Effect of Metformin-Induced Stimulation on the Expression of Insulin Receptor Substrate 1 through Negative Regulation of P70S6k

Objective:The aim is to study the effects of metformin on the expression of 70 kDa ribosomal protein S6 kinase(P70S6k),insulin receptor substrate 1(IRS-1),and IRS-1Ser307 phosphorylation in human luteinized granulosa cells.Methods:Granulosa cells in the experimental group were cultured in M199 medium containing 0.1 mmol/L metformin for 24 h and those in control group were cultured in M199 medium.The expression levels of P70S6k and IRS-1 mRNA were detected by reverse-transcriptiom polymerase chain reaction(RT-PCR)and real-time PCR.P70S6k,IRS-1,p-ser307-IRS-1,and p-thr389-P70S6k protein expression levels were detected by immunofluorescence and western blotting.Results:P70S6k mRNA level was higher and IRS-1 was significantly lower in the experimental group than those in the control group.IRS-1 and p-ser307-IRS-1 were expressed in cell plasma,and P70S6k and p-thr389-P70S6k were expressed in cell nucleus.The results of Western blot analysis indicated that the expression levels of P70S6k,p-thr389-P70S6k,IRS-1,and p-ser307-IRS-1 proteins had significant difference between the experimental group and the control group.Compared to the control group,the relative intensity illustrated that the expression levels of P70S6K and p-thr389-P70S6k significantly increased in the experimental group;however,those of IRS-1 and p-ser307-IRS-1 proteins significantly decreased.Conclusion:Metformin can inhibit the P70S6k mRNA and protein expression levels in the granulosa cells and improve insulin sensitivity by regulating IRS-1 expression through Akt/P70S6k/IRS-1-dependent pathway.

Curcumin Alleviates Hyperandrogenism and Promotes Follicular Proliferation in Polycystic Ovary Syndrome Rats:Insightson IRS1/PI3K/GLUT4 and PTEN Modulations

Objective:To explore the effect of curcuminon the insulin receptor substrate1(IRS1)/phosphatidylinositol-3-kinase(PI3K)/endometrial expression of glucose 4(GLUT4)signalling pathway and its regulator,phosphatase and tensin homolog(PTEN),in a rat model of polycystic ovarian syndrome(PCOS).Methods:PcoS model was induced by letrozole intragastric administration.Sprague-Dawleyrats were randomized into 4groups according to a random number table:(1)control group;(2)PcoS group,which was subjected to PCOS and received vehicle;(3)curcumin group,which was subjected to PCoS and treated with curcumin(200 mg/kg for 2 weeks);and(4)curcumin+LY294002 group,which was subjected to PCOS,and treated with curcumin and LY294002(a specific PI3K inhibitor).Serum hormone levels(17β-estradiol,follicle stimulating hormone,luteinizinghormone,progesterone,and testosterone)were measured by enzyme linked immunosorbentassay,and insulin resistance(IR)was assessed using the homeostasismodel assessment of IR.Ovarian tissues were stained with haematoxylin and eosin for pathological and apoptosis examination.Expression levels of key transcriptional regulators and downstream targets,including IRS1,Pl3K,protein kinase B(AKT),GLUT4,and PTEN,were measured via reverse transcription polymerase chain reaction and Western blot,respectively.Results:The Pcos group showed impaired ovarian morphology and function.Compared with the PCoS group,curcumin treatment exerted ovarioprotective effects,down-regulated serum testosterone,restored IR,inhibited inflammatory cell infiltration in ovarian tissues,decreased IRS1,PI3K,and AKT expressions,and up-regulated GLUT4 and PTEN expressions in PCOS rats(P<0.05orP<0.01).In contrast,IRS1,PI3K,AKT,and PTEN expression levelswerenot significantly different between PCOS and curcumin+LY294002 groups(P>0.05).Conclusion:The beneficial effects of curcumin on PCOS rats included the alteration of serum hormone levels and recovery of morphological ovarian lesions,in which,PTEN,a new target,may play a role in regulating the IRS1/PI3K/GLUT4 pathway.