AIM: To investigate the effects of blockade of insulin receptor substrate-1(IRS-1) on the bio-function of tube formation of human choroidal endothelial cells(HCECs).METHODS: Quantitative reverse transcriptionpolymeras...AIM: To investigate the effects of blockade of insulin receptor substrate-1(IRS-1) on the bio-function of tube formation of human choroidal endothelial cells(HCECs).METHODS: Quantitative reverse transcriptionpolymerase chain reaction(RT-PCR) and Western blot were performed to determine the expression level of IRS-1 and phospho-IRS-1 in HCECs. Tube formation of HCECs was analyzed using three dimensional in vitro Matrigel assay with or without IRS-1 blockage via IRS-1 inhibitor(GS-101) and vascular endothelial growth factor receptor 2(VEGFR2) inhibitor. In addition, cell counting kit(CCK)-8 and Transwell migration assay were exerted to analyze the effects of blockade of IRS-1 on the bio-function of proliferation and migration of HCECs, respectively. The apoptosis of HCECs was examined using flow cytometry(FCM).RESULTS: RT-PCR and Western blot revealed that IRS-1 phospho-IRS-1 were expressed in HCECs and the expression level was enhanced by stimulation of VEGF-A. The number of tube formation was decreased significantly in GS-101 treated groups compared to phosphate buffered saline(PBS) treated control groups. Furthermore, both cell proliferation and migration of HCECs were decreased in the presence of GS-101. FCM analysis showed that the apoptosis of HCECs was enhanced when the cells were treated with GS-101. Western blot also showed that the expression level of cleaved-caspase 3 in GS-101 treated group was higher than that in control group.CONCLUSION: Blockade of IRS-1 can inhibit tube formation of HCECs through reducing cell proliferation and migration and promoting cell apoptosis.展开更多
Type 2 diabetes一associated with impaired insulin/insulin-like growth factor-1 (IGF1) signaling (IIS)一is a risk factor for cognitive impairment and dementia including Alzheimer's disease (AD). The insulin recepto...Type 2 diabetes一associated with impaired insulin/insulin-like growth factor-1 (IGF1) signaling (IIS)一is a risk factor for cognitive impairment and dementia including Alzheimer's disease (AD). The insulin receptor substrate (IRS) proteins are major components of IIS, which transmit upstream signals via the insulin receptor and/or IGF1 receptor to multiple intracellular signaling pathways, including AKT/protein kinase B and extracellular-signal-regulated kinase cascades. Of the four IRS proteins in mammals, IRS1 and IRS2 play key roles in regulating growth and survival, metabolism, and aging. Meanwhile, the roles of IRS1 and IRS2 in the central nervous system with respect to cognitive abilities remain to be clarified. In contrast to IRS2 in peripheral tissues, inactivation of neural IRS2 exerts beneficial effects, resulting in the reduction of amyloid p accumulation and premature mortality in AD mouse models. On the other hand, the increased phosphorylation of IRS 1 at several serine sites is observed in the brains from patients with AD and animal models of AD or cognitive impairment induced by type 2 diabetes. However, these serine sites are also activated in a mouse model of type 2 diabetes, in which the diabetes drug metformin improves memory impairment. Because IRS1 and IRS2 signaling pathways are regulated through complex mechanisms including positive and negative feedback loops, whether the elevated phosphorylation of IRS1 at specific serine sites found in AD brains is a primary response to cognitive dysfunction remains unknown. Here, we examine the associations between IRS 1 /1 RS2-mediated signaling in the central nervous system and cognitive decline.展开更多
Aim: Metabolic syndrome (MetS) is a major risk factor for both diabetes mellitus and cardiovascular disease (CVD). The aims of the study were 1) to investigate the insulin receptor substrate-1 (IRS-1) and insulin rece...Aim: Metabolic syndrome (MetS) is a major risk factor for both diabetes mellitus and cardiovascular disease (CVD). The aims of the study were 1) to investigate the insulin receptor substrate-1 (IRS-1) and insulin receptor substrate-2 (IRS-2) gene polymorphisms in patients with MetS and 2) to examine the relationships between gene polymorphisms and components of MetS. Patients & Methods: The study population included 100 patients with MetS and 30 patients without MetS as control group. Metabolic syndrome (MS) was defined as in ATP III. Entire coding exons of IRS-1 and IRS-2 genes were amplified by polymerase chain reaction (PCR). Insulin resistance (IR) was estimated using the homeostasis model assessment (HOMA). Results: In patients with MetS, 34 (34%), had G972R (rs1801278) gene polymorphism and 66 (66%) had no nucleotide substitutions at the IRS-1 gene (p circumference, blood pressure, triglyceride, HDL-Cholesterol, LDL-Cholesterol and HOMA-IR levels. Conclusion: Insulin receptor substrate-1 and 2 gene polymorphisms were associated with metabolic syndrome but not its components.展开更多
Approximately 170 million people worldwide are chronically infected with hepatitis C virus(HCV).Chronic HCV infection is the leading cause for the development of liver fibrosis,cirrhosis,hepatocellular carcinoma(HCC)a...Approximately 170 million people worldwide are chronically infected with hepatitis C virus(HCV).Chronic HCV infection is the leading cause for the development of liver fibrosis,cirrhosis,hepatocellular carcinoma(HCC)and is the primary cause for liver transplantation in the western world.Insulin resistance is one of the pathological features in patients with HCV infection and often leads to development of typeⅡdiabetes.Insulin resistance plays an important role in the development of various complications associated with HCV infection.Recent evidence indicates that HCV associated insulin resistance may result in hepatic fibrosis,steatosis,HCC and resistance to anti-viral treatment.Thus,HCV associated insulin resistance is a therapeutic target at any stage of HCV infection.HCV modulates normal cellular gene expression and interferes with the insulin signaling pathway.Various mechanisms have been proposed in regard to HCV mediated insulin resistance,involving up regulation of inflammatory cytokines,like tumor necrosis factor-α,phosphorylation of insulin-receptor substrate-1,Akt,up-regulation of gluconeogenic genes like glucose 6 phosphatase,phosphoenolpyruvate carboxykinase 2,and accumulation of lipid droplets.In this review,we summarize the available information on how HCV infection interferes with insulin signaling pathways resulting in insulin resistance.展开更多
Background MicroRNAs （miRNAs） contribute to tumorigenesis by acting as either oncogenes or tumor suppressor genes. In this study, we investigated the role of miR-145 in the pathogenesis of uveal melanoma. Methods Ex...Background MicroRNAs （miRNAs） contribute to tumorigenesis by acting as either oncogenes or tumor suppressor genes. In this study, we investigated the role of miR-145 in the pathogenesis of uveal melanoma. Methods Expression profiles of miRNAs in uveal melanoma were performed using Agilent miRNA array. Quantitative real-time polymerase chain reaction was used to screen the expression levels of miR-145 in normal uveal tissue, uveal melanoma tissue, and uveal melanoma cell lines. Lenti-virus expression system was used to construct MUM-2B and OCM-1 cell lines with stable overexpression of miR-145. Cell proliferation, cell cycle, and cell apoptosis of these miR-145 overexpression cell lines were examined by MTT assay and flow cytometry respectively. The target genes of miR-145 were predicted by bioinformatics and confirmed using a luciferase reporter assay. The expression of insulin-like growth factor-1 receptor （IGF-1R）, insulin receptor substrate-1 （IRS-1） proteins was determined by Western blotting analysis. IRS- 1 was knocked down in OCM-1 cells. TUNEL, BrdU, and flow cytometry assay were performed in IRS-1 knocked down OCM-1 cell lines to analyze its function. Results Forty-seven miRNAs were up regulated in uveal melanoma and 61 were down regulated, miR-145 expression was significantly lower in uveal melanoma sample and the cell lines were compared with normal uveal sample. Overexpression of miR-145 suppressed cell proliferation by blocking the G1 phase entering S phase in uveal melanoma cells, and promoted uveal melanoma cell apoptosis. IRS-1 was identified as a potential target of miR-145 by dual luciferase reporter assay. Knocking down of IRS-1 had similar effect as overexpression of miR-145. Conclusion miR-145 might act as a tumor suppressor in uveal melanoma, and downregulation of the target IRS-1 might be a potential mechanism.展开更多
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 ...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.展开更多
基金Supported by the National Natural Science Foundation in China(No.81671641 No.81970830+6 种基金 No.31600736)Suzhou Municipal Natural Science Foundation(No.SYS201745)Soochow University Doctoral Academic Talents Program(No.5832001313)Jiangsu Provincial Medical Youth Talent(No.QNRC2016718)Jiangsu Provincial Medical Innovation Team(No.CXTDA2017039)Jiangsu Provincial Natural Science Foundation(No.BK20151208)the Soochow Scholar Project of Soochow University(No.R5122001)
文摘AIM: To investigate the effects of blockade of insulin receptor substrate-1(IRS-1) on the bio-function of tube formation of human choroidal endothelial cells(HCECs).METHODS: Quantitative reverse transcriptionpolymerase chain reaction(RT-PCR) and Western blot were performed to determine the expression level of IRS-1 and phospho-IRS-1 in HCECs. Tube formation of HCECs was analyzed using three dimensional in vitro Matrigel assay with or without IRS-1 blockage via IRS-1 inhibitor(GS-101) and vascular endothelial growth factor receptor 2(VEGFR2) inhibitor. In addition, cell counting kit(CCK)-8 and Transwell migration assay were exerted to analyze the effects of blockade of IRS-1 on the bio-function of proliferation and migration of HCECs, respectively. The apoptosis of HCECs was examined using flow cytometry(FCM).RESULTS: RT-PCR and Western blot revealed that IRS-1 phospho-IRS-1 were expressed in HCECs and the expression level was enhanced by stimulation of VEGF-A. The number of tube formation was decreased significantly in GS-101 treated groups compared to phosphate buffered saline(PBS) treated control groups. Furthermore, both cell proliferation and migration of HCECs were decreased in the presence of GS-101. FCM analysis showed that the apoptosis of HCECs was enhanced when the cells were treated with GS-101. Western blot also showed that the expression level of cleaved-caspase 3 in GS-101 treated group was higher than that in control group.CONCLUSION: Blockade of IRS-1 can inhibit tube formation of HCECs through reducing cell proliferation and migration and promoting cell apoptosis.
基金supported by a MEXTGrant-in-Aid for Scientific Research on Innovative Areas(brain environment)(JP24111536 to AT)+3 种基金JSPS KAKENHI(JP24650201,JP26282026,JP17K19951,JP17H02188 to AT)grants from the Mitsubishi Foundation(to AT)NOVARTIS Foundation Japan for the Promotion of Science(to AT)
文摘Type 2 diabetes一associated with impaired insulin/insulin-like growth factor-1 (IGF1) signaling (IIS)一is a risk factor for cognitive impairment and dementia including Alzheimer's disease (AD). The insulin receptor substrate (IRS) proteins are major components of IIS, which transmit upstream signals via the insulin receptor and/or IGF1 receptor to multiple intracellular signaling pathways, including AKT/protein kinase B and extracellular-signal-regulated kinase cascades. Of the four IRS proteins in mammals, IRS1 and IRS2 play key roles in regulating growth and survival, metabolism, and aging. Meanwhile, the roles of IRS1 and IRS2 in the central nervous system with respect to cognitive abilities remain to be clarified. In contrast to IRS2 in peripheral tissues, inactivation of neural IRS2 exerts beneficial effects, resulting in the reduction of amyloid p accumulation and premature mortality in AD mouse models. On the other hand, the increased phosphorylation of IRS 1 at several serine sites is observed in the brains from patients with AD and animal models of AD or cognitive impairment induced by type 2 diabetes. However, these serine sites are also activated in a mouse model of type 2 diabetes, in which the diabetes drug metformin improves memory impairment. Because IRS1 and IRS2 signaling pathways are regulated through complex mechanisms including positive and negative feedback loops, whether the elevated phosphorylation of IRS1 at specific serine sites found in AD brains is a primary response to cognitive dysfunction remains unknown. Here, we examine the associations between IRS 1 /1 RS2-mediated signaling in the central nervous system and cognitive decline.
文摘Aim: Metabolic syndrome (MetS) is a major risk factor for both diabetes mellitus and cardiovascular disease (CVD). The aims of the study were 1) to investigate the insulin receptor substrate-1 (IRS-1) and insulin receptor substrate-2 (IRS-2) gene polymorphisms in patients with MetS and 2) to examine the relationships between gene polymorphisms and components of MetS. Patients & Methods: The study population included 100 patients with MetS and 30 patients without MetS as control group. Metabolic syndrome (MS) was defined as in ATP III. Entire coding exons of IRS-1 and IRS-2 genes were amplified by polymerase chain reaction (PCR). Insulin resistance (IR) was estimated using the homeostasis model assessment (HOMA). Results: In patients with MetS, 34 (34%), had G972R (rs1801278) gene polymorphism and 66 (66%) had no nucleotide substitutions at the IRS-1 gene (p circumference, blood pressure, triglyceride, HDL-Cholesterol, LDL-Cholesterol and HOMA-IR levels. Conclusion: Insulin receptor substrate-1 and 2 gene polymorphisms were associated with metabolic syndrome but not its components.
基金Supported by The National Institutes of Health,NO.DK080812
文摘Approximately 170 million people worldwide are chronically infected with hepatitis C virus(HCV).Chronic HCV infection is the leading cause for the development of liver fibrosis,cirrhosis,hepatocellular carcinoma(HCC)and is the primary cause for liver transplantation in the western world.Insulin resistance is one of the pathological features in patients with HCV infection and often leads to development of typeⅡdiabetes.Insulin resistance plays an important role in the development of various complications associated with HCV infection.Recent evidence indicates that HCV associated insulin resistance may result in hepatic fibrosis,steatosis,HCC and resistance to anti-viral treatment.Thus,HCV associated insulin resistance is a therapeutic target at any stage of HCV infection.HCV modulates normal cellular gene expression and interferes with the insulin signaling pathway.Various mechanisms have been proposed in regard to HCV mediated insulin resistance,involving up regulation of inflammatory cytokines,like tumor necrosis factor-α,phosphorylation of insulin-receptor substrate-1,Akt,up-regulation of gluconeogenic genes like glucose 6 phosphatase,phosphoenolpyruvate carboxykinase 2,and accumulation of lipid droplets.In this review,we summarize the available information on how HCV infection interferes with insulin signaling pathways resulting in insulin resistance.
基金This study was supported by grants from the National Natural Science Foundation of China （No. 81272981） and Natural Sciences Fundation of Beijing, China （No. 7112031）.
文摘Background MicroRNAs （miRNAs） contribute to tumorigenesis by acting as either oncogenes or tumor suppressor genes. In this study, we investigated the role of miR-145 in the pathogenesis of uveal melanoma. Methods Expression profiles of miRNAs in uveal melanoma were performed using Agilent miRNA array. Quantitative real-time polymerase chain reaction was used to screen the expression levels of miR-145 in normal uveal tissue, uveal melanoma tissue, and uveal melanoma cell lines. Lenti-virus expression system was used to construct MUM-2B and OCM-1 cell lines with stable overexpression of miR-145. Cell proliferation, cell cycle, and cell apoptosis of these miR-145 overexpression cell lines were examined by MTT assay and flow cytometry respectively. The target genes of miR-145 were predicted by bioinformatics and confirmed using a luciferase reporter assay. The expression of insulin-like growth factor-1 receptor （IGF-1R）, insulin receptor substrate-1 （IRS-1） proteins was determined by Western blotting analysis. IRS- 1 was knocked down in OCM-1 cells. TUNEL, BrdU, and flow cytometry assay were performed in IRS-1 knocked down OCM-1 cell lines to analyze its function. Results Forty-seven miRNAs were up regulated in uveal melanoma and 61 were down regulated, miR-145 expression was significantly lower in uveal melanoma sample and the cell lines were compared with normal uveal sample. Overexpression of miR-145 suppressed cell proliferation by blocking the G1 phase entering S phase in uveal melanoma cells, and promoted uveal melanoma cell apoptosis. IRS-1 was identified as a potential target of miR-145 by dual luciferase reporter assay. Knocking down of IRS-1 had similar effect as overexpression of miR-145. Conclusion miR-145 might act as a tumor suppressor in uveal melanoma, and downregulation of the target IRS-1 might be a potential mechanism.
基金supported by the Science Technology Research Project of Higher Education of Ningxia Province(NGY2016125)the Open Project of Key Laboratory of Fertility Preservation and Maintenance,Ministry of Education(XY201518)。
文摘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.
