Mechanism Of Guavenoic Acid(GA) On Improving Insulin Resistance In 3T3-L1 IR Adipocytes

Aim: To investigate the effects of Guavenoic Acid(GA) on proliferation and differentiation in 3T3-L1 preadipocytes, and the effects on glucose consumption, free fatty acid(FFA) production, adipocytokines levels in IR 3T3-L1 adipocytes. And then explore mechanisms of drugs by measuring gene expression of PTP1 B, PPARγ and proteins expression of IRS-1, p-IRS-1, akt, p-akt. Methods: 3T3-L1 preadipocytes were cultured and treated with GA(0.3, 1, 3, 10, 30 nmol/L) for 48 h, while medium group(0.1%DMSO), positive drug rosiglitazone(Ros, 10μM) and vanadate(Van, 10μM) were set also, the proliferation ratio was detected by MTT assay. 3T3-L1 preadipocytes were induced to be differentiated into 3T3-L1 adipocytes, the differentiation was observed by oil red O staining. Then 3T3-L1 adipocytes models of insulin resistance were established successfully and treated with drugs, control group and model group were added. Glucose consumption was assayed by glucose oxidase method(GOD-POD) and free fatty acid(FFA) level by colorimetric method. Adipocytokines levels represented by adiponectin, leptin, TNF-α, resistin were assayed by enzyme-linked immunosorbent assays. The gene expression of Protein tyrosine phosphatase1B(PTP1B) m RNA was detected via reverse transcriptase PCR(RT-PCR) analysis. The proteins expression of IRS-1, p-IRS-1, akt, p-akt were determined via Western blot analysis. Results: 1. Effects on proliferation and differentiated of 3T3-L1 preadipocytesCompared with medium group, Ros and Van promoted proliferation of 3T3-L1 preadipocytes significantly(P<0.01); GA treatment promoted proliferation at 3, 10, 30 nmol/L significantly(P<0.01) with dose-dependent manner. Compared with medium group, Ros promoted differentiation of 3T3-L1 preadipocytes significantly(P<0.01) while Van inhibited significantly(P<0.01); GA treatment inhibited differentiation at 0.3-30 nmol/L significantly(P<0.01), and the effects decreased with dosage increasing. 2. Effect on glucose consumption of 3T3-L1 IR adipocytesWhether in basic or insulin stimulated state, compared with IR medium group, Ros and Van improved glucose uptake in 3T3-L1 IR adipocytes significantly(P<0.01); GA improved glucose uptake at 3-30 nmol/L significantly(P<0.05 or P<0.01). 3. Effect on FFA production of 3T3-L1 IR adipocytesCompared with IR medium group, Ros and Van inhibited FFA production significantly in 3T3-L1 IR adipocytes significantly(P<0.01); GA inhibited FFA production at high concentrations(10, 30 nmol/L) significantly(P<0.01). 4. Effects on secretion of adiponection, leptin, TNF-α, resistin in 3T3-L1 IR adipocytesCompared with IR model group, Ros increased the level of adiponection and reduced leptin, TNF-α, resistin significantly(P<0.01) in 3T3-L1 IR adipocytes; Van increased the level of adiponection and reduced TNF-α, resistin significantly(P<0.01),had no significant effect on secretion of leptin; GA(0.3, 3, 30 mol/L) increased the level of adiponection and reduced TNF-α significantly(P<0.01), had no significant effect on secretion of leptin and resistin. 5. Effects on gene expression of PTP1 B m RNA and PPAR γ m RNA in 3T3-L1 IR adipocytesCompared with IR model group, Van down-regulated gene expression of PTP1 B m RNA in 3T3-L1 IR adipocytes significantly(P<0.01); GA down-regulated PTP1 B m RNA at 3, 30 mol/L significantly(P<0.05 or P<0.01). Compared with IR model group, Ros up-regulated gene expression of PPARγ m RNA in 3T3-L1 IR adipocytes significantly(P<0.01); GA had no significant effect on gene expression of PPARγ m RNA. 6. Effects on protein expression of IRS-1、p-IRS-1、akt、p-akt in 3T3-L1 IR adipocytesCompared with IR model group, Van up-regulated protein expression of p-IRS-1 and p-akt in 3T3-L1 IR adipocytes significantly(P<0.01) whereas had no effect on IRS-1 and akt; GA(0.3, 3, 30 mol/L) up-regulated p-IRS-1 and p-akt significantly(P<0.05 or P<0.01) whereas had no effect on IRS-1 and akt. Conclusion:GA significantly promoted the proliferation of 3T3-L1 preadipocytes and inhibited their differentiation. GA significantly promoted the glucose consumption of IR adipocytes and inhibit the production of FFA, improved the metabolism of glucose and lipid; GA significantly promoted the secretion of adiponectin and inhibit TNF-αin IR adipocytes, had the function of improving IR.In conclusion, GA can improve the insulin resistance in 3T3-L1 adipocytes with a dose-dependent manner. The mechanism may be that GA significantly down-regulated gene expression of PTP1 B m RNA and up- regulated protein expression of p-IRS-1, p-Akt in IR adipocytes to improve the insulin signaling pathway.