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Objective To investigate the effects of chronic intermittent hypoxia on the adipose factor and the expressions of insulin receptor substrate 2 (IRS-2),glucose transporter 2 (GLUT-2) and leptin in rat liver.Methods Twenty-four mature SD rats were randomly divided into 3 groups:control group (UC),chronic intermittent hypoxia group (CIH) and reoxygenation group (RH).The arterial blood gas analysis was performed after the establishment of rat model.The serum fasting blood glucose (FBG) and fasting insulin (FINS) in each group were detected by peroxidase method;the concentrations of free fatty acids (FFA) and leptin were detected by ELISA.The expressions ofmRNA and protein of GLUT-2,IRS-2 and leptin were detected by qRT-PCR and Western blotting.Results The serous concentrations of FBG,FINS,FFA and leptin were significantly higher in CIH group than in UC group (P＜0.05),and were dramatically higher in RH group than in both CIH group (P=0.003) and UC group (P=0.000).Western blotting and qRT-PCR detection showed that the protein and mRNA expressions of GLUT-2 and IRS-2 were significantly lower in CIH group than in RH group of rat liver (P＜0.05),while were markedly lower in RH group than in UC group (P＜0.05);the expressions of leptin protein and mRNA were significantly higher in CIH group than in RH group (P＜0.05),while were obviously higher in RH group than in UC group of rat liver (P＜0.05).Conclusion Insulin resistance induced by chronic intermittent hypoxia may be associated with the elevation of serum FFA and leptin,and be related to the decreased expression of GLUT-2 and IRS-2 and increased expression of leptin in liver.
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Objective To investigate the effects of chronic intermittent hypoxia and on GLUT-2, IRS-2 expression in the rat kidney .Methods Rats were randomly divided into control group ( control) , chronic intermittent hypoxia group (CIH), chronic intermittent hypoxia reoxygenation group (RH).The chronic intermittent hypoxia animal models were developed , arterial blood gas analysis was immediately carried out .Serum glucose was measured by peroxidase and serum insulin was detected by radioimmunoassay .After Removing the kidney tissue of rats ,protein expression of GLUT-2, IRS-2 were detected with Western blot and immunohistochemical , mRNA expression of GLUT-2,IRS-2 were observed by qPCR .Results Oximetry in the control group was ≥95%, oxygen saturation in the CIH group was ≤85%, oxygen saturation in the RH group was ≥86%; fasting blood glucose , serum insulin and insulin resistance index in the CIH group were significantly higher those of control group and RH group ( P<0.05);RH group was higher than control group (P<0.05).Protein and mRNA expression of GLUT-2, IRS-2 in the CIH group were higher than the control group and RH group ( P<0.05 ); RH group was higher than control group (P<0.05).Conclusions Chronic intermittent hypoxia can increase blood glucose ,upregulate the expres-sion of GLUT-2 , IRS2 in the rat kidney and enhance insulin resistance and decrease insulin sensitivity .
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Diversos aditivos químicos han sido utilizados para garantizar la polimerización de genes con islas CpG. El objetivo de este trabajo fue diseñar una mezcla potenciadora de PCR para amplificar genes con islas CpG. Con ese fin se analizaron fragmentos de los genes IRS2 y HNF1a con el programa EMBOSS CpG Report. Los iniciadores se diseñaron con el programa Primer 3 y se analizaron con el programa e-PCR. Se usaron tres aditivos químicos: Albúmina sérica bovina (0,1µg/µL), dimetilsulfóxido (5%) y formamida (5%) para 5 ensayos de PCR: dos usando un solo aditivo, dos combinando dos aditivos y uno combinando tres aditivos. Las amplificaciones con las mezclas se realizaron con las enzimas Taq Nativa, taq Recombinante y Taq Platinum. La calidad de los amplicones se probó por secuenciación. Fragmentos sin islas CpG (HNF-1a) amplificaron con las tres enzimas, sin el uso de los aditivos pero presentaron problemas de pureza en la secuenciación. Los fragmentos del gen IRS2 con islas CpG amplificaron sólo con la combinación de tres aditivos dimetilsulfóxido, albúmina sérica bovina y formamida, independientemente de la enzima usada, las secuencias fueron limpias. Se concluye que la mezcla de tres aditivos es una solución que permite obtener amplicones de alta calidad en genes con islas CpG, con cromatogramas limpios en la secuenciación.
Several chemical additives have been used to assure polymerization in CpG islands.The aim of the present work was to design a PCR enhancer mixture in order to amplify GC-rich genes. Fragments of IRS2 and HNF1a genes were analyzed using EMBOSS CpG Report Software. Primers were designed with the Primer3 Software and were tested with ePCR Software. Three additives were used: BSA (0.1µg/µL), DMSO (5%) and formamide (5%), in five PCR assays, two using one additive, two combining two additives and one with all additives. DNA sequences were amplified with the following enzymes: Native Taq, recombinant Taq and platinum Taq DNA polymerase. Amplicon quality was examined by sequencing. HNF1a gene was amplified without additives; however, the sequences were not amplified and showed purity problems in sequencing. The gene fragments IRS2 with CpG islands were amplified with additives DMSO, BSA and Formamida mixture, notwithstanding the enzyme used. These sequences were clean. DMSO-BSA-Formamide mixture can be a solution to obtain GC-rich DNA amplicons with such a high quality that it generates neat chromatograms during sequencing.
Diversos aditivos químicos têm sido utilizados para garantir a polimerização de genes com ilhas CpG. O objetivo do trabalho foi desenhar uma mistura que potencie PCR para amplificar genes com ilhas CpG. Para esta finalidade foram analisados fragmentos dos genes IRS2 e HNF1a com o programa EMBOSS CpG Report. Os iniciadores foram desenhados com o programa Primer 3 e se analisaram com o programa e-PCR. Foram utilizados três aditivos químicos: Albumina sérica bovina (0,1µg/µlL, Dimetilsulfóxido (5%) e formamida (5%) para 5 ensaios de PCR: dois usando um único aditivo, dois combinando dois aditivos e um combinando três aditivos. As amplificações com as misturas se realizaram com as enzimas, Taq Nativa, taq Recombinante e Taq Platinum. A qualidade das ampliações foi provada por sequenciação. Fragmentos sem ilhas CpG (HNF-1a) amplificaram com as três enzimas, sem o uso dos aditivos porém apresentaram problemas de pureza na sequenciação. Os fragmentos do gene IRS2 com ilhas CpG amplificaram apenas com a combinação de três aditivos dimetilsulfóxido, albumina sérica bovina e formamida, independentemente da enzima usada, as sequências foram limpas. A conclusão que a mistura de três aditivos é uma solução que permite obter ampliações de alta qualidade em genes com ilhas CpG, com cromatogramas limpos na secuenciação.
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Animals , Cattle , Polymerase Chain Reaction , CpG Islands/genetics , DNA/blood , Polymerase Chain Reaction/veterinary , Genetic Diseases, Inborn/diagnosisABSTRACT
Objective In order to investigate the effect of SH2B1 on leptin signal transduction JAK2/IRS2 and its biological function.Methods Vitro kinase assay and Western blot were used to analyse tyrosine phosphorylatin of key molecule JAK2 and insulin receptor substrate-2 (IRS2). ELISA was used to measure the plasma leptin levels in mice. The postnatal growth of mice was monitored over 27 weeks. Results SH2B1 dramatically enhanced the leptin-stimulated tyrosine phosphorylation of JAK2 and IRS2 in HEK293 cells stably expressing LRb (HEK239~(LRb)). Leptin-stimulated activation of hypothalamic JAK2 and phosphorylation of hyphothalamic IRS2 were significantly impaired in SH2B1~(-/-) mice. The deletion of SH2B1 led to leptin resistance,and fasting and randomly fed plasma leptin levels were respectively 3.2 times and 5.1 times higher in SH2B1~(-/-) males than wild-type littermates at 15 weeks of age. SH2B1~(-/-) males gained body weight rapidly and exceeded wild-type littermates from 5~(th) week. SH2B1(-/-) (at 21 weeks) was approximately twice heavier than wild-type littermates.Conclusion SH2B1 is an endogenous enhancer of leptin sensitivity and required for maintaining normal bodyweight in mice via leptin JAK2/IRS2 pathway.
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Objective To study the effects of inhibiting forkhead transcription factor O1 (FoxO1)expression by small interference RNA (siRNA) on the glucose utilization of insulin resistant HepG-2 cell line and the mechanism.Methods FoxO1 gene-targeted siRNA vector,which carried a red fluorescence,was constructed and then confirmed by DNA sequencing.HepG-2 cells were induced to a status of insulin resistance by being exposed to 10-6 mol/L insulin for 24 h.The study included 4 groups: HepG-2 cell group cultured with normal medium (group A) ; insulin resistant HepG-2 cell group (group B) ; insulin resistant HepG-2 cell group into which FoxO1 siRNA vector was transfected (group C) ; insulin resistant HepG-2 cell group into which Lipofectamine2000 was added (group D).The transfection efficiency could be estimated by observing the expression of red fluorescence.The expression of FoxO1 mRNA was analyzed by RT-PCR.The expressions of IRS-2 and tyrosine phosphorylation were detected by Western blot and immunoprecipitation.Results FoxO1 gene-targeted siRNA vector was built successfully.The expression of red fluorescence was the strongest at 48 h after transfection.Compared with group A,the glucose consumption and the expression of IRS-2 tyrosine phosphorylation of group B were decreased (P<0.01),and expression of FoxO1 mRNA was increased (P<0.05) in group B.There was no difference in the expressions of IRS-2 protein between A and B groups.Compared with group B,the expression of FoxOl mRNA was decreased (P < 0.01),and the glucose consumption and expression of IRS-2 tyrosine phosphorylation were significantly increased (P<0.05) in group C.There was no difference between group D and group B in glucose consumption, FoxO1 mRNA, IRS-2 protein and IRS-2 tyrosine phosphorylation expressions.Conclusions Inhibiting the expression of FoxO1 gene in insulin resistant HepG-2 cells seems to improve insulin sensitivity by feedback regulating the IRS-2 tyrosine phosphorylation expressions.
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RT-PCR and immunohistochemistry were used to measure the mRNA and protein expression of insulin receptor substrate 2 (IRS-2) in the muscle of smoking rats. The mRNA and protein expressions of IRS-2 in the smoking subgroups fed with normal diet or high-fat diet were significantly lower than those in corrsponding control groups (0.27±0.02 vs 0.41±0.25, 0.40±0.04 vs 0.51±0.02 for mRNA; 2.91±0.42 vs 4.90±0.29, 2.43±0.36 vs 3.80±0.30 for protein, all P<0.01). There were no differences in mRNA and protein expressions of IRS-2 between the smoking group of diabetic rats and the matched control rats (P>0.05). The variation of gene expression of IRS-2 may be involved in the mechanism of insulin resistance caused by smoking.
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BACKGROUND: Insulin receptor substrate 2 (IRS-2) is a key regulator of beta cell proliferation and apoptosis. This study was aimed to investigate effect of the glucolipotoxicity on apoptosis in INS-1 cell, and the effect of Exendin-4, a GLP-1 receptor agonist, on IRS-2 expression in the glucolipotoxicity induced INS-1 cell. The goal was to discover the new action mechanism and function of Exendin-4 in beta cell apoptosis. METHOD: INS-1 cells were cultured in glucolipotoxic condition for 2, 4 or 6 days and were categorized as G groups. Another group in which 50 nM Exendin-4 was added to INS-1 cells, cultured in glucolipotoxic condition, were named as Ex-4 groups. We investigated the expression of IRS-2 by RT-PCR, phosphorylated IRS-2 and phosphorylated Akt protein levels by western blot. We measured the apoptosis ratio of INS-1 cell in glucolipotoxic condition by TUNEL staining in both groups. RESULT: IRS-2 expression of INS-1 cells decreased with correlation to the time of exposure to glucolipotoxic condition. pIRS-2 and pAkt protein levels decreased in the similar pattern in glucolipotoxicity group. However, this effect of glucolipotoxicity on INS-1 cell was inhibited by the Exendin-4 treatment. In the Ex-4 groups, IRS-2 expression, pIRS-2 and pAkt protein levels remained at the similar level to low glucose condition state. Also, apoptosis induced by glucolipotoxicity was suppressed by Exendin-4 treatment significantly. CONCLUSION: We showed that the long-term treatment of Exendin-4 inhibited the apoptosis of beta cells significantly in glucolipotoxic condition and that this effect of Exendin-4 was related with IRS-2 and Akt among the beta cell's intracellular signal transduction pathway.
Subject(s)
Apoptosis , Blotting, Western , Cell Proliferation , Cells, Cultured , Glucagon-Like Peptide 1 , Glucagon-Like Peptide-1 Receptor , Glucose , In Situ Nick-End Labeling , Insulin Receptor Substrate Proteins , Peptides , Phosphorylation , Receptors, Glucagon , Signal Transduction , VenomsABSTRACT
0.05);(2) Comparing with the control group,GDM group had higher levels of FPG,FINS,and HOMA-IR(P
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Objective To investigate the expression of insulin receptor substrate-2(IRS-2)in the liver of nonalcoholic fatty liver(NAFL)mice.Methods Ten female C57BL6J mice fed with normal diet were served as controls,while fifteen fed with high fat and sugar diet for either 8 weeks(group-A)or 16 weeks(group B)were as NAFL mice models.Abdominal fat mass,total cholesterol(TC),triglycerides(TG),high density lipoprotein cholesterol(HDL-C),fasting blood glucose(FBG)insulin(INS)and liver function,liver weight were measured.And the expression of IRS-2 and the content of lipid in the liver were also detected.Results The abdominal fat mass serum TC,and FBG were significantly higher in high fat and sugar diet fed mice than that in controls(P
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The levels of protein expressions o f insulin receptor substrate-1 and -2 (IRS-1 and IRS-2) in abdominal subcuta neous adipose tissue from type 2 diabetic patients were measured by Western blot technique. The expression of IRS-1 protein was reduced and the expression of I RS-2 protein was unchanged in adipose tissue of type 2 diabetic patients. IRS- 2 may be the main docking protein and one of the factors causing hyperinsulinemi a and insulin resistance in type 2 diabetes mellitus.
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Objective To investigate the effects of insulin on the mRNA expression of insulin receptor substrate-2 (IRS-2) in MG-63 cells. Methods Semi-quantitative RT-PCR was used to study the action of insulin on the mRNA expression of IRS-2 in MG-63 cells. Results Insulin regulated the mRNA expression of IRS-2 in a dose- and time-dependent manners in MG-63 cells. Insulin up-regulated the expression of IRS-2 mRNA at 10~ -10～10~ -6mol/L(P
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Objective To study the genotype distribution of insulin receptor substrate-2(IRS-2)gene 1057G/A polymorphism in Han population from Southwest China,and to explore its association with the metabolism of glucose and lipids,insulin resistance and islet?-cell function in type 2 diabetic patients and subjects with impaired glucose tolerance(IGT).Methods A total of 929 Hans[462 patients with type 2 diabetes(DM group) and 164 subjects with IGT(IGT group)and 303 normal controls(NC group)]from Chongqing and nearby regions were screened for 1057G/A polymorphism of IRS-2 gene by PCR-RFLP assay.Body mass index(BMI),plasma glucose,serum insulin and lipid profile,high-sensitive C-reactive protein(hsCRP)and non-esterified fatty acid were measured.Homeostasis model assessment of insulin resistance(HOMA-IR)and disposition index(DI)were used to estimate insulin resistance and?-cell function respectively.Results In DM group,A allele frequency was significantly lower than that in NC group(0.326 vs 0.388,X~2=6.19,P=0.01).Compared with NC group,AA genotype frequeney was lower and GG genotype frequeney was higher in DM group(0.104 vs 0.135 and 0.452 vs 0.360 respectively,X~2=6.80,P