Blockade of insulin receptor substrate-1 inhibits biological behavior of choroidal endothelial cells

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.

Insulin receptor substrate 1 may play divergent roles in human colorectal cancer development and progression

BACKGROUND Despite effective prevention and screening methods,the incidence and mortality rates associated with colorectal cancer(CRC)are still high.Insulin receptor substrate 1(IRS-1),a signaling molecule involved in cell proliferation,survival and metabolic responses has been implicated in carcinogenic processes in various cellular and animal models.However,the role of IRS-1 in CRC biology and its value as a clinical CRC biomarker has not been well defined.AIM To evaluate if and how IRS-1 expression and its associations with the apoptotic and proliferation tumor markers,Bax,Bcl-xL and Ki-67 are related to clinicopathological features in human CRC.METHODS The expression of IRS-1,Bax,Bcl-xL and Ki-67 proteins was assessed in tissue samples obtained from 127 patients with primary CRC using immunohistochemical methods.The assays were performed using specific antibodies against IRS-1,Bax,Bcl-xL,Ki-67.The associations between the expression of IRS-1,Bax,Bcl-xL,Ki-67 were analyzed in relation to clinicopathological parameters,i.e.,patient age,sex,primary localization of tumor,histopathological type,grading,staging and lymph node spread.Correlations between variables were examined by Spearman rank correlation test and Fisher exact test with a level of significance at P<0.05.RESULTS Immunohistochemical analysis of 127 CRC tissue samples revealed weak cytoplasmatic staining for IRS-1 in 66 CRC sections and strong cytoplasmatic staining in 61 cases.IRS-1 expression at any level in primary CRC was associated with tumor grade(69%in moderately differentiated tumors,G2 vs 31%in poorly differentiated tumors,G3)and with histological type(81.9%in adenocarcinoma vs 18.1%in adenocarcinoma with mucosal component cases).Strong IRS-1 positivity was observed more frequently in adenocarcinoma cases(95.1%)and in moderately differentiated tumors(85.2%).We also found statistically significant correlations between expression of IRS-1 and both Bax and Bcl-xL in all CRC cases examined.The relationships between studied proteins were related to clinicopathological parameters of CRC.No significant correlation between the expression of IRS-1 and proliferation marker Ki-67,excluding early stage tumors,where the correlation was positive and on a high level(P=0.043,r=0.723).CONCLUSION This study suggests that IRS-1 is co-expressed with both pro-and antiapoptotic markers and all these proteins are more prevalent in more differentiated CRC than in poorly differentiated CRC.

葛根芩连汤通过IRS-1/PI3K/AKT通路对胃肠湿热型2型糖尿病大鼠糖脂代谢的影响

目的探究葛根芩连汤通过胰岛素受体底物-1(IRS-1)/磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(AKT)通路对胃肠湿热型2型糖尿病大鼠糖脂代谢的影响。方法将40只SD大鼠随机分为正常组(2 mL生理盐水灌胃)、造模组(2 mL生理盐水灌胃)、二甲双胍组(4.17 mg/100 g二甲双胍灌胃)和葛根芩连汤组(1 g/100 g葛根芩连汤灌胃),每组10只。采用高脂高糖饲料加腹腔注射链脲佐菌素(STZ)构建2型糖尿病大鼠模型,随后喂食油脂、42°白酒及蜂蜜水构建胃肠湿热型2型糖尿病大鼠模型。测量各组大鼠不同时间节点体质量,血糖仪测定空腹血糖(FBG);ELISA检测空腹胰岛素(FINS)、三酰甘油(TG)、总胆固醇(TC)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)水平变化、计算胰岛素抵抗指数(HOMA-IR);HE染色检测肝组织病理学变化;检测肝组织过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)及丙二醛(MDA)含量变化。Western blot检测肝组织IRS-1、PI3K、p-PI3K、AKT及p-AKT蛋白变化。结果与正常组比较,造模组大鼠体质量、FBG、FINS及HOMA-IR、GSH-Px、CAT、SOD、IRS-1、p-PI3K/PI3K及p-AKT/AKT水平均明显下降(P<0.05)、TG、TC、IL-6、TNF-α及MDA含量均显著升高(P<0.05),可见局灶性肝实质损失。与造模组比较,二甲双胍组及葛根芩连汤组大鼠体质量、FBG、FINS及HOMA-IR、GSH-Px、CAT、SOD、IRS-1、p-PI3K/PI3K及p-AKT/AKT水平均明显升高(P<0.05)、TG、TC、IL-6、TNF-α及MDA含量均显著降低(P<0.05),显示正常的肝实质。结论葛根芩连汤可明显改善胃肠湿热型2型糖尿病糖脂紊乱,可能是通过IRS-1/PI3K/AKT通路发挥作用。

Glucagon-like peptide-1 receptor agonists as a possible intervention to delay the onset of type 1 diabetes:A new horizon

Type 1 diabetes(T1D)is a chronic autoimmune condition that destroys insulinproducing beta cells in the pancreas,leading to insulin deficiency and hyperglycemia.The management of T1D primarily focuses on exogenous insulin replacement to control blood glucose levels.However,this approach does not address the underlying autoimmune process or prevent the progressive loss of beta cells.Recent research has explored the potential of glucagon-like peptide-1 receptor agonists(GLP-1RAs)as a novel intervention to modify the disease course and delay the onset of T1D.GLP-1RAs are medications initially developed for treating type 2 diabetes.They exert their effects by enhancing glucose-dependent insulin secretion,suppressing glucagon secretion,and slowing gastric emptying.Emerging evidence suggests that GLP-1RAs may also benefit the treatment of newly diagnosed patients with T1D.This article aims to highlight the potential of GLP-1RAs as an intervention to delay the onset of T1D,possibly through their potential immunomodulatory and anti-inflammatory effects and preservation of beta-cells.This article aims to explore the potential of shifting the paradigm of T1D management from reactive insulin replacement to proactive disease modification,which should open new avenues for preventing and treating T1D,improving the quality of life and long-term outcomes for individuals at risk of T1D.

Effect of insulin and metformin on methylation and glycolipid metabolism of peroxisome proliferator-activated receptor γcoactivator-1A of rat offspring with gestational diabetes mellitus

Objective:To discuss the effect of insulin and metformin on amethylation and glycolipid metabolism of peroxisome proliferator-activated receptor γ coactivator-1A(PPARGC1A) of rat offspring with gestational diabetes mellitus(GDM).Methods:A total of 45 pregnant rats received the intraperitoneal injection of streptozotocin to establish the pregnant rat model of GDM.A total of 21 pregnant rats with GDM were randomly divided into three groups,with 7ruts in each group,namely the insulin group,metformin group and control group.Rats in the insulin group received the abdominal subcutaneous injection of 1 mL/kg recombinant insulin glargine at 18:00 every day.Rats in the metformin group received the intragastric infusion of metformin hydrochloride at 18:00 every day,with the first dose of 300 mg/kg.The doses of two groups were adjusted every 3 d to maintain the blood glucose level at 2.65-7.62 mmol/L.Rats in the control group received the intragastric infusion of 1 mL normal saline at 18:00 every day.After the natural delivery of pregnant rats.10 offspring rats were randomly selected from each group.At birth,4 wk and 8 wk after the birth of offspring rats,the weight of offspring rats was measured.The blood glucose level of offspring rats was measured at 4wk and 8 wk,while the level of serum insulin,triglyceride and leptin was measured at 8 wk.Results:The weight of offspring rats at birth in the insulin group and metformin group was significantly lower than the one in the control group(P<0.05),and there was no significant difference at 4 wk and 8 wk among three groups(P>0.05).The fasting blood glucose and random blood glucose in the insulin group and metformin group at 4 wk and 8 wk were all significantly lower than ones in the control group(P<0.05);there was no significant difference between the insulin group and metformin group(P>0.05).The expression of PPARGC1 A mRNA in the insulin group and metformin group was significantly higher and the methylation level of PPARGC1 A was significantly lower than the one in the control group(P<0.05),but there was no significant difference between the insulin group and metformin group(P>0.05).Insulin and leptin at 8 wk in the insulin group and metformin group were significantly higher,while triglyceride was significantly lower than the one in the control group(P<0.05);triglyceride level of rats in the insulin group was significantly higher than the one in the metformin group(P<0.05).There was no significant difference in insulin and leptin level of offspring rats between the insulin group and metformin group(P>0.05).Conclusions:GDM can induce the methylation of PPARGC1 A of offspring rats to reduce the expression of PPARGC1 A mRNA and then cause the disorder of glycolipid metabolism when the offspring rats grow up;the insulin or metformin in the treatment of pregnant rats with GDM can reduce the methylation level of PPARGC1 A and thus improve the abnormal glycolipid metabolism of offspring rats.

Involvement of insulin receptor substrates in cognitive impairment and Alzheimer’s disease

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.

Glucagon-like peptide-1/glucose-dependent insulinotropic polypeptide dual receptor agonist DA-CH5 is superior to exendin-4 in protecting neurons in the 6-hydroxydopamine rat Parkinson model

Patients with Parkinson's disease(PD) have impaired insulin signaling in the brain. Incretin hormones, including glucagon-like peptide-1(GLP-1) and glucose-dependent insulinotropic polypeptide(GIP), can re-sensitize insulin signaling. In a recent phase II clinical trial, the first GLP-1 mimic, exendin-4, has shown reliable curative effect in patients with PD. DA-CH5 is a novel GLP-1/GIP receptor unimolecular coagonist with a novel peptide sequence added to cross the blood-brain barrier. Here we showed that both exendin-4 and DA-CH5 protected against 6-hydroxydopamine(6-OHDA) cytotoxicity, inhibited apoptosis, improved mitogenesis and induced autophagy flux in SH-SY5Y cells via activation of the insulin receptor substrate-1(IRS-1)/alpha serine/threonine-protein kinase(Akt)/c AMP response element-binding protein(CREB) pathway. We also found that DA-CH5(10 nmol/kg) daily intraperitoneal administration for 30 days post-lesion alleviated motor dysfunction in rats and prevented stereotactic unilateral administration of 6-OHDA induced dopaminergic neurons loss in the substantia nigra pars compacta. However, DA-CH5 showed curative effects in reducing the levels of α-synuclein and the levels of pro-inflammatory cytokines(tumor necrosis factor-α, interleukin-1β). It was also more effective than exendin-4 in inhibiting apoptotic process and protecting mitochondrial functions. In addition, insulin resistance was largely alleviated and the expression of autophagy-related proteins was upregulated in PD model rats after DA-CH5 treatment. These results in this study indicate DA-CH5 plays a therapeutic role in the 6-OHDAunilaterally lesioned PD rat model and is superior to GLP-1 analogue exendin-4. The study was approved by the Animal Ethics Committee of Shanxi Medical University of China.

Insulin-like growth factor receptor-1 overexpression is associated with poor response of rectal cancers to radiotherapy

AIM: To explore the potential correlation between insulin-like growth factor receptor-1 (IGF-1R) expression and rectal cancer radiosensitivity.

Roles of sulfonylurea receptor 1 and multidrug resistance protein 1 in modulating insulin secretion in human insulinoma

BACKGROUND:Sulfonylurea receptor 1(SUR1)and multidrug resistance protein 1(MRP1)are two prominent members of multidrug resistance proteins associated with insulin secretion. The aims of this study were to investigate their expression in insulinomas and their sole and synergistic effects in modulating abnormal insulin secretion. METHODS:Fasting glucose,insulin and C-peptide were measured in 11 insulinoma patients and 11 healthy controls. Prolonged oral glucose tolerance tests were performed in 6 insulinoma patients.Insulin content,SUR1 and MRP1 were detected in 11 insulinoma patients by immunohistochemistry. SUR1 and MRP1 were also detected in 6 insulinoma patients by immunofluorescence. RESULTS:Insulinoma patients presented the typical demons-trations of Whipple’s triad.Fasting glucose of each insulinoma patient was lower than 2.8 mmol/L,and simultaneous insulin and C-peptide were increased in insulinoma patients. Prolonged oral glucose tolerance tests showed that insulin secretion in insulinoma patients were also stimulated by high glucose.Immunohistochemistry and immunofluorescence staining showed that SUR1 increased,but MRP1 decreased in insulinoma compared with the adjacent islets. CONCLUSIONS:The hypersecretion of insulin in insulinomas might be,at least partially,due to the enrichment of SUR1. In contrast,MRP1,which is down-regulated in insulinomas, might reflect a negative feedback in insulin secretion.

Effects of Insulin-like Growth Factor 1 Receptor and Its Inhibitor AG1024 on the Progress of Lung Cancer

Summary： The type 1 insulin-like growth factor receptor （IGF-1R） and its downstream signaling com- ponents have been increasingly recognized to drive the development of malignancies, including non-small cell lung cancer （NSCLC）. This study aimed to investigate the effects of IGF-1R and its in- hibitor, AG1024, on the progression of lung cancer. Tissue microarray and immunohistochemistry were employed to detect the expressions of IGF-1 and IGF-1R in NSCLC tissues （n=198）. Western blotting was used to determine the expressions oflGF-1 and phosphorylated IGF-1R （p-IGF-1R） in A549 human lung carcinoma cells, and MTT assay to measure cell proliferation. Additionally, the expressions of IGF-1, p-IGF-1R and IGF-1R in a mouse model of lung cancer were detected by Western blotting and real-time fluorescence quantitative polymerase chain reaction （FQ-PCR）, respectively. The results showed that IGF-1 and IGF-1R were overexpressed in NSCLC tissues. The expression levels of IGF-1 and p-IGF-1R were significantly increased in A549 cells treated with IGF-1 as compared to those treated with IGF-1 ＋AG 1024 or untreated cells. In the presence of IGF-1, the proliferation of A549 cells was significantly increased. The progression of lung cancer in mice treated with IGF-1 was significantly increased as compared to the group treated with IGF-l＋AG1024 or the control group, with the same trend mirrored in IGF-1/p-IGF-1R/IGF-1R at the protein and/or mRNA levels. It was concluded that IGF- 1 and IGF inhibitor AG 1024 promotes lung cancer progression.

Fixed-ratio combinations of basal insulin and glucagon-like peptide-1 receptor agonists as a promising strategy for treating diabetes

The maintenance of appropriate glycemic control is important for the prevention of diabetic complications in people with type 2 diabetes(T2D). Numerous oral antidiabetic drugs are now clinically available, but in particular, the introduction of injection regimens using insulin and/or glucagon-like peptide-1 receptor agonist(GLP-1RA)s represents promising step-up options for oral antidiabetic drug treatment. The recently licensed fixed-ratio combination(FRC) products,which comprise basal insulin and a GLP-1RA, have potent anti-hyperglycemic effects and reduce the undesirable side-effects of each component, such as body weight gain, hypoglycemia, and gastrointestinal symptoms. Two FRCs-insulin degludec/Liraglutide and insulin glargine/Lixisenatide-are now clinically available and, to date, several phase Ⅱ/Ⅲ trials have been conducted in particular groups of subjects with T2D. However, their utility in real-world clinical settings is of interest for most clinicians. Recently reported real-world clinical trials of these two FRCs in various situations have demonstrated their efficacy regarding glycemic control and the quality of life of people with T2D. Their long-term safety and efficacy require confirmation, but a treatment strategy that includes an FRC may be compatible with the concept of “well-balanced” therapy in certain groups of patients with T2D who have inadequate glycemic control.

Insulin receptor substrate gene polymorphisms are associated with metabolic syndrome but not with its components

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.

Tumor necrosis factor alpha receptor 1 deficiency in hepatocytes does not protect from non-alcoholic steatohepatitis, but attenuates insulin resistance in mice

BACKGROUND End-stage liver disease caused by non-alcoholic steatohepatitis(NASH)is the second leading indication for liver transplantation.To date,only moderately effective pharmacotherapies exist to treat NASH.Understanding the pathogenesis of NASH is therefore crucial for the development of new therapies.The inflammatory cytokine tumor necrosis factor alpha(TNF-α)is important for the progression of liver disease.TNF signaling via TNF receptor 1(TNFR1)has been hypothesized to be important for the development of NASH and hepatocellular carcinoma in whole-body knockout animal models.AIM To investigate the role of TNFR1 signaling in hepatocytes for steatohepatitis development in a mouse model of diet-induced NASH.METHODS NASH was induced by a western-style fast-food diet in mice deficient for TNFR1 in hepatocytes(TNFR1ΔHEP)and their wild-type littermates(TNFR1fl/fl).Glucose tolerance was assessed after 18 wk and insulin resistance after 19 wk of feeding.After 20 wk mice were assessed for features of NASH and the metabolic syndrome such as liver weight,liver steatosis,liver fibrosis and markers of liver inflammation.RESULTS Obesity,liver injury,inflammation,steatosis and fibrosis was not different between TNFR1ΔHEP and TNFR1fl/fl mice.However,Tnfr1 deficiency in hepatocytes protected against glucose intolerance and insulin resistance.CONCLUSION Our results indicate that deficiency of TNFR1 signaling in hepatocytes does not protect from diet-induced NASH.However,improved insulin resistance in this model strengthens the role of the liver in glucose homeostasis.

Simiao Wan alleviates obesity-associated insulin resistance via PKCε/IRS-1/PI3K/Akt signaling pathway based on network pharmacology analysis and experimental validation

Background:The purpose of the study was to investigatethe active ingredients and potential biochemicalmechanisms of Simiao Wan(SMW)in obesity-associated insulin resistance.Methods:An integrated network pharmacology method to screen the active compoundsand candidate targets,construct the protein-protein-interaction network,and ingredients-targets-pathways network was constructed for topological analysis to identify core targets and main ingredients.To find the possible signaling pathways,enrichment analysis was performed.Further,a model of insulin resistance in HL-7702 cells was established to verify the impact of SMW and the regulatory processes.Results:An overall of 63 active components and 151 candidate targets were obtained,in which flavonoids were the main ingredients.Enrichment analysis indicated that the PI3K-Akt signaling pathway was the potential pathway regulated by SMW in obesity-associated insulin resistance treatment.The result showed that SMW could significantly ameliorate insulin sensitivity,increase glucose synthesis and glucose utilization and reduce intracellular lipids accumulation in hepatocytes.Also,SMW inhibited diacylglycerols accumulation-induced PKCεactivity and decreased its translocation to the membrane.Conclusion:SMW ameliorated obesity-associated insulin resistance through PKCε/IRS-1/PI3K/Akt signaling axis in hepatocytes,providing a new strategy for metabolic disease treatment.

RNA interference affects tumorigenicity and expression of insulin-like growth factor-1,insulin-like growth factor-1 receptor,and basic fibroblast growth factor-2 in rat C6 glioma cells

BACKGROUND： Human gliomas are more likely to express basic fibroblast growth factor-2 （FGF-2） insulin-like growth factor-1（IGF-1）, and IGF-1 receptor （IGF-1R） than normal brain tissue. These factors activate signal transduction systems of Ras/MAPK and PI3K/Akl, which promote glioma growth. OBJECTIVE： To utilize RNA interference （RNAi） technique to down-regulate FGF-2, IGF-1, and IGF-1R gene expression, and to investigate the effects of these genes on rat C6 glioma cells, as well as the feasibility of RNAi for treating glioma. DESIGN, TIME AND SETTING： This neurooncological, randomized, controlled, in vivo and in vitro experiment, which used RNAi methodology, was performed at the Laboratory of Molecular Biology, Institute of Biochemistry, Chinese Academy of Sciences between August 2005 and February 2008. MATERIALS： Rat C6 cell lines were purchased from Shanghai Institute of Cellular Biology Affiliated to Chinese Academy of Sciences. Small interfering RNA （siRNA） was synthesized by Shanghai GenePharma. Anti-IGF-1, anti-IGF-1R, anti-FGF-2, anti-mouse and anti-rabbit IgG G1-HRP antibodies were provided by Santa Cruz Biotechnology, USA. Four to six week-old BALB/c nude mice were purchased from the Laboratory Animal Center, Chinese Academy of Sciences. METHODS： C6 glioma cells were transfected with siRNA, which was chemically synthesized in vitro to correspond to endogenous FGF-2, IGF-1, and IGF-1R genes. The inhibition ratio of targeting mRNA expression was detected by semiquantitative RT-PCR, and protein expression was determined by Western blot analysis. C6 glioma cell proliferation was observed using a growth curve C6 glioma cell apoptosis rate and cell cycle were detected by flow cytometry. C6 glioma cell growth regression was observed by transwell migration assay. In addition, nude mouse subcutaneous tumor models were used in this study. For studying the anti-tumor effects of IGF-1 and IGF-1R siRNA, two blank control groups, with six mice each, were set up： A （2.5 μg siRNA was injected one week after C6 cells were inoculated, Le., when tumor volume reached 8 mm × 8 mm） and B （siRNA was injected at the same time with C6 cells were inoculated. To study the effects of FGF-2 siRNA, the groups consisted of a blank control group, negative control group, 2.6 μg siRNA group, 4 μg siRNA group, and 5.3 μg siRNA group, with six mice each. MAIN OUTCOME MEASURES： mRNA and protein inhibition ratio of FGF-2, IGF-1, and IGF-1 R; C6 glioma cell proliferation, apoptosis, and cycle growth arrest; C6 glioma cell growth regression and subcutaneous tumorigenicity rates. RESULTS： All siRNA constructs proved to be effective. After 48 hours, transfection of 200 nmol/L siRNA resulted in a FGF-2 or IGF-1R gene inhibition ratio 〉 80% and an IGF-1 gene inhibition ratio of approximately 70%. Protein expression levels for FGF-2, IGF-1, and IGF-1R decreased in a dose-dependent manner following siRNA transfection, with an inhibition rate 〉 85%, 60%, and 50%, respectively. C6 glioma cell proliferation and apoptosis rates increased in proportion to siRNA. The apoptosis rate of C6 glioma cells induced by FGF-2, IGF-1, and IGF-1R siRNA was 39.96%, 15.07% and 22.47%, respectively （P 〈 0.01）. Transfection of 200 nmol/L IGF or IGF-1R siRNA for 48 hours suppressed C6 glioma cell migration. At 30 days after intratumoral injection of 2.6, 4, and 5.3 tJg FGF-2 siRNA, tumor growth regression rate of FGF-2 siRNA was 56%, 67%, and 86%, respectively. The tumor growth regression rate was 71.88% and 45.71%, respectively, when IGF-1 or IGF-1R siRNA was intratumorally injected 1 week after C6 glioma cell transplantation. When IGF-1 or IGF-1 R siRNA was intratumorally injected during C6 glioma cell transplantation, the tumor growth regression rate was 78.13% and 74.29%, respectively. CONCLUSION： siRNA transfection downregulated gene expression of FGF-2, IGF-1, and IGF-1R In addition, siRNA treatment markedly suppressed glioma cell proliferation, growth, and migration, and concomitantly reduced subcutaneous tumorigenicity.

APP17肽对糖尿病脑病小鼠海马IRS-1、IGF-1R表达的影响

目的 通过对胰岛素受体底物 1(IRS 1)、胰岛素样生长因子 1受体 (IGF 1R)在海马表达的观察 ,研究APP17肽对糖尿病脑病小鼠海马神经元IRS 1、IGF 1R的影响。 方法 用链脲佐菌素 (STZ)诱发小鼠糖尿病模型 ,并皮下注射APP17肽对糖尿病小鼠进行治疗 ,4周后取脑组织做IRS 1、IGF 1R免疫组织化学染色。 结果 糖尿病小鼠海马内IRS 1、IGF 1R阳性反应神经元胞浆与突起深染 ,而正常小鼠及APP17肽保护的糖尿病小鼠海马阳性细胞数目少 ,染色淡。 结论 糖尿病小鼠海马IRS 1、IGF 1R广泛表达 ;APP17肽能影响糖尿病脑病小鼠脑内IRS 1,IGF 1R的表达 。

长期酒精摄入对雄性大鼠胰岛素敏感性及肝脏IR、IRS-1、IRS-2 mRNA表达的影响

目的从基因表达水平探讨长期酒精摄入影响肝脏胰岛素敏感性的分子机制。方法清洁级Wistar雄性大鼠40只,随机分为对照组和低、中、高剂量酒精组,每天摄入酒精剂量分别为0、0·8、1·6和2·4g/kg bw。给予酒精19周后,测定空腹血糖、血胰岛素,计算胰岛素抵抗指数(HOMA-IR)。提取肝脏总RNA,通过RT-PCR测定胰岛素受体(IR)、胰岛素受体底物1(IRS-1)、胰岛素受体底物2(IRS-2)的mRNA表达水平。结果与对照组相比,高剂量组血糖升高(P<0·05);各剂量组血胰岛素浓度均升高,低、中剂量组升高有显著性差异(P<0·05);各剂量组HOMA-IR均显著高于对照组(P<0·05)。各剂量组IR mRNA表达均降低;IRS-1及IRS-2mRNA表达在低、中剂量组升高,高剂量组降低。结论长期过量酒精摄入可以引起雄性大鼠胰岛素抵抗,肝脏IR、IRS-1、IRS-2mRNA表达降低是酒精影响胰岛素敏感性的分子机制之一。

覆盆子酮对HepG2细胞胰岛素信号转导通路中SHP-1和IRS-1表达的影响

目的观察覆盆子酮对HepG2细胞糖消耗的影响及胰岛素信号转导通路中SHP-1和人胰岛素受体底物(IRS-1)表达的影响。方法体外培养人肝癌HepG2细胞,采用MTT法检测细胞活力;葡萄糖氧化酶法检测细胞糖消耗;RT-PCR法检测蛋白酪氨酸磷酸酶SHP-1的基因表达;Western-blot法检测IRS-1的表达量。结果覆盆子酮可明显促进HepG2细胞对葡萄糖的消耗,明显降低HepG2细胞SHP-1 mRNA基因表达量,并能促进IRS-1蛋白表达量。结论覆盆子酮可能通过影响胰岛素信号转导通路中IRS-1和SHP-1的表达发挥降糖作用。

IRS-1的表达和泛素化在KKAy及C57BL/6J小鼠糖尿病发展过程中的意义

目的:探讨胰岛素受体底物-1(IRS-1)蛋白表达和泛素化水平在糖尿病发展过程中的作用。方法:20只8周龄C57BL/6J小鼠随机分为正常对照组(正常饲料喂养,n=10)和胰岛素抵抗组(高脂饲料喂养,n=10)。8周龄KKAy小鼠8只,高脂饲料喂养,为2型糖尿病组。检测各组小鼠体重、血糖和血胰岛素水平。12周后免疫印迹检测各组动物肝组织IRS-1的表达量,同时采用剥离免疫印迹法检测肝组织IRS-1的泛素化水平。结果:高脂喂养12周后C57BL/6J小鼠出现胰岛素抵抗,KKAy小鼠出现肥胖和糖尿病。2型糖尿病组IRS-1的表达显著低于正常对照组和胰岛素抵抗组(P<0.05),而IRS-1的泛素化水平则显著高于胰岛素抵抗组(P<0.05),与对照组无显著性差异(P>0.05)。结论:2型糖尿病时IRS-1的表达下调,IRS-1泛素化水平增加是导致胰岛素信号转导障碍的重要原因。

脂联素对自发性2型糖尿病大鼠肝脏IRS-1磷酸化的影响及其可能机制

目的探讨脂联素对自发性2型糖尿病(OLETF)大鼠肝脏胰岛素信号通路的影响及其分子信号机制。方法自发性2型糖尿病大鼠OLETF鼠20只,同系健康对照LETO鼠10只。于8周和32周龄分别宰杀。检测空腹血糖、血浆胰岛素、脂联素、血脂水平,并以ELISA、免疫印迹和免疫组化法检测肝脏脂联素、胰岛素受体底物-1酪氨酸磷酸化水平(py-IRS-1)以及IKKβ和NFkB表达水平。结果 OLETF鼠血浆脂联素水平在8周龄和32周龄均显著低于LETO鼠(P<0.05)。32周龄时OLETF鼠血清胰岛素、TG、FFA、ISI均显著升高(P<0.05)。脂联素水平与FBG、血清胰岛素、TG、FFA、ISI数显著相关(P均<0.01)。OLETF鼠肝脏脂联素和py-IRS-1水平在8周龄和32周龄均显著低于LETO鼠,IKKβ和NFkB水平也显著升高(P<0.01)。py-IRS-1与肝组织脂联素、IKKβ和NFkB变化显著相关(P<0.01)。32周龄时脂联素水平与NF-κB和py-IRS-1显著负相关(P<0.05)。结论 OLETF鼠脂联素可能通过NFkB通路影响肝脏IRS-1酪氨酸磷酸化水平,导致肝脏的胰岛素信号传导障碍及糖、脂代谢紊乱,参与2型糖尿病的发生发展。