To Explore The Effects Of Cajanonic Acid A On The Insulin Signaling Pathway In HepG2 Cells With PTP1B Over Expression

Type 2 diabetes mellitus, formerly called non-insulin-dependent diabetes mellitus, is a serious and costly disease affecting more and more people including children, adolescents, and young adults because of bad ways of life and genetic factors. Insulin resistance (IR) is the most important pathogenesis and pathological feature, which play a vital role in the progression of type 2 diabetes and its complications. Thus the prevention and treatment against to IR will reduce the incidence of type 2 diabetes and its complications.ObjectiveOur research team found that Cajanonic Acid A (CAA), a new stilbenes, had the effects on inhibiting preadipocyte differentiation in 3T3-L1, improving insulin sensitivity in type 2 diabetes rats and insulin resistance cells induced by dexamethasone. To further unravel the hypoglycemic mechanism of CAA, we focused on Proteintyrosine Phosphatase 1B (PTP1B), a negative regulator in insulin signaling pathway. HepG2 hepatocyte overexpressing PTP1B was used as a model to evaluate the roles of CAA on the expression and activity of PTP1B, and the regulation of insulin signal transduction. We expect this research will provide experimental basis for CAA pharmaceutical development for type 2 diabetes.MethodsTo identify the effect of CAA on PTP1B activity, a commercial PTP1B assay kit was used to measure PTP1B activity. The kit included human recombinant PTP1B expressed in E. coli. The phosphopetide (IR5) composed of amino acid 1142-1153 in insulin receptor β subunit domain was supplied as PTP1B substrate. The release of free phosphate was detected by colorimetric assay. Suramin was choosed as a control for inhibitor.To increase the expression of PTP1B, a human ptplb gene expression vector (pCMV-PTPN1) was transfeced into HepG2 hepatocyte via lipofectamine 2000 reagent. The expression level of ptplb was monitored by real time fluorescence quantitative-polymerase chain reaction (RTFQ-PCR). Insulin-stimulated glucose consumption in both untransfeced (HepG2) and transfeced (HepG2/PTPlB) cells was tested with a Glucose Assay Kit (glucose oxidase-peroxidase method).To assess the reverse effect of CAA on insulin resistance induced by overexpression of PTP1B, the cells were treated with high, middle and low dose (25,12.5 and 6.25 μmol/L) of CAA and 10 nmol/L of insulin for 24h or 48h. Rosiglitazone (ROS) was used as a positive control. Glucose consumption was measured with Glucose Assay Kit.To explore the mechanism of CAA on insulin signaling pahway, the cells were treated for 24h with CAA (0,6.25,12.5 and 25 μmol/L) in 0.5% fetal calf serum and stimulated for 30 min with 100 nmol/L insulin prior to being collected. The mRNA levels of genes encoding insulin signal transducer, such as irs-1 (insulin receptor substrate-1), irs-2 (insulin receptor substrate-2), gsk3β (glucogen sythasekinase 3β) and glut-1 (glucose transporter-1) were quantified by using RTFQ-PCR technology. The protein levels of PTP1B and GLUT-1, and the tyrosyl phosphorylation of key proteins for insulin signal transduction, such as IRS-1 and p85 regulatory subunit of PI3K (phoaphatidyl inositol 3 kinase) were deteced by Western Blot analysis.ResultsThe assay of PTP1B activity showed that CAA effectively reduced the dephosphorylation reaction catalyzed by PTP1B. The half inhibitive concentration (IC50) was 63.19μmol/L. With 200 μmol/L of CAA treatment, the PTP1B activity level was sharply droped to 1.8% of the original one.After PTP1B expression vector was transfected into HepG2, we got the stable overexpressing cell named as HepG2/PTPlB via hygromycin screening. The transcript level was 4 times higher than that in the HeG2 cells. Without insulin stimulation, there was no difference in glucose consumption between HepG2 and HepG2/PTP1B cells. However, comparing the dose-dependent increment of insulin-stimulated glucose consumption in HepG2 cells (P＜0.05), it had no obvious change in HepG2/PTPlB (P>0.05). These results indicated that high PTP1B level may induce insulin resistance in hepatocytes.After the treatment with CAA, the sensitivity to insulin in HepG2/PTPlB cells was significantly improved (P<0.01). Relative to untreated cells, cells in 25,12.5 and 6.25 μmol/L of CAA for 24h or 48h indreased the insulin-stimulated glucose consumption dose-dependently by 154.3%,127.5%, 58.8% and 92.1%,53.7%,28.1% respectively. The cells treated with 30,20 and 10 μmol/L of ROS were used as positive controls, in which glucose consumption stimulated by insulin were upgraded 224.2%,213.7%,168.4%(24h) and 112.3%, 104.3%,98.4%(48h).The results of RTFQ-PCR illustrated that there was obvious difference in irs-1 and glut-1 expression but not in ptplb, irs-2 and gsk3β expression response to insulin stimulation between the cells basal-and over-expressing PTP1B. In HepG2 cells, the mRNA level of irs-1 and glut-1 after insulin stimulation had a one-fold and two-fold increase over that of the control without insulin. But in the HepG2/PTP1B cells with 4-fold increment of PTP1B at the mRNA level, the expression of glut-1 was insensitive to insulin, and mRNA level of irs-1 had a drop of 60% after insulin stimulation. It revealed that PTP1B overexpression down-regulated insulin-stimulated genes transcription.Simultaneously, the insulin-stimulated mRNA level of irs-1, irs-2, gsk3 & and glut-1 in the HepG2/PTP1B cells were raised by CAA in dose-dependent manner. In the cells treated with 25μmol/L of CAA, the mRNA expression of irs-1, irs-2, gsk3β and glut-1 were increased up to 5.0,2.0,1.5,2.6 times.CAA treatment didn’t affect the ptplb gene expression at the mRNA level, but significantly suppressed the PTP1B at the protein level in the HepG2/PTP1B cells, suggesting that CAA reduced the expression of ptplb gene by post transcriptional control.With the inhibition of PTP1B, there was a maximal 5-fold increase in insulin-stimulated PI3K phosphorylation, a 4-fold increase in insulin-sti mulated IRS-1 phosphorylation, and a 42-fold increase in insulin-stimulat ed GLUT-1 expression.ConclusionThe overexpression of PTP1B which act as a down-regulating factor in insulin signaling pathway remarkably debased the sensitivity of HepG2 cells to insulin. CAA may reverse hepatocellular insulin resistance induced by PTP1B overexpression via inhibition of PTP1B with up-regulating the expression and activity of insulin signal transduction factors.