m RNA(p <0.05).The hepatic AC3 m RNA levels in obese mice increased significantly higher than that in normal contral mice(p <0.05).5.The hepatic levels of AC3 protein was significantly lower in obese mice than in normal control mice(p <0.01).Liraglutide treatment can significantly increased the hepatic levels of AC3 protein(p <0.01).The hepatic AC3 protein expression levels in obese mice increased significantly higher than that in normal contral mice(p <0.05).6.Statistical analysis showed that the serum AC3 levels were negatively correlated with BW(r=-0.682,p=0.000)and the HOMA-IR score(r=-0.716,p=0.000).The levels of the AC3 m RNA were negatively correlated with BW(r=-0.441,p=0.031)and the HOMA-IR score(r=-0.654,p=0.001).The levels of the AC3 protein were negatively correlated with the BW(r=-0.672,p=0.000)and the HOMA-IR score(r=-0.665,p=0.000).Conclusions:Liraglutide treatment decreased BW,HOMA-IR,triglycerides and FFA and increased glycerol.Liraglutide treatment can promote lipolysis.The effects of liraglutide on BW,HOMA-IR,triglycerides,FFA and glycerol in obese mice were significantly higher than that in normal contral mice.Liraglutide treatment can upregulation AC3 levels in mice.AC3 levels in obese mice increased significantly higher than that in normal contral mice.AC3 levels were negatively correlated with BW and HOMA-IR.Part two Effects of Liraglutide on the AC3/c AMP/PKA/HSL Lipolysis Pathway in mice liver and Hepa1-6 CellsObjectives: Investigate the effect of liraglutide on the AC3/c AMP/PKA/HSL lipolysis pathway in mice liver and hepa1-6 cells.Methods:1.Animal studies Mice feeding and intervention were same as part one.After 8 weeks,the mice were fasted overnight and anaesthetized with sodium pentobarbital(30 mg/kg,i.p.).The livers were immediately dissected,frozen in liquid nitrogen and stored at-80°C until analysed.Hepatic GLP-1R,AC3 and HSL m RNA expression levels were measured by real-time quantitative polymerase chain reaction(RT-q PCR).Hepatic glucagon-like peptide-1 receptor(GLP-1R),AC3,HSL and phosphorylated HSL Ser-660(p-HSL(s660))protein expression levels and cyclic adenosine monophosphate(c AMP)levels were measured by Western bloting.Liver samples were prepared according to the instructions provided.PKA activity in liver samples was measured with a PKA Kinase Assay Kit.2.Cells studies The mouse hepatocyte cellline(hepa1-6)was purchased from Fu Dan IBS cell centre and was maintained in Dulbecco's modified Eagle's medium.Cells were cultured with liraglutide in different concentrations(0,100 and 200 nmol/l),and after 48 hours,the cells were harvested for western blotting,RT-q PCR and measuring PKA activity.GLP-1R,AC3 and HSL m RNA expression levels were measured by RT-q PCR.GLP-1R,AC3,HSL and p-HSL(s660)protein expression levels and c AMP levels were measured by western bloting.PKA activity was measured with a PKA Kinase Assay Kit.Results:1.The hepatic levels of GLP-1R m RNA was significantly higher in obese mice than in normal control mice(p <0.01).Liraglutide treatment can significantly increased the hepatic levels of GLP-1R m RNA(p <0.01).The hepatic levels of AC3 m RNA was significantly lower in obese mice than in normal control mice(p <0.01).Liraglutide treatment can significantly increased the hepatic levels of AC3 m RNA(p <0.01).The hepatic GLP-1R m RNA and AC3 m RNA levels in obese mice increased significantly higher than that in normal contral mice(p <0.05).No significant differences in the hepatic levels of HSL m RNA were observed between normal control mice and obese mice or between liraglutide-treated mice and saline-treated mice.2.The hepatic levels of GLP-1R protein was significantly higher in obese mice than in normal control mice(p <0.01).Liraglutide treatment can significantly increased the hepatic levels of GLP-1R protein(p <0.01).The hepatic levels of AC3 protein was significantly lower in obese mice than in normal control mice(p <0.01).Liraglutide treatment can significantly increased the hepatic levels of AC3 protein(p <0.01).No significant differences in the hepatic levels of HSL protein were observed between normal control mice and obese mice or between liraglutide-treated mice and saline-treated mice.The hepatic levels of p-HSL(s660)protein was significantly lower in obese mice than in normal control mice(p <0.01).Liraglutide treatment can significantly increased the hepatic levels of p-HSL(s660)protein(p <0.01).The hepatic levels of c AMP was significantly lower in obese mice than in normal control mice(p <0.01).Liraglutide treatment can significantly increased the hepatic levels of c AMP(p <0.01).The effects of liraglutide on GLP-1R,AC3,p-HSL(s660)protein levels and c AMP levels in obese mice were significantly higher than that in normal contral mice(p <0.05).3.Liver PKA activity was significantly higher in normal control mice than in obese mice(p <0.01).Liraglutide treatment significantly increased liver PKA activity in normal control and obese mice(p <0.01).The hepatic PKA activity in obese mice increased significantly higher than that in normal contral mice(p <0.05).4.GLP-1R and AC3 m RNA levels were increased with the increasing of liraglutide concentration.No significant differences in HSL m RNA level were observed with the higher liraglutide concentrations.Spearman correlation analysis showed that GLP-1R m RNA levels were positive correlated with the concentrations of liraglutide(r=0.843,p=0.004)and AC3 m RNA levels were positive correlated with the concentrations of liraglutide(r=0.843,p=0.004).5.GLP-1R,AC3 and p-HSL(s660)protein levels an c AMP level were increased with the increasing of liraglutide concentration.No significant differences in HSL protein level were observed with the higher liraglutide concentrations.Spearman correlation analysis showed that GLP-1R protein levels were positive correlated with the concentrations of liraglutide(r=0.791,p=0.011),AC3 protein levels were positive correlated with the concentrations of liraglutid(r=0.843,p=0.004),p-HSL(s660)protein levels were positive correlated with the concentrations of liraglutide(r=0.738,p=0.023),and c AMP levels were positive correlated with the concentrations of liraglutid(r=0.738,p=0.023).6.PKA activity was increased with the increasing of liraglutide concentration.Spearman correlation analysis showed that PKA activity levels were positive correlated with the concentrations of liraglutide(r=0.843,p=0.004).Conclusions:Liraglutide treatment can upregulate hepatic AC3/c AMP/PKA/HSL pathway in mice.The effect of liraglutide on hepatic AC3/c AMP/PKA/HSL pathway in obese mice was significantly higher than that in normal contral mice.Liraglutide can upregulate AC3/c AMP/PKA/HSL pathway in hepa1-6 cells. |
PDF Full Text Request