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ObjectiveThis study aims to investigate the therapeutic effect of Tangbikang granules（TBK） on type 2 diabetes mellitus （T2DM） complicated with non-alcoholic fatty liver disease （NAFLD） and to elucidate the underlying mechanism. MethodT2DM and NAFLD were induced in ZDF rats， which were then respectively treated （ig） with low-dose （0.625 g·kg-1）， medium-dose （1.25 g·kg-1）， and high-dose （2.5 g·kg-1） TBK for 12 weeks. Fasting blood glucose （FBG） and body mass were recorded every 4 weeks during the treatment. One week before sampling， the feed intake of rats was detected， and after 12 h night fasting， oral glucose tolerance test （OGTT） was performed. The area under the curve （AUC） was used to evaluate glucose tolerance， and the homeostatic model assessment for insulin resistance （HOMA-IR） was calculated. Blood in abdominal aorta and liver were collected for determination of blood glucose and lipid metabolism indexes： Fasting serum insulin （FINS）， serum total cholesterol （TC）， triglyceride （TG）， low density lipoprotein cholesterol （LDL-C）， high density lipoprotein cholesterol （HDL-C）， and nonesterified fatty acids （NEFA）. The liver was weighed to calculate the liver index， and the liver tissue morphology was observed and analyzed based on hematoxylin-eosin （HE） staining and periodic acid-Schiff （PAS） staining. The protein levels of insulin receptor substrate （IRS）， phosphatidylinositol 3-kinase （PI3K）， protein kinase B （Akt） and phosphorylated IRS and Akt were detected by Western blotting. All data were analyzed by SPSS 20.0. ResultThe feed intake of the model group was higher than that in the normal group （P<0.01）， and the feed intake the administration groups was lower than that in the model group （P<0.05， P<0.01）. At the 8th and 12th week， the body mass in the model group was lower than that in the normal group （P<0.01）. Compared with the model group， TBK reduced FBG in a concentration-dependent manner. The blood glucose level in OGTT and AUC in the model group were higher/larger than those in the normal group （P<0.01）. The blood glucose value in OGTT was decreased in TBK groups and the metformin group compared with that in the model group， and AUC in the administration groups was significantly different from that in the model group （P<0.01）. The serum level of FINS and HOMA-IR in the model group were higher than those in the normal group （P<0.01）， and they were lower in the TBK groups than in the model group （P<0.01）. Serum levels of TG， TC， HDL-C， NEFA （P<0.05， P<0.01）， and LDL-C were higher in the model group than in the normal group. Serum levels of TG， TC， LDL-C， and NEFA in the TBK groups were lower than those in the model group， and the levels of TG， LDL-C， and NEFA in TBK groups were concentration-dependent （lowest levels in high-dose TBK group）. Compared with the model group， high-dose TBK significantly increased the level of HDL-C （P<0.05）. Liver index of the model group was higher than that in the normal group （P<0.01）. The liver index of the administration groups showed a decreasing trend with no significant difference from that in the model group. As for the HE staining result of liver， the model group had unclear structure of liver lobule， enlarged cells of different sizes， and obvious steatosis of hepatocytes. TBK of all doses alleviated liver injury， particularly the high dose. For the PAS staining， compared with the normal group， the model group demonstrated significant fat vacuoles and significant reduction in purplish red glycogen granules in the cytoplasm. The staining results of high- and medium-dose groups of TBK were more similar to the normal group. Western blot was used to detect the protein expression of liver tissue. The expression of PI3K protein， p-IRS1/IRS1， and p-Akt/Akt in the model group were lower than those in the normal group （P<0.01）， and they were higher in the high-dose TBK group than in the model group （P<0.01）. ConclusionTBK exerts therapeutic effect on T2DM combined with NAFLD in ZDF rats by activating the typical PI3K signaling pathway.
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BACKGROUND@#MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression, influence cellular processes, and promote disease development. Variations in miRNA expression have been observed in many diseases, including hepatitis, cardiovascular disease, and cancer. The aim of this study is to investigate the effect of miR-144-3p on the invasion and metastasis of lung adenocarcinoma by targeting recombinant insulin receptor substrate 1 (IRS1).@*METHODS@#The expression of miR-144-3p in patients with lung adenocarcinoma was queried through bioinformatics database. MirTarPathway was used to analyze the KEGG enrichment pathway of miRNA. The expression and plasmid transfection efficiency of miR-144-3p in lung adenocarcinoma cell lines were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Transwell assay was used to detect the changes of cell invasion and migration ability in different groups. Bioinformatics determined the key genes (Hub genes) of miR-144-3p; Double luciferase target assay was used to detect the mutual binding of miR-144 and IRS1. Western blot assay was used to detect the expression of IRS1 in different cell lines and the expression of after overexpression of miR-144.@*RESULTS@#The expression of miR-144-3p in lung adenocarcinoma tissues was decreased, qRT-PCR results indicated that the expression of miR-144-3p in lung adenocarcinoma cell A549 was significantly decreased (P<0.05), and the overexpressed plasmid was successfully transfected (P<0.05). Overexpression of miR-144 decreased the ability of cell migration and invasion (P<0.05). The expression of IRS1 was up-regulated in lung adenocarcinoma tissues. Survival analysis showed that patients with lung adenocarcinoma with high IRS1 expression had a poor prognosis (P<0.05). Double luciferase assay results showed that miR-144 could specifically identify 3'-UTR of IRS1 and inhibit reporter enzyme expression (P<0.05). Western blot indicated that the expression of IRS1 was increased in A549 cells (P<0.05). After overexpression of miR-144, the expression level of IRS1 protein was decreased (P<0.05). Transwell experiment proved that miR-144-3p could inhibit invasion and metastasis of lung adenocarcinoma cells by targeting IRS1 (P<0.05).@*CONCLUSIONS@#MiR-144-3p inhibits the invasion and migration of A549 cells through targeted regulation of IRS1, thus playing an anticancer role in tumors.
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Objective:To study the effect of Baihutang on blood glucose， blood lipid metabolism and vascular remodeling in type 2 diabetic rats and its regulation on insulin receptor substrate-1（IRS-1）/ phosphatidylinositol-3 kinase（PI3K）/ protein kinase B（Akt） signal pathway. Method:The 90 rats were randomly divided into normal group， model group， Baihutang low， middle and high dose groups and metformin group， with 15 rats in each group. Except for normal group， the other rats were injected intraperitoneally with streptozotocin to establish the model of type 2 diabetes. The rats in the low， middle and high dose groups were given Baihutang formula granules of 5， 10， 20 g·kg-1 respectively according to their body weight. The positive control group was given metformin （100 mg·kg-1） by intragastric administration， while those in the control group and model group were given the same amount of normal saline once a day for 12 weeks. The levels of fasting blood glucose， glycosylated hemoglobin， serum tumor necrosis factor-α（TNF-α）， interleukin-6（IL-6）， interleukin-1 β（IL-1β）， total cholesterol（TC）， triglyceride（TG） and low-density lipoprotein cholesterol（LDL-C） were measured after administration. The levels of sterol regulatory element binding protein 1C （SREBP1C）， acetyl CoA carboxylase （ACC）， fatty acid synthase gene （FASN） and carnitine palmitoyl transferase 1A （CPT1A）， acylcoa oxidase 1（ACOX1）， recombinant human acylcoa dehydrogenase （ACADM） mRNA in liver of rats were detected by Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）， Western blot was used to detect the protein levels of IRS-1， PI3K and Akt in liver of rats. Hematoxylin-eosin（HE） staining was used for histopathological examination of rat thoracic aortic vessels. The migration ability of vascular smooth muscle cells in rat thoracic aorta was detected by scratch test. Result:Compared with the normal group， the fasting blood glucose， glycosylated hemoglobin， serum TNF-α， IL-6，IL-1β， TC，TG and LDL-C levels， liver lipid synthesis gene mRNA level and vascular smooth muscle cell migration ability of thoracic aorta in model group were significantly higher than those in normal group （P<0.05）， while fatty acid oxidation gene mRNA level and IRS-1，PI3K，Akt protein level in liver were significantly decreased in model group （P<0.05）. The vascular wall thickness of thoracic aorta increased significantly in rats （P<0.05）. Compared with model group， the levels of fasting blood glucose， glycosylated hemoglobin， serum TNF-α，IL-6， IL-1β， TC， TG and LDL-C， the level of lipid synthesis gene mRNA in liver and the migration ability of vascular smooth muscle cells in thoracic aorta of rats in all Baihutang groups were significantly lower than those in model group （P<0.05）. The mRNA level of fatty acid oxidation gene and the protein levels of IRS-1， PI3K and Akt in liver were significantly increased（P<0.05）， and the histopathology of thoracic aorta was significantly improved and the vascular wall thickness decreased significantly（P<0.05）. Conclusion:Baihutang can reduce the levels of blood glucose， blood lipid and serum inflammatory factors in type 2 diabetic rats， regulate the expression of genes related to lipid metabolism in liver， and improve the histopathology and vascular remodeling of thoracic aorta. The mechanism may be related to the regulation of IRS-1/PI3K/Akt signal pathway.
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Objective: The present study was designed to investigate the modulating effect of Zhenxin Xingshui Yizhi Fang and its essential oil extract on cognitive deficits in mice.Method: For the purpose of this study 5 months old APP/PS1 double transgenic mice and wild-type C57BL/6JNju were selected as experimental animals.Then APP/PS1 double transgenic mice were randomly divided into model group,essential oil low and high-dose groups (12.13,48.50 mg·L-1),Zhenxin Xingshui Yizhi Fang group (0.46 g·kg-1).Meanwhile,wild-type C57BL/6JNju mice were used as a normal group.APP/PS1 double transgenic mice were treated with Zhenxin Xingshui Yizhi Fang and its essential oil extract for 22 consecutive days.Mice were subjected to a Morris water maze test and a platform test in order to determine their cognitive effect.Nissl's staining was used to observe pathological changes in brain tissue.Meanwhile,senile plaques (SP) were observed by employing Thioflavin-S staining.The expression of glucose transporter 1(GLUT1) and insulin receptor substrate-1(IRS-1) were analyzed using immunohistochemistry techniques.The levels of neurotransmitters such as acetylcholine (ACH),glutamate (GLU) and γ-aminobutyric acid (GABA) in the hippocampus were quantified by enzyme-linked immunosorbent assay (ELISA).Result: The memory function was significantly reduced in model group,and severe brain injury and neuronal apoptosis were also observed in comparison to normal group (PPPPPPPConclusion: These results indicate that Zhenxin Xingshui Yizhi Fang and its essential oil extract could ameliorate cognitive deficits and GLUT1 and IRS-1 could be a possible therapeutic target for AD.It may be an interesting approach to the treatment of Alzheimer's disease.
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OBJECTIVE: To observe the effect of electroacupuncture (EA) on insulin signaling pathway in liver tissues of central neuronal specific signal transduction and activator of transcription 5 conditional-knockout (Stat 5 NKO) mice, so as to explore its mechanism underlying improvement of insulin resistance (IR)．. METHODS: Twenty-four male Stat 5 NKO mice were randomly divided into model and EA groups (n＝12 mice/group), and 12 Stat 5 fl/fl mice were used as the normal control group. EA (2 Hz/15 Hz, 0.8－1.0 mA) was alternatively applied to ipsilateral "Zusanli" (ST 36) and "Neiting" (ST 44) for 20 min, once a day, 6 times a week for 4 weeks. The glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed, and the values of fasting plasma glucose (FPG) and fasting insulin (FINS) were measured by glucometer and ELISA, separately. The insulin sensitivity index (ISI) was calculated. The phosphorylation protein expressions of insulin receptor substrate 1 (IRS 1), insulin receptor β (IRβ) and protein kinases B (Akt) in the liver tissues were detected by Western blot. RESULTS: In Stat 5 NKO mice (model group), FPG level and glucose area under the curve (GAUC) of ITT and GTT were significantly increased (P0.05). Compared with the normal group, the protein expression levels of liver p-IRS 1 and p-IRβ were significantly up-regulated (P<0.001), and the p-Akt expression was significantly down-regulated (P<0.01) in the model group. Following EA treatment, the increased p-IRS 1 and p-IRβ protein expression and the decreased p-Akt expression were apparently reversed in the EA group relevant to the model group (P<0.001, P<0.01)．. CONCLUSION: EA can improve the IR induced by central neuronal Stat 5-knockout in mice, which may contribute to its effectiveness in regulating hepatic IRβ/IRS 1/Akt signaling pathway.
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Objective To evaluate the effect of Ji Tang Zhi on glucose metabolism in insulin resistance (IR) HepG 2 cell line,and to explore the related mechanism.Methods The HepG2 cells were incubated in culture medium addition of 10-7 mol/L insulin for 24 h to establish the IR cell model.Effect of Ji Tang Zhi on rate of glucose absorption in HepG2 cell was detected by the method of glucose oxidase-peroxidase (GOD-POD).We performed an MTT assay to determine cytotoxicity effects of Ji Tang Zhi on HepG2 cell line.The expression of p-IRS-1 Ser307,PI3K and GLUT-4 were detected by Western blotting.Results Incubated with 10-7 mol/L insulin for 24 h,the insulin resistance cell model had been built.Compared with model group,the rate of glucose absorption of cell treated with JTZ (30 ～ 120 μg/mL) was significantly improved.According to model cells,the expression of GLUT-4 and PI3K decreased significantly compared to control cells.While the expression of p-IRS-1 Ser 307 was inhibited and GLUT-4 and PI3K expression were increased in IR cells after treated with JTZ (30 ～ 120 μtg/mL).Conclusion JTZ exert beneficial effects on hyperglycosemia in IR cell line possibly through regulating the levels of GLUT-4,p-IRS-1 Ser307 and PI3K in HepG2 cell.
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Objective This study aims to investigate the effects of high-fat diet rich in perilla oil on the insulin sensitivity-related gene expression in skeletal muscle in insulin resistant rats.Methods The insulin resistant ( IR) rat models were randomly divided into 2 groups, including high fat group ( HF) and perilla oil ( PO) intervention group fed with 20%substitution of lard energy in the HF.The insulin sensitivity of rats was measured after 4 weeks.Theα-linolenic acid ( ALA) content of PO in the rat plasma were analyzed by gas chromatograph.Real-time PCR was applied to measure glucose transporter 4 ( GLUT4 ) and insulin receptor substrate-1 ( IRS-1 ) mRNA, and Western blot assay was used for detecting the expression of GLUT4 and IRS-1 in the skeletal muscle.Results At the gene and protein levels, PO remarkably reduced the level of IRS-1 and upregulated the level of GLUT4 with increasing intake of ALA and serum ALA content in IR rats.The results of hyperinsulinemic-euglycemic clamp test showed no significant difference between the two groups.Conclusions The results of our study suggest that consumption of n-3 PUFA at levels that can typically be found in the diet fed to IR rats in the form of ALA (0.556 g/d) may not improve insulin sensitivity, even though regulating the expression of GLUT4 and IRS-1 in the skeletal muscle.
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Objective To construct an eukaryotic expression vetor of ring finger protein 6 (RNF6)and to investigate its ubiqu-itylation on insulin receptor substrate-1(IRS-1).Methods Human RNF6 coding sequence was amplified by polymerase chain reac-tion (PCR)method with human cDNA as template.The pcDNA3.1-CHA-RNF6 was constructed with routine molecular methods and transfected into hepatocarcinoma cell HepG2.Relative quantification of IRS-1 mRNA was preformed by real-time reverse tran-scription PCR.Western blot was applied to detect the expression levels of IRS-1.Results 48 h after the plasmid carring RNF6 gene transfected HepG2 cells,the mRNA level of ISR-1 gene reduced to 69% of control group.The expression of IRS-1 was markedly lower than control group (P <0.01).Conclusion The expression of IRS-1 is markedly down-regulated in HepG2 and enhancement of the ubiquitylation level of IRS-1 contribute to the disorder in insulin signal transferring.
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Objective:To explore the effects of lanthanum nitrate low-dose on 2 type diabetic rat pancreas INSR ,IRS1 and apoptosis of cells.Methods:The 50 male Wistar rats were randomly divided into normal control group (10) and model group(40),and were given normal diet and high-fat high-sugar diet.After 8 weeks,making the module intraperitoneal injection of streptozotocin (STZ,30 mg/kg) rats to induce type 2 diabetes model ,after a successful modeling experimental animals were divided into three groups namely the control group,diabetic model group and La(NO3)3 treatment group,three groups of rats were given daily saline,saline,lanthanum (0.2 mg/kg) nitrate administered a month ,then were sacrificed blood and pancreas.By radioimmunoassay to detect glucose ,insulin levels(in rat serum);by ELISA to detect the protein content of INSR and IRS 1(in rat serum);by immunohistochemistry to detect the protein expression levels of INSR and IRS1(in rat pancreas tissue);TUNEL apoptosis detection kit was used to detect pancreatic tissue percentage of apoptosis;HE staining was used to detect pathological changes of the pancreas.Results: Compared with the normal control group,diabetic model rats blood glucose and pancreatic tissue apoptosis rate were significantly increased ,blood insulin,INSR and IRS1 protein content and pancreatic tissue the protein expression levels of INSR and IRS 1 were significantly reduced ,difference was statistically significant ( P<0.01 );La( NO3 ) 3 treatment group blood glucose and pancreatic tissue apoptosis rate were slightly elevated ,blood insulin , INSR and IRS1 protein content and pancreatic tissue the protein expression levels of INSR and IRS 1 were slightly reduced significantly(P<0.05);Light microscopy showed pancreatic tissue in the control group closely arranged ,plump,large volume islet pancreatic tissue of diabetic rats in groups evacuate more smaller islet volume ,pancreatic tissue lanthanum nitrate basic set tight,plump,slightly islet volume small.Conclusion:Oral lanthanum nitrate can be lowered blood sugar levels and inhibiting apoptosis of pancreatic tissue ,increased insulin levels and the protein expression levels of INSR and IRS 1 in pancreatic tissue ,have a protective effect on diabetic rat pancreas .
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3T3-L1 adipocytes transfected with TSH receptor (TSHR) shRNA were incubated with bovine TSH.The concentration of tumor necrosis factor (TNF)-α in culture medium was measured by enzyme linked immunosorbent asssy.Protein level of insulin receptor substrate 1 (IRS-1) was quantified by Western blotting.Tyrosine phosphorylation of IRS-1 was measured by immunoprecipitation.The results showed that 1 mIU/ml TSH significantly sitmulated TNF-α release in 3T3-L1 adipocytes [(341.85 ± 12.00 vs 522.67 ± 36.22) ng/L,P＜0.01],along with the decreases in IRS-1 protein expression and its tyrosine phosphorylation (P＜ 0.01).These effects disappeared when TSHR expression was down-regulated with RNA interference in 3T3-L1 adipocytes.In addition,WP9QY,a TNF-α antagonist,blocked TSH-decreased IRS-1 expresssion.These results suggest that TSH downregulates IRS-1 protein expression and its tyrosine phosphorylation through stimulating production of TNF-α,and thus contributes to the development of insulin resistance.
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Objective To investigate the short term effect of insulin on proinsulin gene expression of HIT-T15 insu-linnoma cells(pancreatic isletβ-cell). Methods The HIT-T15 cells were randomly divided into four groups.Blank Con-trol Group (LG):complete medium contain 1.4 mmol/L glucose. Control group (LGC):co-cultured nifedipine with medium in order to restain endogenous insulin release. Experimental group (LINS or HINS) add 0.5 U/L insulin or 5 U/L insulin on top of LGC. After being stimulated for 0, 30, 60, 90, 120 mins, proinsulin (PI) mRNA level were assessed by semi-quantitative RT-PCR. Insulin receptor substrate1 (IRS1) tyrosine phosphorylation was detected by immunocytochemistry. Results (1) Expression of PI was up regulated by both LINS and HINS, and peak at 60 mins. (2) After stimulation for 30 mins, the level of IRS1 tyrosine phosphorylation in the experimental group was significantly higher than control group, and the peak time be-tween LINS and HINS was different. (3) Between group of LG and LGC, the expression of PI mRNA and IRS1 tyrosine phos-phorylation show no difference. Conclusion Short term exogenous insulin stimulation can promote expression of proinsulin genes,which is concentration dependent. The expression and regulation of PI were related with IRS1 signal transduction.
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BACKGROUND: The insulin receptor substrate-1 (IRS-1) is the primary docking molecule for the insulin-like growth factor I receptor (IGF-IR), and is required for activation of the phosphatidylinositol 3'-kinase (PI3K) pathway. IRS-1 activation of the (PI3K) pathway regulates IGF-mediated survival, enhancement of cellular motility and apoptosis. Therefore, we attempted to ascertain whether IRS-1 genetic variations affect an individual's risk for non-small cell lung cancer (NSCLC). METHODS: Two-hundred and eighteen subjects, either diagnosed with NSCLC or control subjects, were matched by age, gender and smoking status. Genomic DNA from each subject was amplified by PCR and analyzed according to the restriction fragment length polymorphism (RFLP) profile to detect the IRS-1 G972R polymorphism. RESULTS: The frequencies of each polymorphic variation, in the control population, were as follows: GG=103 (94.5%) and GR=6 (5.5%); for the NSCLC subjects, the genotypic frequencies were as follows: GG=106 (97.2%) and GR=3 (2.8%). We could not demonstrate statistically significant differences in the genotypic distribution between the NSCLC and the control subjects (p=0.499, Fisher's Exact test). The relative risk of NSCLC, associated with the IRS-1 G972R polymorphic variation, was 1.028 (95% CI; 0.63~9.90). In addition, we found no differences between polymorphic variants with regard to the histological subtype of NSCLC. CONCLUSION: We did not observe any noteworthy differences in the frequency of the IRS-1 G972R polymorphism in NSCLC patients, compared to control subjects. These results suggest suggesting that, in our study population, the IRS-1 G972R polymorphism does may not appear to be associated with an increased risk of NSCLC.
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Humans , Apoptosis , Carcinoma, Non-Small-Cell Lung , DNA , Genetic Variation , Insulin , Insulin Receptor Substrate Proteins , Insulin-Like Growth Factor I , Phosphatidylinositols , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Receptor, Insulin , Smoke , SmokingABSTRACT
Objective To investigate the expression and tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and in the adipose tissue of patients with gestational diabetes mellitus (GDM), and to explore molecular mechanisms of insulin resistance in GDM. Methods The serum and adipose tissue were sampled from patients with GDM (GDM group, n = 20) and normal pregnant women (control group, n = 20). Fasting plasma glucose was measured by glucose oxidase assay. The expressions of IRS-1 protein and mRNA were determined by Western blot and semi-quantitative RT-PCR. The tyrosine phosphorylation of IRS-1 was measured by immunoprecipitation. Results Compared with control group, in GDM group, the expression of IRS-1 mRNA was markedly decreased (0.61 ±0.06 vs 1.12 ± 0.17, P < 0.01), the expression of IRS-1 protein was significantly decreased (0.57 ±0.08 vs O. 83 ±0.07, P <0.01) and tyrosine phosphorylation was significantly reduced (0.23 ± O. 06 vs O. 62 ±0.04, P < 0.01) in the adipose tissue. Conclusion The decline of protein expression and tyrosine phosphorylation of IRS-1 in the adipose tissue of gestational diabetes appears to be one of the moleculemechanisms of insulin resistance in patients with GDM.
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Objective To investigate the mechanism of Chinese herbal medicine compound (Kaiyuqingwei Decoction) improving islet cells function of type 2 diabetes. Methods The models of type 2 diabetic rats were established by feeding with high-fat-diet and injecting low dosage of streptozotocin (15 mg/kg BW). Rats were randomly divided into model group, Kaiyuqingwei group, and Rosiglitazone group and normal control was established, at the same time, fasting and post-glucose loading 2 hours blood glucose, serum fructosamine and basal insulin were determined. Euglycemic hyperinsulin clamp technique was adopted to evaluate insulin sensitivity of periphery tissues, and intravenous glucose tolerance test to evaluate islets function. Adopting immunohistochemistry stain (EnVision) and computer image analysis technique to determine the expression of insulin, insulin receptor and insulin receptor substrate-1 in islets. Results There were insulin resistance, dysfunction of islet cells and obvious increase of blood glusose in model rats. All these could be improved by Kaiyuqingwei Decoction and Rosiglitazone. Meanwhile, immunohistochemistry stain of islets demonstrated that Kaiyuqingwei Decoction could increase expression of IRc and IRS-1. Conclusion Kaiyuqingwei Decoction have a certain positive role in improving glucose metabolism of type 2 diabetic rats. The mechanism include two aspects, one is elevation of insulin sensitivity, another is amelioration of dysfunction of islet cells. One of the important mechanisms of amelioration of dysfunction in islet cells is amelioration of insulin signal transduction in pancreatic islets.
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Objective To determine the effect of a variation of CAG-rich region,which was fond in the 5'-regulatory sequence of insulin receptor substrate-1(IRS-1)gene in Type 2 diabetes mellitus(T2DM)patients,on gene expression and its mechanism.Methods The recombinants,pGL2.P-T3 and pGL2.P-T5,were constructed with luciferase reporter vector,pGL2 promoter.T3 and T5 were wild-type and variant alleles,respectively.The recombinants were cotransfected with pSV-β-galactosidase control vector to Hela cells.Luciferase assay was performed to assess transcriptional actiVity.The electrophoresis mobility shift assay(EMSA)and DNA footprint assay were applied to determine the interaction between the DNA regulatory sequences and nuclear proteins of Hela cells.Results The relative transcription activity of T5 was lower than that of T3[(7.76±1.05)%vs(9.98±1.40)%,P＜0.05];EMSA showed both T3 and T5 formed a single retarded band in gel with the same mobility with nnclear proteins;T5 had 2 binding sites for transacting factors,CGCGCCCGCGGGCGGCGGC and GGGCGGCTGGTGGCGGCTG,which was the same as T3.Conclusion Although the variation in T5 do not alter the DNA-binding sites for Hela cell nuclear extracts,the notable decrease in gene transcrip tionactivity induced by it may be an important factor to the development T2DM in the carrier.
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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 glucose uptake and IRS-1-associated signaling pathway by stimulated insulin under TNF-? treatment.Methods:3T3-L1 adipocytes were treated with TNF-? within 6 hours and 24 hours respectively. 2-deoxy ~3H glucose was used to measure glucose uptake and western blot was used to measure IRS-1, PKB protein, tyrosine and serine307 phosphorylation on IRS-1, and PKB phosphorylation.Results:On basal status, glucose uptake of 3T3-L1 cells and phospho-tyrosine of IRS-1, PKB phosphorylation, and serine307 phosphorylation on IRS-1 were all low. Insulin stimulation induced glucose uptake and IRS-1 tyrosine phosphorylation, serine307 phosphorylation, PKB phosphorylation rapidly. TNF-? inhibited insulin-induced glucose uptake, tyrosine phosphorylation of IRS-1 and PKB phosphorylation. Rapamycin reversed the effects of TNF-?. Treated with TNF-? within 6 hours increased serine307 phosphorylation but had no effect on IRS-1 protein level. TNF-?-induced serine307 phosphorylation of IRS-1 was not affected by rapamycin. IRS-1 level was decreased under 24 hours TNF-? treatment and rapamycin can reverse the effect.Conclusion:TNF-? induced insulin resistance in 3T3-L1 adipocytes mightbe related to impaired IRS-1 tyrosine phosphorylation, rapamycin could reverse the effects of TNF-?. Treated with TNF-? within 6 hours stimulate phosphrylation of serine307 of IRS-1 and 24 hours treatment decreased IRS-1 protein level. Rapamycin antagonist TNF-?-induced loses of IRS-1.
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Objective To investigate the molecular mechanism of metformin. Methods The changes of protein expression of IRS-1 in liver, skeletal muscle and adipose tissues after treatment with metformin for OLETF rats, a model of spontaneous type 2 diabetes mellitus, were measured by Western blot analysis, and compared with those before treatment. Results 22 weeks after the treatment with metformin, the protein expression of IRS-1 was significantly increased in liver (P0. 05) ;and the protein expression of IRS-1 in adipose tissue was significantly decreased (P
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Insulin receptor substance-1 (IRS-1) mRNA and protein were assayed in rat muscle of hindlamb by RT-PCR and immunohistochemistry respectively.Smoking decreased the expressions of IRS-1 mRNA and protein in rat muscle of normal chow smoking group,high fat chow smoking group and diabetic smoking group as compared with matched control groups (P
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Objective To investigate the effect of a peptide,APP17,on regulating the expression of insulin receptor substrate\|1(IRS\|1) and insulin\|like growth factor (IGF\|1R) in neurons of the hippocampus from diabetic mouse. Methods Diabetic mouse models were established by injection of streptozotion.In experimental group,these models were injected with APP17 peptide subcutaneously and their brain sections were taken after 4 weeks of survival. The immunohistochemical stainning of these sections were then performed with IRS\|1 and IGF\|1R antibody.With regard to control groups,the mouse models were only injected saline and gone through the same procedure of immunohistochemistry together with normal mice. Results IRS\|1 and IGF\|1R positive neurons were widely distributed in the hippocampus of the diabetic mice,and the cytoplasm was darkly stained.In the contrast,positive cells in the hippocampus were lightly stained in those normal mice and the APP17 peptide\|treated diabetic mice. Conclusion The expression of IRS\|1 and IGF\|1R could increase in the hippocampus of dabetic mice.The APP17 can regulate the distribution of IRS\|1 and IGF\|1R in the brain of diabetic mice and return them to normal situation.