Anti-diabetic Effects And Mechanisms Of The Oleanolic Acid Derivative DKS26

Background and ObjectivesDiabetes mellitus is a chronic metabolic disorder characterized by hyperglycemia due to insulin secretion deficiency or/and insulin resistance,and seriously endangers human health with its incidence increasing year by year.Because of the adverse reaction or/andpoor long-term efficacy of current antidiabetic drugs,it is imperative to develop more safe and effective medicines for diabetes control.The natural compound oleanolic acid(OA)has various biological activities mainly including hypoglycemic,hypolipidemic,hepatoprotective and anti-inflammatory effects,and its anti-hyperglycemic effect is associated with the increased incretin GLP-1 release.Nevertheless,the weak in vivo pharmacological effects and potential hepatotoxicity of OA limit its application in diabetes therapy.This study was designed to investigate the antidiabetic effects of the novel oleanolic acid derivative DKS26 in vitro and in vivo,as well as its hepatoprotective effect in diabetic mice and medication safety in normal mice,and explore its GLP-1 secretory effect in vitro and in vivo to further elucidate the potential anti-diabetic mechanisms,providing an important guideline for DKS26 to develop a new potential therapeutic for the treatment of diabetes.Methods1.Pharmacodynamics of DKS26 in vitro(1)Effects of DKS26 on glucose consumption in human hepatic HepG2 cellsInfluences of DKS26 on glucose consumption in HepG2 cells were detected by the glucose oxidase method.Experimental groups were established as solvent control group,1 mM of metformin positive control group,0.1,1 and 10 μM of OA control groups and 0.1,1 and 10 μM of DKS26 tested groups.(2)Effects of DKS26 on lipid levels in free fatty acids(FFAs)-induced lipid accumulated HepG2 cellsInfluences of DKS26 on lipid levels in FFAs-induced lipid accumulated HepG2 cells were investigated by triglyceride(TG),total cholesterol(TC)kit testing and oil red O staining.Experimental groups were established as normal control,0.25 mM FFAs model control,10μM bezafibrate positive control,1 mM metformin,10 μM OA and 10μM DKS26.2.Pharmacodynamics of DKS26 in vivo(1)Therapeutic effects of DKS26 in STZ-induced diabetic miceThe STZ-induced diabetic mouse model(fasting blood glucose levels ≥11.1 mmol/L)was established by a single intraperitoneal injection of STZ at a dose of 130 mg/kg in ICR mice.The diabetic mice were randomly divided into 4 groups according to the blood glucose levels,which were STZ model control group(n=7),100 mg/kg metformin positive control group(n=8),100 mg/kg OA(n=8)control group and 100 mg/kg DKS26 tested group(n=8).Besides,the normal(n=5)and model control groups mice were administrated equally with 0.5%CMC-Na.During 33 days’ intragastric administration,effects of DKS26 on fasting blood glucose,oral glucose tolerance(OGT),water intake,food intake and body weight in STZ-induced diabetic mice were monitored.After the last administration,influences of DKS26 on levels of plasma glycated serum protein(GSP),insulin,TG,TC,high density lipoprotein cholesterol(HDL-C),low density lipoprotein cholesterol(LDL-C),alanine aminotransferase(ALT),aspartate aminotransferase(AST),alkaline phosphatase(ALP),total bile acid(TBA),direct bilirubin(D-BIL),and total bilirubin(T-BIL)of mice were assessed;the islet and liver histopathological morphology were observed by HE staining;the hepatic lipid accumulation was measured by TG,TC kits testing and oil red O staining;changes of hepatic inflammatory related genes(TNF-a,IL-6,IL-1β,CD68 and F4/80)were measured by RT-PCR assays;the gallbladder size was estimated by the vernier caliper and weighing methods.(2)Therapeutic effects of DKS26 in db/db diabetic miceDb/db diabetic mice with blood glucose levels ≥11.1 mmol/L were randomly divided into 4 groups(n=7-8)according to blood glucose levels,namely db/db model control group(0.5%CMC-Na),100 mg/kg metformin positive control group,100 mg/kg OA control group and 100 mg/kg DKS26 tested group.During 30 days’ intragastric administration,effects of DKS26 on fasting and random blood glucose,OGT,water intake,food intake and body weight in db/db diabetic mice were monitored.After the last treatment,influences of DKS26 on levels of plasma GSP,insulin,TG,TC,HDL-c,LDL-c,ALT,AST,ALP,TB A,D-BIL and T-BIL of mice were measured,and effects of it on the islet and liver histopathological morphology,hepatic lipid accumulation,hepatic inflammatory and gallbladder size were estimated.3.Anti-diabetic molecular mechanisms of DKS26 in vitro and in vivo(1)Effects of DKS26 on GLP-1 expression and secretionLevels of GLP-1 in the culture supernatant of enteroendocrine NCI-H716 L cells and the plasma of mice,and levels of cAMP in NCI-H716 cells and mouse intestine were assessed by the ELISA;expressions of GLP-1 protein in culture cells and mouse intestine were detected by immunofluorescence assays;expressions of GLP-1 related genes(gcg and PC3)in samples of all the models in vitro and in vivo were detected by RT-PCR assays;alterations of p-PKA and PKA protein in the same samples were assessed by western blotting tests.(2)Effect of DKS26 on human G protein-coupled bile acid receptor(TGR5)Effect of DKS26 on human TGR5(hTGR5)was assessed by an in vitro model of HER293/pGL4.29/hTGR5 cells.Experimental groups were established as blank control group,INT-777 positive control groups,OA control groups and DKS26 tested groups.(3)Effects of DKS26 on hepatic AMPK protein and glucolipid metabolism related gene expressionsExpressions of protein p-AMPK,AMPK,p-ACC and ACC in normal HepG2 cells,FFAs-induced lipid accumulated HepG2 cells and mouse liver tissues were detected by western blotting analyses.Expressions of glucolipid metabolism related genes(G6Pase,PEPCK,GLUT2,SREPB-1c,FAS,PPARα and CPT1)in HepG2 cells and mouse liver were measured by RT-PCR assays.4.Effects of DKS26 on hepatic function in normal miceLevels of plasma ALT,AST and TBA were measured after the continuous administration of DKS26 to investigate its effect on liver function in normal mice.Experimental groups were established as solvent control group,100 mg/kg metformin control group,100 mg/kg OA control group and 100 mg/kg DKS26 tested group.Results1.Pharmacodynamics of DKS26 in vitro(1)Effects of DKS26 on glucose consumption in human hepatic HepG2 cellsDKS26 significantly increased glucose consumption in HepG2 cells,which was similar to that of metformin.(2)Effects of DKS26 on lipid levels in FFAs-induced lipid accumulated HepG2 cellsDKS26 decreased the levels of TG,TC and lipid droplets in FFAs-induced lipid accumulated HepG2 cells.2.Pharmacodynamics of DKS26 in vivo(1)Therapeutic effects of DKS26 in STZ-induced diabetic miceIn STZ-induced diabetic mice,DKS26 significantly lowered the levels of plasma glucose,GSP and lipid profiles,improved OGT,and reduced food and water intake.Moreover,the hypoglycemic effect of DKS26 was equivalent to metformin,and stronger than OA.DKS26 obviously increased plasma insulin levels,and ameliorated islet morphology,and reduced the levels of plasma ALT,AST,and ALP,decreased hepatic lipids,improved liver histopathologic morphology and inhibited hepatic inflammatory related gene expressions in diabetic mice.Besides,DKS26 notablely decreased the levels of plasma TBA,D-BIL and T-BIL and the ratio of D-BIL/T-BIL of mice,and reduced the gallbladder sizes.(2)Therapeutic effects of DKS26 in db/db diabetic miceIn db/db diabetic mice,DKS26 significantly lowered the levels of plasma glucose,GSP and lipid profiles,improved OGT,and reduced water intake.Moreover,the hypoglycemic effect of DKS26 was equivalent to metformin,and stronger than OA.DKS26 markedly enhanced plasma insulin levels,and improved islet morphology,as well as decreased the levels of plasma ALT,AST,and ALP,reduced hepatic lipids,ameliorated liver histopathologic morphology and inhibited hepatic inflammatory related gene expressions in diabetic mice.Besides,DKS26 exerted no significant influences on the plasma TBA levels and gallbladder sizes in diabetic mice,but caused a decrease in the levels of plasma D-BIL and T-BIL although with no significant effect on the ratio of D-BIL/T-BIL.3.Anti-diabetic molecular mechanisms of DKS26 in vitro and in vivo(1)Effects of DKS26 on GLP-1 expression and secretionDKS26 sharply increased the GLP-1 levels in the culture supernatant of NCI-H716 cells,and enhanced the expressions of GLP-1 protein and related genes(gcg and PC3)in NCI-H716 cells accompanied with an increase of cAMP levels and p-PKA protein expression.It also enhanced the levels of plasma GLP-1 and gene expressions of gut gcg and PC3 in both STZ-induced and db/db diabetic mice accompanied by an increase of intestinal cAMP levels and p-PKA protein expression.Besides,DKS26 promoted the in situ expression of GLP-1 protein in the gut of STZ-induced diabetic mice.(2)Effect of DKS26 on hTGR5DKS26 showed no TGR5 agonistic activity on hTGR5 at the highest concentration of 100 μM,while OA weakly exerted weak agonistic effects on hTGR5(OA EC50=13.659±1.812 μM,positive control INT-777 ECs50=1.765±0.452 μM).(3)Effects of DKS26 on hepatic AMPK protein and glucolipid metabolism related gene expressionsIn the in vitro study,DKS26 increased the protein expressions of phosphorylated p-AMPK and its downstream target p-ACC in normal HepG2 cells in an concentration and time dependent manner accompanied with the inhibited expressions of gluconeogenesis key enzymes(G6Pase and PEPCK)and increase of glucose transporter GLUT2.DKS26 also reversed the weakened expression of protein p-AMPK and p-ACC in FFAs-induced lipid accumulated HepG2 cells accompanied by inhibiting expressions of SREPB-1c and FAS which were related to fat synthesis,and promoting expressions of PPARa and CPT1 related to lipolysis.In the in vivo study,DKS26 up-regulated the protein expressions of liver p-AMPK and p-ACC in both STZ-induced and db/db diabetic mice accompanied with the down-regulated expressions of G6Pase and PEPCK and up-regulation of GLUT2 as well as the inhibition of SREPB-1c and FAS and promotion of PPARa and CPT1.4.Effects of DKS26 on hepatic function in normal miceThe continuous administration of DKS26 exerted no significant effects on the levels of plasma ALT,AST and TBA in normal mice,but OA at the same dose increased the plasma levels of these hepatic damage related indexes.ConclusionsThe oleanolic acid derivative DKS26 exerted good anti-diabetic effects in vitro and in vivo.The anti-diabetic mechanism of DKS26 might be related to the promotion of GLP-1 expression and secretion,and the activation of hepatic AMPK to regulate glucolipid metabolism.In addition,DKS26 exerted the hepatoprotective effect in diabetic mice,and was safer than OA.1.DKS26 increased glucose consumption in hepatic HepG2 cells and decreased lipid levels in FFAs-induced lipid accumulated HepG2 cells,and its mechanism probably was related to activating the hepatic AMPK signaling pathway,leading to the down-regulated expression of PEPCK and G6Pase to reduce gluconeogenesis,and upregulation of GLUT2 to increase glucose uptake as well as the down-regulation of SREBP-1c and FAS to inhibit fatty acid synthesis,and up-regulation of PPARa and CPT1 to promote fatty acid oxidation.2.In STZ-induced diabetic mice,DKS26 reduced plasma glucose and lipids and improved OGT,and its hypoglycemic effect was equivalent to that of metformin,and stronger than OA;it reduced water and food intake improving the diabetic symptoms of polydipsia and polyphagia;it increased the plasma insulin level and ameliorated the islet histopathology morphology exerting islet protective effects;and it ameliorated liver function,histopathology morphology,fat accumulation and inflammation exerting hepatoprotective effects.Besides,DKS26 decreased plasma TBA levels and gallbladder sizes of mice,indicating that it had no risks of cholestasis or gallbladder filling in STZ-induced diabetic mice.3.In db/db diabetic mice,DKS26 significantly lowered levels of fasting and random blood glucose and plasma lipids,and its hypoglycemic effect was equivalent to that of metformin,and stronger than OA;it reduced water intake improving the diabetic symptom of polydipsia;it increased plasma insulin levels and ameliorated islet morphology exerting islet protective effects;and it ameliorated function,histopathology morphology,fat accumulation and inflammation exerting hepatoprotective effects.Besides,it exerted no significant influences on plasma TBA levels and gallbladder sizes of mice,indicating that it had no risks of cholestasis or gallbladder filling in db/db diabetic mice.4.DKS26 promoted GLP-1 expression and secretion in enteroendocrine NCI-H716 cells which probably was related to the activation of cAMP/PKA signaling pathway.It increased plasma GLP-1 levels,promoted GLP-1 related genes(gcg and PC3)expression,and activated the cAMP/PKA signalling pathway in both STZ-induced and db/db diabetic mice,indicating that its hypoglycaemic,hypolipidaemic and islet protective effects in diabetic mice were probably mediated by the promotion of the GLP-1 secretion and expression.5.DKS26 increased the protein expression of p-AMPK and p-ACC accompanied with the inhibited gene expression of PEPCK、G6Pase、SREBP-1c and FAS and promotion of GLUT2.PPARa and CPT1 in both STZ-induced and db/db diabetic mice,prompting that the hypoglycaemic and hypolipidaemic effects in diabetic mice might also be mediated by the activation of hepatic AMPK signaling pathway to regulate glycolipid metabolism related enzymes and nuclear transcription factors,leading to a reduce in gluconeogenesis and fatty acid synthesis and an increase in glucose uptake and fatty acids oxidation.6.In normal mice,DKS26 showed no influences on liver function,but OA could damage liver function,prompting that DKS26 was safer than OA.