Pharmacodynamics Studies Of A Novel Indole Compound GY3 Based On Insulin Sensitizing Effect

Diabetes mellitus is a metabolic disorder characterized by hyperglycemia that is caused by absolute or relative insulin secretion insufficiency. Long-term hyperglycemia induces several systemic chronic syndromes based on a series of systemic pathological changes in blood vessels and nerves, which is threatening the human health.Diabetes mellitus can be mainly divided into type 1(insulin-dependent)diabetes and type 2(non-insulin dependent)diabetes,with type 2 diabetes accounting for more than 90%of all patients.Thiazolidinediones(TZDs),well-known peroxisome proliferator-activated receptorγ,(PPARγ)agonists,have been shown to play a pivotal role in therapy of type 2 diabetes,and they can ameliorate insulin resistance.Because of the adverse reactions of TZDs such as body weight gain,hepatic toxicity and fluid retention,the research of non-thiazolidinedione insulin sensitizers have attracted much more attention in recent years.Recent studies have shown that a number of non-steroid anti-inflammatory drugs can act as agonists of PPARγ,which is important nuclear receptor to regulat insulin sensitivity.GY3,named 1-(4-chlorobenzoyl)-5-hydroxy-2-methyl-3-indoleacetitic acid,is a novel synthesized indole compound that derived from indomethacin.It has been founded that GY3 could significantly enhance the differentiation of 3T3-L1 preadiopocytes into adipocytes.In this study,we aimed to identify the insulin sensitizing effectss and mechanisms of GY3,and detected the antidabetic effects in vivo.Aims.To identify the insulin sensitizing effectss and mechanism of GY3,and detecte the antidabetic effects in vivo.Methods:1.The effects of GY3 on 3T3-L1 adipocytes differentiation was detected by oil red staining analysis.And the proteins of glucose transporter 4 (GLUT4)and adiopnectin during the 3T3-L1 cells differentiation when treated with GY3 were determined by Western blot analysis.2.Activation of GY3 on PPARγand PPARαwere determined by transient transfection assay.3.The protein levels of adiponectin and GLUT4 in mature adipocytes were detected by Western blot analysis.Real time PCR was used to detect the gene expression of resistin and leptin.Elisa analysis was used to determine the release of adiponectin in culture media.4.The level of adiponectin and proteins included in the insulin pathway were detected by Western blot analysis.5.The effects of GY3 on insulin mediated glucose consumption were determined in HepG2 human hepatocelular carcinoma cell line by glucose oxidation analysis.6.The effects of GY3 on serum blood,glycosy lated serum protein,total cholesterol,triglyceride,free fat acid,food up-take and water up-take was detected in db/db mice during 3 weeks administration.Results:1.The effect of GY3 on 3T3-L1 adipocytes differentiation. As oil red staining analysis shown,1μmol/L and 10μmol/L GY3 significantly increased the lipid accumulating of 3T3-L1 adipocytes induced by insulin,dexamethasone and isobutylmethylxanthine mixture,GY3 increass the accumulation of lipid insignificantly induced by insulin only, whereas rosiglitazone could.However,Western blot analysis showed that GY3 could significantly increase the expression of GLUT4 and adiponectin as well as rosiglitazone did in both conditions mentioned above.2.Receptor activativity assayAs transient transfection assay presented,GY3 rarely showed the activation on PPARγ,whereas the known PPARγagonist rosiglitazone could activated PPARγsignificantly.And it was founded GY3 weakly activate on PPARα.3.The effects of GY3 in mature adipocytes. a)The effects of GY3 on the insulin pathway proteins in mature adipocytes.As western blot analysis showed,100 nmol/L insulin,10μmol/L GY3 or 10μmol/L rosiglitazone elevated the expression of PI3K,p-AKT and GLUT4 in mature adipocytes after 24h treatment.b)The effect of GY3 on adiponectin expression in mature adipocytesAs Real time PCR shown,the mRNA levels of adiponectin were both elevated in the presence of 10μmol/L GY3,10μmol/L rosiglitazone or 100 nm insulin(6.6±0.3-fold(P＜0.001),3.1±0.4-fold(P＜0.001),2.0±0.2-fold(P＜0.05),respectively)for 24h's treatment.Meanwhile,the protein expression of adiponectin was also increased by GY3.Elisa analysis also showed that the adiponectin release of mature adipocytes treated with 10μmol/L GY3,10μmol/L rosiglitazone or 100 nm insulin was increased by 81.2%(P＜0.001),84.8%(P＜0.001)and 56.8%(P＜0.05) respectively。c)The effects of GY3 on leptin and resistin expression in mature adipocytesLeptin mRNA were up-regulated by 10μmol/L GY3 with 2.0±0.5-fold(P＜0.001)and 100 nmol/L insulin with 3.7±0.5-fold(P＜0.001), whereas rosiglitazone down-regulated this marker with 0.7±0.06-fold (P＜0.05)).The mRNA of resistin were increased in the presence of 10μmol/L GY3 with 1.6±0.12-fold(P＜0.05).In contrast,rosiglitazone or insulin decreased the resistin mRNA level with 0.57±0.17-fold(P＜0.05), and 0.33±0.13-fold(P＜0.05)respectively.4.The effects of GY3 on some proteins in TNF-αinduced insulin resistant adipocytes.a)The reproduction of TNF-αinduced insulin resistant adipocytes and the effect of GY3 on GLUT4 in this insulin resistant cell model.It was founded that the GLUT4 expression was suddenly decreased after 24h culture in the presence of TNF-α.Whereas the expression of GLUT4 was not down-regulated until 96h culture when co-treating TNF-αwith GY3.b)The effects of GY3 on the expression of adiponectin in TNF-αinduced insulin resistant adipocytes.Western blot analysis showed that 0.1,1 and 10μmol/L GY3 up-regulated the adiponectin level in a dose-related manner in TNF-αinduced insulin resistant adipocytes.c)The effects of GY3 on the expression of protein within insulin pathway in TNF-αinduced insulin resistant adipocytes.Western blot analysis showed that 10μmol/L GY3 up-regulated the expression of p-IRS-1 and GLUT4 and decreased p-JNK in TNF-αinduced insulin resistant adipocytes.5.The effects of GY3 on Glucose consumption in HepG2 cells.GY3 exhibited different efficiency of the glucose-lowering effects in HepG2 cells when different concentration of insulin was suppenlemented into the culture medium.Compared with vehicle control,GY3 showed the significant enhancement effect on glucose consumption in a dose-related manner,when the cells were in or not in the presence of 1 nmol/L insulin.However,when 100 nmol/L insulin presented in the medium,only metformin increased the glucose consumption in HepG2 cells.6.The influence of GY3 in db/db mice.a)The influence of GY3 on serum glucose in db/db mice.After gastric intubation daily,the values of serum glucose in db/db mice treated with CMC-Na,10 mg/kg rosiglitazone or 30 mg/kg GY3 were 22.3±2.4 mmol/L,7.6±1.4 mmol/L and 12.4±3.1 mmol/L,and the glucose values were decreased by rosiglitazone group or GY3 by 66.0%(P＜0.001)and 44.3%(P＜0.001),respectively after 1 week. After 2 weeks,Serum glucose values in db/db mice treated with CMC-Na,rosiglitazone or GY3 were 20.7±1.2 mmol/L,8.9±2.4 mmol/L and 17.1±2.0 mmol/L,and the glucose values were decreased by rosiglitazone group or GY3 by 57.0%(P＜0.001) and17.1%(P＜0.05),respectively.After 3 weeks,Serum glucose values in db/db mice treated with CMC-Na,rosiglitazone or GY3 were 23.6±3.2 mmol/L,11.5±1.2 mmol/L,18.2±3.1 mmol/L,and the glucose values were decreased by rosiglitazone group or GY3 by 51.37%(P＜0.001)and 23.07%(P＜0.05),respectively.b)The influence of GY3 on glycosylated serum protein in db/db mice.After gastric intubation daily,the value of GSP(GSP)in db/db mice treated with CMC-Na,10 mg/kg rosiglitazone or 30 mg/kg GY3 were 3.76±0.52 mmol/L,3.16±0.55 mmol/L and 3.57±0.14 mmol/L,and the GSP levels were decreased by rosiglitazone group or GY3(P＞0.05) after 1 week.After 2 weeks,GSP values in db/db mice treated with CMC-Na,rosiglitazone or GY3 were 4.14±0.26 mmol/L,3.43±0.11 mmol/L and 3.87±0.21 mmol/L,And the GSP levels were decreased by rosiglitazone group or GY3 by 17.2%(P＜0.001)and 6.7%(P＜0.01), respectively.After 3 weeks,GSP values in db/db mice treated with CMC-Na,rosiglitazone or GY3 were 3.97±0.05 mmol/L,3.13±0.08 mmol/L and 3.51±0.16 mmol/L,and the levels of GSP were decreased by rosiglitazone or GY3 by 21.1%(P＜0.001)and 11.6% (P＜0.001),respectively.c)The influence of GY3 on serum triglyceride(TG)in db/db mice.After gastric intubation daily,the values of serum triglyceride in db/db mice treated with CMC-Na,10 mg/kg rosiglitazone or 30 mg/kg GY3 were 1.00±0.21 mmol/L,0.63±0.15 mmol/L and 0.69±0.12 mmol/L,and the TG values were decreased by rosiglitazone group or GY3(37%(P＜0.001)and 31%(P＜0.001),respectively)after 1 week. After 2 weeks,Serum TG values in db/db mice treated with CMC-Na, rosiglitazone or GY3 were 0.92±0.08 mmol/L,0.64±0.05 mmol/L and 0.70±0.12 mmol/L,and the values were decreased by rosiglitazone group or GY3 by 31%(P＜0.001)and 23%(P＜0.05),respectively.After 3 weeks,Serum TG values in db/db mice treated with CMC-Na, rosiglitazone or GY3 were 20.80±0.11 mmol/L,0.45±0.15 mmol/L and 0.64±0.07 mmol/L,and the triglyceride values were decreased by rosiglitazone or GY3 by 44%(P＜0.01)and 20%(P＜0.05),respectively.d)The influence of GY3 on serum total cholesterol(T-CHO)in db/db mice.After gastric intubation daily,the values of serum T-CHO in db/db mice treated with CMC-Na,10 mg/kg rosiglitazone or 30 mg/kg GY3 were 6.78±0.49 mmol/L,5.65±0.81 mmol/L and 6.25±0.41 mmol/L, and the values were decreased by rosiglitazone by 9.6%(P＜0.05)after 1 week's administration.After 2 weeks,Serum T-CHO values in db/db mice treated with CMC-Na,rosiglitazone or GY3 were 5.39±0.59 mmol/L,4.81±0.53 mmol/L and 4.41±0.42 mmol/L,and the values were decreased by GY3 by 18.2%(P＜0.05).After 3 weeks,Serum T-CHO in db/db mice treated with CMC-Na,rosiglitazone or GY3 were 7.49±1.17 mmol/L,7.17±0.99 mmol/L and 6.91±0.79 mmol/L,and no significant difference was shown..e)The influence of GY3 on serum insulin in db/db mice.After 3 weeks gastric intubation daily,the values of serum insulin in db/db mice treated with CMC-Na,10 mg/kg rosiglitazone or 30 mg/kg GY3 were 9.79±4.85μIU/ml,9.58±2.63μIU/ml and 18.25±7.11μIU/ml,and the insulin levels were decreased by rosiglitazone group or GY3 by 67.8%(P＜0.01)and 38.8%(P＜0.05),respectively.f)The influence of GY3 on serum and hepatic free fat acid(FFA)in db/db mice.After 3 weeks gastric intubation daily,the FFA values in db/db mice treated with CMC-Na,10 mg/kg rosiglitazone or 30 mg/kg GY3 were 888.3±216.7μmol/L,387.7±111.3μmol/L and 684.1±198.8μmol/L. And the serum FFA levels were decreased by rosiglitazone group or GY3 by 56.4%(P＜0.05)and 23%(P＞0.05),respectively.The Hepatic FFA were weakly up-regulated by rosiglitazone or GY3(P＞0.05).g)The influence of GY3 on food and water up-take in db/db mice.After 1 weeks gastric intubation daily,the value of water up-take in db/db mice treated with 10 mg/kg rosiglitazone or 30 mg/kg GY3 decreased by 42%(P＜0.001)and 27%(P＜0.001)respectively,and by 45%(P＜0.001)and 16.4%(P＜0.05)respectively after 2 weeks, (P＜0.001),and by 33%(P＜0.05)and 17.6%(P＜0.01)respectively after 3 weeks,compared to vehicle control.After 1 weeks gastric intubation daily,the value of value of water up-take decreased only in db/db mice treated with 10 mg/kg rosiglitazone(P＜0.001).Conclusions:1.GY3 promoted the differentiation of 3T3-L1 preadipocytes independent on the activation of PPARγ.2.GY3 could enhance insulin sensitivity dependent on the activation of insuliln signal path way and enhancment of adiponectin expression in mature adipocytes.3.GY3 reversed insulin resistance induced by TNF-αin mature adipocytes.4.GY3 enhanced glucose consumption in HepG2 cells that might contributed to its antidiabetic action.5.GY3 decreased serum glucose,insulin,glycosylated serum protein, total cholesterol,triglyceride,free fat acid,and food and water up-take contents in db/db mice.GY3 could improve the morphologic exhibition of liver in db/db mice and diabetic symptoms of excessive drinking and eating.