Effects Of Protein Tyrosin Phosphatase-1B On Beta Cell Insulin Secretion And Insulin Signaling Transduction

IntroductionObetsity and Type 2 Diabetes become the most popular diseases all over the world. The epidemic data showed that there had been 1,100,000,000 people who were overweight, and even 312,000,000 of them had suffered from obesity. Type 2 Diabetes is also popular and harmful to people. It is estimated that the population of type 2 DM will up to 366,000,000 in 2030. As we know, the common pathogenesis of obesity and type 2 DM is insulin resistance and data showed that people with insulin resistance had 5-fold higher risk of type 2 Diabetes as well as 2-fold higher risk of cardiovascular events than people without insulin resistantance. Insulin resistance become a key point in type 2 diabetes and obesity treatment. Most studies focused on insulin resistance of the typical insulin-sensitive tissue such as adipose, muscle, liver and hypothalamus.These studies concluded that increasing exercises, modifying daily-life style can increase fat comsuptoin, improve glucose utilization of muscle and thus ameliorate peripheral insulin resistance. However, beta cell also had insulin resistance which was characterized as impaired insulin secretion by glucose stimulated insulin secretion test (GSIS) and insulin signaling obstruction. The new concept of beta-cell insulin resistance act as a supplement of the theory of typical insulin resistance.Previous study indicate that insulin receptor-βsubunit conditional knockout mice(βIRKO) lost more than 85% insulin secretion by GSIS and moved on to impaired glucose torlence (IGT) in 2 -month -years old. This demonstrate that insulin signaling is essential toβcell itself for insulin secretion. However, the exact mechanism ofβcell insulin resistance remained unclear.At the molecular level, the insulin signaling begins with activation of insulin receptor (IR) resulted in tyrosine phosphorylation of serials of substrates, including the IR substrate 1 (IRS-1) and IRS-2. After tyrosine phosphorylation of IRS-1 and IRS-2, they bind and activate the enzyme- phosphatidylinositol 3-kinase (PI3-K). The activation of PI3-K increases serine phosphorylation of protein kinase B (Akt), which in turn stimulates the glucose transport in the muscle and adipose tissue, glycogen synthesis in the liver and muscle, and lipogenesis in the adipose tissue. Recent studies suggest that protein tyrosine phosphatase-1B (PTP-1B) was an important negative regulator of insulin and leptin signal transduction. However, whether PTP-1B has a similar effect on beta cell insulin resistance remained unclear.Part One: Expression of PTP-1B in beta cell insulin resistance induced by glucose and palmitic acid.ObjectiveIn this study, we investigated the causative factors ofβcell insulin resistance and the expression of PTP-1B. We also observed the relationship between PTP-1B andβcell insulin secreting function.MethodsMIN6 cells under serial subcultivation were divided into four groups:①glucose treatment: the final concentration of glucose was 33.3mmol/L and 40mmol/L, respectively; incubated for 24 hours;②palmitic acid treatment (PA group): the final concentration of PA was 125umol/L, 250umol/L, respectively; incubated for 24 hours;③dehydrated-alcohol control group: added identical volume of dehydrated alcohol compared with PA group; incubated for 24 hours;④control group: nothing treated. We performed trizol kit to extract total RNA of each group and synthesized cDNA from RNA using reverse transcriptase.Realtime-PCR was used to detect the expression of PTP-1B mRNA level.Immunocytochemical stain and western-bloting were performed to observe the expression of PTP-1B protein. We also investigated MIN6 cell insulin secreting function by performing the insulin releasing test and serum levels of insulin were determined by RIA assay.Results:1. glucose concentration of 40mmol/L treatment induce beta cell insulin resistance successfully and the glucose stimulated insulin secretion reduced significantly.Compared with the control group, the basic insulin secretion decreased significantly(P<0.05) while insulin levels were much lower in high glucose concentration(P<0.01).However, this phenomenon was not seen in group of 33.3mmol/L glucose treatment . In contrast, the base line of insulin secretion in this group increased slightly and no differences of insulin secretion were seen comparing with control group. 2. Palmitic acid treatment also induce beta cell insulin resistance, resulted in the decrease of insulin secretion. Although the base line insulin releasing was similar to control group in PA of 125umol/L,the high glucose stimulated insulin secretion was much lower than control group. In group of 250umol/L PA,decreasing insulin secretion was only seen in high glucose stimulation(P<0.05). And there no differences of insulin secretion between dehydrated-alcohol control group and normol control.3. The expressions of protein tyrosine phosphatase-1B were much higher in either glucose or PA group than in control group.(1) Immunocytochemical stain showed that the expressions of PTP-1B obviously increased in glucose treatment (40mmol/L) and PA (125umol/L) group, respectively (P=0.000).Furthermore, the expression of PTP-1B was much higher in glucose treatment than in PA group.However, Neither 33.3mmol/L glucose treatment nor 250umol/L PA group had increased expression of PTP-1B,compared with control group.(2) Realtime- Polymerase Chain Reaction (Realtime-PCR) is a simple and powerful method to amplify PTP-1B gene of a tiny amount. Amplified products are monitored in real time.In both glucose treatment (40mmol/L) and PA (125umol/L) group, the ratio of PTP-1B /β-actin were higher.Compared with control, the expression of PTP-1B mRNA level increased 50 fold in glucose treatment and 25 fold in PA group.(3)Western-bloting showed that the expression of PTP-1B protein in each group was much higher than control.And there were similar results consistent wiht realtime-PCR results.Conclusion:Both glucose concentration of 40mmol/L treatment and palmitic acid treatment induce beta cell insulin resistance, resulted in the decrease of insulin secretion. The expressions of protein tyrosine phosphatase-1B were much higher in these groups than in control group.PTP-1B may be an important regulator in the development of beta cell insulin resistance induced by glucose or PA. Part Two: Expression of PTP-1B in beta cell insulin resistance induced by chronic stimulation of inflammatory factor.Objective : In this study, we investigated the causative factors ofβcell insulin resistance and the expression of PTP-1B. We also observed the relationship between PTP-1B andβcell insulin secreting function.MethodsMIN6 cells under serial subcultivation were divided into four groups:①Tumor necrosis factor-α(TNF-α)treatment group A :with final concentration of 1.0 ug/L; incubated for 24 hours;②TNF-αtreatment group B :with final concentration of 10 ug/L; incubated for 24 hours;③TNF-αtreatment group C :with final concentration of 20 ug/L ;incubated for 24 hours;④control group: nothing treated. We performed trizol kit to extract total RNA of each group and synthesized cDNA from RNA using reverse transcriptase. PCR was used to detect the expression of PTP-1B mRNA level.Western-bloting were performed to observe the expression of PTP-1B protein. We also investigated MIN6 cell insulin secreting function by performing the insulin releasing test and serum levels of insulin were determined by RIA assay.Results:1.Different concentrations of TNF-αinduced beta cell insulin resistance and the insulin secretion reduced sinificantly in these three groups vs control group. However, TNF-αdid not have concentration-dependent effects on beta cell. Both TNF-αB group and A group showed the same effects on the severity of beta cell dysfunction.2. The expression of PTP-1B mRNA increased in MIN6 cells induced by TNF-α.3.The expression of PTP-1B protein increased in MIN6 cells induced by TNF-α. Western-bloting showed that PTP-1B protein expression was much higher in TNF-αintervention group.Conclusion:TNF-αinduce beta cell insulin resistance, resulted in the decrease of insulin secretion. However, TNF-αdid not have concentration-dependent effects on beta cell.Meanwhile, the expressions of protein tyrosine phosphatase-1B were much higher in TNF-αtreatment groups than in control group. PTP-1B may be an important regulator in the development of beta cell insulin resistance induced by stimulation of inflammatory factor.Part Three: Effects of overexpression of PTP-1B on beta cell insulin secretion.Objective :In this part, we investigated the effects of overexpression of PTP-1B on beta cell insulin secretion.Methods:HEK293 cell line were serial subcultivated to amplify Ad-PTP-1B adenovirus. Cesium chloride centrifugal purification was performed to purify Ad-PTP-1B adenovirus and virus titre was detected. MIN6 cells under serial subcultivation were divided into 3 groups:①MIN6 cells transfected with Ad-PTP-1B;②MIN6 cells transfected with Ad-EGFP;③control group: nothing treated. We performed trizol kit to extract total RNA of each group and synthesized cDNA from RNA using reverse transcriptase. PCR was used to detect the expression of PTP-1B mRNA level.Western-bloting were performed to observe the expression of PTP-1B protein. We also investigated MIN6 cell insulin secreting function by performing the insulin releasing test and serum levels of insulin were determined by RIA assay.Results:1. Virus titre of Ad-PTP-1B was 3.19×1010pfu/ml.2.MOI 1:100 was the most effective concentration for transfection.3.Overexpression of PTP-1B in MIN6 cells resulted in the siginificant decrease of insulin secretion by GSIS vs. control. And there were no effects on MIN6 cell insulin secretion by transfected with Ad-EGFP.Conclusions:PTP-1B had direct negative effects on beta cell function and induced beta cell insulin resistance.Therefore, PTP-1B plays an important role in the development of beta cell insulin resistance. Part Four: Effects of overexpression of PTP-1B on beta cell insulin signaling.Objective:In this part, we investigated the effects of overexpression of PTP-1B on beta cell insulin signaling.MethodsHEK293 cell lines were serial subcultivated to amplify Ad-PTP-1B adenovirus. Cesium chloride centrifugal purification was performed to purify Ad-PTP-1B adenovirus and virus titre was detected. MIN6 cells under serial subcultivation were divided into 3 groups:①M IN6 cells transfected with Ad-PTP-1B;②MIN6 cells transfected with Ad-EGFP;③control group: nothing treated. We performed trizol kit to extract total RNA of each group and synthesized cDNA from RNA using reverse transcriptase. PCR was used to detect the expression of PTP-1B mRNA level.Western-bloting were performed to observe the expression of IR-βand IRS-1. The tyrosine phosphorylations of IRS-1, IRβwere detected by immuno-precipitation. We also investigated MIN6 cell insulin secreting function by performing the insulin releasing test and serum levels of insulin were determined by RIA assay.Results:1. The insulin-stimulated tyrosine phosphorylation of IRβin MIN6 cells in Ad-PTP-1B group were significantly lower than that in control group (P < 0.05).2. The insulin-stimulated tyrosine phosphorylation of IRS-1 in MIN6 cells in Ad-PTP-1B group were significantly lower than that in control group (P < 0.05).Conclusions:The expression of PTP-1B increased to suppress the insulin signal transduction in beta cell and insulin-stimulated tyrosine phosphorylation of both IRβand IRS-1 decreased in beta cell.According to these results, it is supposed that the signal transduction pathway of insulin is the key part of beta cell insulin resistance induced by PTP-1B.