The Effect And Mechanism Of Sang-Tong-Jian On Type 2 Diabetes Mellitus Model Via Glucose Transporter 4

Objective:In our previous study,Sang-Tong-Jian(STJ)was reported to have a good hypoglycemic effect.However,little is known about the mechanisms by which STJ exerts its anti-diabetic effects.In the present study,we used KKAy mice and insulin-resistant 3T3-L1 adipocytes to investigate the effects of STJ on glycolipid metabolism,insulin resistance and IRS1-PI3K-AKT-GLUT4 signaling pathway,and to elucidate the underlying mechanisms.Methods and results:1.The effect and mechanism of STJ on hypoglycemic action in KKAy mice:1.1 The effect of STJ on glucose metabolism in KKAy mice:Fifty male KKAy mice with high random blood glucose levels(>13.7 mmol/L)were considered diabetic,and then were divided randomly into 5 groups according to blood glucose levels to make the five groups had similar average blood glucose levels.These groups were termed model,metformin at 260 mg/kg,and STJ at 105,210 and 420 mg/kg.The C57BL/6J mice were considered non-diabetic as the control group.Mice in medication group were orally administrated with metformin or STJ for 13 weeks,and the control and model mice were orally given the same volume of distilled water.Body weight and residual food were weighed and the food intake was calculated weekly during experiments.RBG levels were tested every 10 days during the experiments.At the end of the experiments,all mice were fasted for 8 h,and blood samples were collected for analysis of glycosylated hemoglobin(GHb)according to a colorimetric method.Mouse liver and skeletal muscle tissues were washed with cold normal saline.Glycogen levels were measured by colorimetric assay using the anthracenone method.The results showed that the STJ-treated KKAy mice exhibited obviously decreased RBG levels and GHb levels,and significantly elevated the hepatic and muscle glycogen contents.1.2 The effect of STJ on insulin resistance in KKAy mice:In the 10th and 13th week during the experimental period,the KKAy mice were subjected to OGTT and ITT,respectively.At the end of the experiments,all mice were fasted for 8 h and blood samples were collected.Blood samples were also immediately centrifuged(4℃,3000 rpm,10 min)to obtain serum for analysis of fasting insulin(FINS)levels using kits for enzyme-linked immunosorbent assay(ELISA).Homeostasis model assessment of insulin resistance(HOMA-IR)and insulin sensitivity index(ISI)were calculated according to the following formulas,respectively:HOMA-IR=Fasting blood glucose(mmol/L)× Fasting serum insulin(mIU/L)/22.5,ISI=1/[Fasting blood glucose(mmol/L)× Fasting serum insulin(mIU/L)].The results showed that STJ markedly ameliorated the tolerance of glucose and insulin,decreased HOMA-IR and enhanced ISI in diabetic KKAy mice.1.3 The effect of STJ on IRS1-PI3K-AKT-GLUT4 signaling pathway in adipose tissue of KKAy mice:At the end of the experiments,mice were fasted for 8 h and blood samples were taken from the orbital venous plexus of each mouse.The mice were then sacrificed.Epididymal fat was weighed and coefficient was calculated.Mouse pancreas and adipose tissues were fixed in 10%formalin and embedded in paraffin,and tissue slices of 4 μm thickness were prepared.The morphology and structure of these tissues were visualized using H&E staining according to standard methods.The protein expression of P-IRS1、P-PI3K、P-Aktser473、IRS1、PI3K、Akt、GLUT4、β-actin in adipose tissue of mice and the protein expression of GLUT4 and Na+K-ATPase α1 on the cell membrane were examined by western blot analyses.The mRNA expression of GLUT4 in adipose tissue of mice was analyzed by real-time PCR.The results showed that STJ improved the morphology and function of pancreas and adipose tissue,activated IRS1-PI3K-AKT signaling molecules,increased the total GLUT4 protein and mRNA expression and promoted translocation of GLUT4 in KKAy mice.2.The effect and mechanism of STJ on hypoglycemic action in insulin-resistant 3T3-L1 adipocytes:2.1 The effect of STJ on glycolipid metabolism and adipocytokine secretion in insulin-resistant 3T3-L1 adipocytes:Mouse 3T3-L1 pre-adipocyte cells were identificated with oil red O staining.To induce insulin resistance,the differentiated adipocytes were incubated in 10%FBS DMEM with 1μM dexamethasone(Dex)for 24 h.After the model copy is successful,the glucose content in supernatant of cell culture was detected by glucose oxidase-peroxidase method.Free fatty acid content in supernatant of cell culture was detected by enzymatic method.The content of adiponectin and leptin in supernatant of cell culture was detected by enzyme-linked immunosorbent assay.The results showed that STJ could promote glucose consumption,inhibit free fatty acid production,promote adiponectin secretion and inhibit leptin secretion in insulin-resistant 3T3-L1 adipocytes.2.2 The effect of STJ on IRS1-PI3K-AKT-GLUT4 signaling pathway in insulin-resistant 3T3-L1 adipocytes:The protein expression of P-IRS1、P-PI3K、P-Aktser473、IRS1、PI3K、Akt、GLUT4、β-actin in 3T3-L1 adipocytes and the protein expression of GLUT4 and Na+K-ATPaseα1 on the cell membrane were examined by western blot analyses.The protein expression of P-IRS1 and P-Aktser473 were examined by immunofluorescence.The results showed that STJ activated IRS1-PI3K-AKT signaling molecules,increased the total GLUT4 protein expression and promoted translocation of GLUT4 in insulin-resistant 3T3-L1 adipocytes.2.3 STJ improved glucose uptake through IRS1-PI3K-AKT-GLUT4 signaling pathway in insulin-resistant 3T3-L1 adipocytes:After the cells were treated with 100 nM wortmannin(a PI3K-specific inhibitor),the effect of STJ on glucose consumption was observed in insulin-resistant 3T3-L1 adipocytes.The protein expression of P-Aktser473、Akt、GLUT4、β-actin in 3T3-L1 adipocytes and the protein expression of GLUT4 and Na+K-ATPase al on the cell membrane were examined by western blot analyses.The results showed that wortmannin reverses the effect of STJ on promotion of glucose uptake and GLUT4 translocation in insulin-resistant 3T3-L1 adipocytes.Conclusions:1.STJ could decrease RBG levels and GHb levels,and significantly elevate the hepatic and muscle glycogen contents,and markedly ameliorate the tolerance of glucose and insulin,and enhance insulin sensitivity in KKAy mice.The mechanism could be related to the activation of IRS1-PI3K-AKT signaling molecules,promotion of CLUT4 protein expression and GLUT4 translocation.2.STJ could promote glucose consumption,inhibit free fatty acid production,promote the adiponectin secretion and inhibit the leptin secretion through activating IRS1-PI3K-AKT-GLUT4 signaling pathway in insulin-resistant 3T3-L1 adipocytes.