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Insulin signalling is tightly controlled by various factors, but the exact molecular mechanism remains incompletely understood. We have previously reported that phospholipase C-related but catalytically inactive protein (PRIP; used here to refer to both PRIP-1 and PRIP-2, also known as PLCL1 and PLCL2, respectively) interacts with Akt1, the central molecule in insulin signalling. Here, we investigated whether PRIP is involved in the regulation of insulin signalling in adipocytes. We found that insulin signalling, including insulinstimulated phosphorylation of the insulin receptor (IR), insulin receptor substrate-1 (IRS-1) and Akt, and glucose uptake were impaired in adipocytes from PRIP double-knockout (PRIP-KO) mice compared with those from wild-type (WT) mice. The amount of IR expressed on the cell surfacewas decreased in PRIP-KO adipocytes. Immunoprecipitation assays showed that PRIP interacted with IR. The reduced cell surface IR in PRIP-KO adipocytes was comparable with that inWT cells when Rab5 (Rab5a, -5b and -5c) expression was silenced using specific siRNA. In contrast, the dephosphorylation of IRS-1 at serine residues, some of which have been reported to be involved in the internalisation of IR, was impaired in cells from PRIPKO mice. These results suggest that PRIP facilitates insulin signalling by modulating the internalisation of IR in adipocytes. [ABSTRACT FROM AUTHOR]

The IGF signaling pathway plays critical role in regulating skeletal myogenesis. We have demonstrated that KIF5B, the heavy chain of kinesin‐1 motor, promotes myoblast differentiation through regulating IGF‐p38MAPK activation. However, the roles of the kinesin light chain (Klc) in IGF pathway and myoblast differentiation remain elusive. In this study, we found that Klc1 was upregulated during muscle regeneration and downregulated in senescence mouse muscles and dystrophic muscles from mdx (X‐linked muscular dystrophic) mice. Gain‐ and loss‐of‐function experiments further displayed that Klc1 promotes AKT–mTOR activity and positively regulates myogenic differentiation. We further identified that the expression levels of IRS1, the critical node of IGF‐1 signaling, are downregulated in Klc1‐depleted myoblasts. Coimmunoprecipitation study revealed that IRS1 interacted with the 88‐154 amino acid sequence of Klc1 via its PTB domain. Notably, the reduced Klc1 levels were found in senescence and osteoporosis skeletal muscle samples from both mice and human. Taken together, our findings suggested a crucial role of Klc1 in the regulation of IGF‐AKT pathway during myogenesis through stabilizing IRS1, which might ultimately influence the development of muscle‐related disorders. [ABSTRACT FROM AUTHOR]

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Objective: The aim of this research was to probe the effect of isobavachalcone inhibiting the high-fat diet (HFD) and streptozotocin (STZ) induced hyperglycemia and hyperlipemia in mice and its underlying mechanisms. Methods: A mouse model with type 2 diabetes mellitus (T2DM) was established by the HFD and low-dose streptozotocin. T2DM mice were treated with 10 mg/kg of isobavachalcone for 4 weeks. The levels of fasting blood glucose (FBG), blood lipids, and relative proteins in the PI3K/AKT signaling pathway as well as the histological structural changes in the liver were determined. Results: The results showed that the expression levels of FBG (42%, P<.001), triglyceride (70%, P<.001), and glucose transporter 2 were significantly reduced by isobavachalcone. The proteinic and mRNA expressions of IRS1, PI3K, and AKT genes of T2DM mice were dramatically increased with the isobavachalcone treatment. Furthermore, isobavachalcone ameliorated the morphology of the liver in T2DM model mice. Conclusion: The results suggested that isobavachalcone controls the development of T2DM as well as the regulatory mechanism involved in glucose and lipid metabolism through the PI3K/AKT signal pathway in the liver. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is an age-related, multifactorial progressive neurodegenerative disorder manifested by cognitive impairment and neuronal death in the brain areas like hippocampus, yet the precise neuropathology of AD is still unclear. Continuous failure of various clinical trial studies demands the utmost need to explore more therapeutic targets against AD. Type 2 Diabetes Mellitus and neuronal insulin resistance due to serine phosphorylation of Insulin Receptor Substrate-1 at 307 exhibits correlation with AD. Dipeptidyl Peptidase-4 inhibitors (DPP-4i) have also indicated therapeutic effects in AD by increasing the level of Glucagon-like peptide-1 in the brain after crossing Blood Brain Barrier. The present study is hypothesized to examine Linagliptin, a DPP-4i in intracerebroventricular streptozotocin induced neurodegeneration, and neuroinflammation and hippocampal insulin resistance in rat model of AD. Following infusion on 1st and 3rd day, animals were treated orally with Linagliptin (0.513 mg/kg, 3 mg/kg, and 5 mg/kg) and donepezil (5 mg/kg) as a standard for 8 weeks. Neurobehavioral, biochemical and histopathological analysis was done at the end of treatment. Dose-dependently Linagliptin significantly reversed behavioral alterations done through locomotor activity (LA) and morris water maze (MWM) test. Moreover, Linagliptin augmented hippocampal GLP-1 and Akt-ser473 level and mitigated soluble Aβ (1–42), IRS-1 (s307), GSK-3β, TNF-α, IL-1β, IL-6, AchE and oxidative/nitrosative stress level. Histopathological analysis also exhibited neuroprotective and anti-amylodogenic effect in Hematoxylin and eosin and Congo red staining respectively. The findings of our study concludes remarkable dose-dependent therapeutic potential of Linagliptin against neuronal insulin resistance via IRS-1 and AD-related complication. Thus, demonstrates unique molecular mechanism that underlie AD. [ABSTRACT FROM AUTHOR]

Insulin signalling is tightly controlled by various factors, but the exact molecular mechanism remains incompletely understood. We have previously reported that phospholipase C-related but catalytically inactive protein (PRIP; used here to refer to both PRIP-1 and PRIP-2, also known as PLCL1 and PLCL2, respectively) interacts with Akt1, the central molecule in insulin signalling. Here, we investigated whether PRIP is involved in the regulation of insulin signalling in adipocytes. We found that insulin signalling, including insulinstimulated phosphorylation of the insulin receptor (IR), insulin receptor substrate-1 (IRS-1) and Akt, and glucose uptake were impaired in adipocytes from PRIP double-knockout (PRIP-KO) mice compared with those from wild-type (WT) mice. The amount of IR expressed on the cell surfacewas decreased in PRIP-KO adipocytes. Immunoprecipitation assays showed that PRIP interacted with IR. The reduced cell surface IR in PRIP-KO adipocytes was comparable with that inWT cells when Rab5 (Rab5a, -5b and -5c) expression was silenced using specific siRNA. In contrast, the dephosphorylation of IRS-1 at serine residues, some of which have been reported to be involved in the internalisation of IR, was impaired in cells from PRIPKO mice. These results suggest that PRIP facilitates insulin signalling by modulating the internalisation of IR in adipocytes. [ABSTRACT FROM AUTHOR]

Insulin signalling is tightly controlled by various factors, but the exact molecular mechanism remains incompletely understood. We have previously reported that phospholipase C-related but catalytically inactive protein (PRIP; used here to refer to both PRIP-1 and PRIP-2, also known as PLCL1 and PLCL2, respectively) interacts with Akt1, the central molecule in insulin signalling. Here, we investigated whether PRIP is involved in the regulation of insulin signalling in adipocytes. We found that insulin signalling, including insulin-stimulated phosphorylation of the insulin receptor (IR), insulin receptor substrate-1 (IRS-1) and Akt, and glucose uptake were impaired in adipocytes from PRIP double-knockout (PRIP-KO) mice compared with those from wild-type (WT) mice. The amount of IR expressed on the cell surface was decreased in PRIP-KO adipocytes. Immunoprecipitation assays showed that PRIP interacted with IR. The reduced cell surface IR in PRIP-KO adipocytes was comparable with that in WT cells when Rab5 (Rab5a, -5b and -5c) expression was silenced using specific siRNA. In contrast, the dephosphorylation of IRS-1 at serine residues, some of which have been reported to be involved in the internalisation of IR, was impaired in cells from PRIP-KO mice. These results suggest that PRIP facilitates insulin signalling by modulating the internalisation of IR in adipocytes. [ABSTRACT FROM AUTHOR]

Despite rigorous endeavors, existing attempts to handle type 2 diabetes (T2DM) are still a long way off, as a substantial number of patients do not meet therapeutic targets. Insulin resistance in skeletal muscle is discerned as a forerunner in the pathogenesis of T2DM and can be detected years before its progress. Studies have revealed the antidiabetic properties of Carica papaya (C. papaya), but its molecular mechanism on insulin receptor substrate-1 (IRS-1)/Akt signaling mechanisms is not yet known. The present study aimed to evaluate the role of C. papaya on IRS1 and Akt in high-fat-diet–streptozotocin-induced type 2 diabetic rats and also to analyze the bioactive compounds of C. papaya against IRS-1 and Akt via in silico analysis. Ethanolic extract of the leaves of C. papaya (600 mg/kg of body weight) was given daily for 45 days postinduction of T2DM up to the end of the study. Gluconeogenic enzymes, glycolytic enzymes, gene expression, and immunohistochemical analysis of IRS-1 and Akt in skeletal muscle were evaluated. C. papaya treatment regulated the levels of gluconeogenic and glycolytic enzymes and the levels of IRS-1 and Akt in skeletal muscle of type 2 diabetic animals. In silico studies showed that trans-ferulic acid had the greatest hit rate against the protein targets IRS-1 and Akt. C. papaya restored the normoglycemic effect in diabetic skeletal muscle by accelerating the expression of IRS-1 and Akt. [ABSTRACT FROM AUTHOR]

Jidong He,1 Junpeng Zhang,1 Lijuan Dong,1 Xuefeng Dang,1 Li Wang,2 Long Cheng,3 Yunxiang Huang4 1Department of Gastroenterology, Baoji People’s Hospital, Baoji, Shanxi 721000, People’s Republic of China; 2Department of Diabetic Nephropathy, Baoji Central Hospital, Baoji, Shanxi 721008, People’s Republic of China; 3Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, People’s Republic of China; 4Department of R&D, Asparagus Engineering Research Center of Hebei Province, Qinhuangdao 066008, People’s Republic of ChinaCorrespondence: Li WangBaoji Central Hospital, No. 8, Jiangtan Road, Baoji, Shanxi 721008, People’s Republic of ChinaEmail 53080690@qq.comLong ChengInstitute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Peking Union Medical College, No. 151, Malianwa North Road Haidian District, Beijing 100094, People’s Republic of ChinaTel/Fax +86 10 57833013Email 13581972102@139.comPurpose: Dihydromyricetin (DHM), the main bioactive flavonoid in vine tea, exerts multiple health beneficial effects. This work aimed to identify whether a naturally derived flavonoid product, DHM, can significantly attenuate metabolic syndrome and improve insulin sensitivity.Methods: 10-week-old db/db mice were randomly assigned to receive the antidiabetic agent metformin (Met, 50 mg/kg BW), DHM (1.0 g and 0.5 g/kg BW) or placebo and were simultaneously fed a high-fat diet for 8 weeks. The general status of the animals was observed and recorded daily, body weight and blood glucose levels were measured weekly, during the experimental period. On day 55, the oral glucose tolerance test (OGTT) was performed. After OGTT, all animals were anesthetized and sacrificed by cervical decapitation. Blood samples were collected in tubes to detect plasma insulin and the biochemical parameters of lipid metabolism. Pancreas histological changes and islet fibrosis were demonstrated by H&E staining and Masson staining, respectively. Moreover, the expression of insulin receptor substrate-1 and phosphorylated insulin receptor substrate-1 in the insulin signaling pathway was detected by Western blot assay.Results: The oral administration of DHM (1.0 g and 0.5 g/kg BW) reduced the fasting blood glucose, serum insulin, and glycated hemoglobin levels and the insulin resistance (HOMA-IR) index. Furthermore, DHM intervention decreased body weight and the serum lipid profile. In addition, DHM treatment also markedly decreased the relative abdominal fat weight. Western blot analysis indicated that DHM upregulated the IRS-1 (Y612) tyrosine phosphorylation, improving insulin resistance. Treatment with dihydromyricetin attenuated the progression of insulin resistance and pancreatic fibrosis in fatty db/db mice.Conclusion: In summary, we determined the antimetabolic syndrome effect of DHM in db/db obese mice. DHM upregulates the IRS-1 (Y612) tyrosine phosphorylation, improving insulin resistance. Therefore, DHM is a promising therapeutic candidate for the control of metabolic syndrome.Keywords: dihydromyricetin, metabolic syndrome, hypoglycemic function, insulin resistance, insulin receptor substrate-1

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 transcription-polymerase 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.

IntroductionDiabetes increases the risk of Parkinson’s disease (PD). The phosphorylation of type 1 insulin receptor substrate (IRS-1) determines the function of insulin signaling pathway. Extracellular vesicles (EVs) are emerging as biomarkers of human diseases. The present study investigated whether PD patients exert altered phosphorylation IRS-1 (p-IRS-1) inside the blood neuron-derived extracellular vesicles (NDEVs).Research Design and MethodsIn total, there were 94 patients with PD and 63 healthy controls recruited and their clinical manifestations were evaluated. Blood NDEVs were isolated using the immunoprecipitation method, and Western blot analysis was conducted to assess total IRS-1, p-IRS-1, and downstream substrates level in blood NDEVs. Statistical analysis was performed using SPSS 19.0, and p < 0.05 was considered significant.ResultsThe isolated blood EVs were validated according to the presence of CD63 and HSP70, nanoparticle tracking analysis and transmission electron microscopy. NDEVs were positive with neuronal markers. PD patients exerted significantly higher level of p-IRS-1S312 in blood NDEVs than controls. In addition, the p-IRS-1S312 levels in blood NDEVs was positively associated with the severity of tremor in PD patients after adjusting of age, sex, hemoglobin A1c, and body mass index (BMI).ConclusionPD patients exerted altered p-IRS-1S312 in the blood NDEVs, and also correlated with the severity of tremor. These findings suggested the association between dysfunctional insulin signaling pathway with PD. The role of altered p-IRS-1S312 in blood NDEVs as a segregating biomarker of PD required further cohort study to assess the association with the progression of PD.

Aim and backgrounds: sedentary lifestyle and high-calorie foods lead to obesity, hence the aim of the study was to investigate the effects of different doses of exercise and caloric restriction on insulin receptor substrate-1 in obese male rats. Methods and materials: 48, eight-week-old male Wistar rats (190 ± 16 g) fed high-fat palate for seventeen weeks. After obesity induction, the obesity group was randomly assigned to one of the six groups: EXE, CR, EXCR1, EXCR2, and EXCR3 as experimental groups and OC as the control group. Then 10% of food intake was reduced. Exercise intensity was adjusted about 70-75% vo2max, 28 m/min for 48 to 55 minute. Negative energy balance was applied through caloric restriction (CR) (6d/w), exercise training (EXE) (6d/w), and in three combinations: EXCR1 (2d/w exercise + 4d/w CR), EXCR2 (3d/w exercise + 3d/w CR) and EXCR3 (4d/w exercise + 2d/w CR) for two month. One way ANOVA and Tukey post-hoc was applied for Data analysis by SPSS version 16 (p

Colorectal cancer (CRC) is a common human malignant tumor, and the fourth most common cause of cancer-associated mortality in China. However, the pathogenesis of CRC is not yet fully understood. The present study aimed to investigate the expression and clinical significance of microRNA (miR)-126 and insulin receptor substrate-1 (IRS-1), as well as the role of miR-126 in the prognosis of patients with CRC. A total of 86 colorectal tissue specimens, including 40 CRC and adjacent normal tissue, 26 colorectal adenoma tissue and 20 normal colorectal tissue samples, were collected for the present study. Reverse transcription-quantitative PCR analysis was performed to determine miR-126 and IRS-1 mRNA expression levels, while western blotting and immunohistochemistry (IHC) analyses were performed to determine IRS-1 protein expression levels. The correlation between miR-126 and IRS-1 expression, as well as the association between altered miR-126 and IRS-1 expression levels and clinicopathological characteristics, and the overall survival time of patients with CRC were assessed. The results demonstrated that miR-126 expression was significantly downregulated, while IRS-1 protein expression was upregulated in CRC tissues compared with that in adjacent normal tissues, colorectal adenoma tissues and normal colorectal tissues, respectively. IHC analysis exhibited strong positive staining of IRS-1 protein in CRC tissues, while absent or weak staining of IRS-1 protein was detected in adjacent normal tissues, colorectal adenoma tissues and normal colorectal tissues. miR-126 expression was inversely correlated with IRS-1 protein expression in CRC tissues (r=−0.420; P<0.05). Furthermore, downregulated miR-126 expression was associated with advanced clinicopathological characteristics of the disease and a shorter overall survival time in patients with CRC. Taken together, the results of the present study suggest that miR-126 downregulation may be a candidate molecular marker predictive of poor prognosis of patients with CRC. [ABSTRACT FROM AUTHOR]

Insulin is important for brain function and neuronal survival. Insulin signaling is initiated by the phosphorylation of insulin receptor substrate‐1 (IRS‐1) at tyrosine (pTyr) residue. However, IRS‐1 is inhibited by phosphorylation at serine (pSer). In Alzheimer's disease (AD), oxidative stress and accumulation of amyloid beta (Aβ) induce neuroinflammation, which augments pSer‐IRS‐1 and reduces pTyr‐IRS‐1 disturbing insulin signaling pathway. Coenzyme Q10 (CoQ10) and biotin possess antioxidant and anti‐inflammatory properties, and, in this study, their impact on insulin signaling is investigated in an aluminium chloride (AlCl3) model of AD. AD was induced by oral administration of AlCl3 (75 mg/kg) for 60 days. Biotin (2 mg/kg), CoQ10 (10 mg/kg), and their combination were supplemented concomitantly with AlCl3 for 60 days. Memory test and histological examination were performed. Brain levels of lipid peroxides, antioxidants (reduced glutathione and superoxide dismutase), inflammatory markers (tumor necrosis factor‐α, interleukin‐6 [IL‐6], IL‐1, and nuclear factor κB), and phosphorylated Akt (survival kinase) as well as protein levels of Aβ, IRS‐1 (pTyr and pSer), and caspase‐3 (apoptotic marker) were determined. AlCl3 resulted in impaired memory, significant increase in Aβ, lipid peroxides, inflammatory markers, caspase‐3, and pSer‐IRS‐1, with significant reduction of the antioxidants, pTyr‐IRS‐1, and p‐Akt reflecting Aβ‐induced inflammation and defective insulin signaling. Histological examination revealed focal aggregations of inflammatory cells and neuronal degeneration. The biochemical deviations and histological changes were attenuated by the concomitant treatment with biotin and, to greater extent, with CoQ10 and the combination. In conclusion, biotin and CoQ10 could protect against AD via attenuating inflammatory response and enhancing insulin signaling. [ABSTRACT FROM AUTHOR]

Patients with Her2-positive breast cancer exhibit de novo resistance or develop acquired resistance in less than one year after Her2 targeting treatment, but the mechanism is not fully elucidated. Compensatory pathways such as the IGF-1R/IRS-1 pathway, are activated, leading to aberrant enhanced PI3K/Akt/mTOR pathway activity to attenuate the efficacy of trastuzumab. Cullin7 could participate in the degradation of IRS-1 in a mTOR/S6K dependent manner. Whether Cullin7 participates in trastuzumab resistance needs to be further investigated. Here, we reveals that Cullin7 is overexpressed in trastuzumab-resistant Her2 positive breast cancer cells. Knockdown of Cullin7 reduces degradation of Ser phosphorylation of IRS-1, attenuates activation of the PI3K/AKT pathway, and partly restores trastuzumab sensitivity in trastuzumab-resistant Her2 positive breast cancer cells. IGFBP-3 expression is decreased in trastuzumab-resistant Her2 positive breast cancer cells, which leads to release of the Wnt signaling pathway inhibition and an increase in Cullin7 expression, as mediated by TCF7L2. Overexpression of Cullin7 in Her2-amplified breast cancer tissues has clinical implications because it positively correlates with shorter disease-free survival (DFS) and inadequate response to trastuzumab. Thus, our results suggest a critical role for Cullin7 in response to trastuzumab, which has significant implications for selection of the optimal therapeutic strategy for Her2 positive breast cancers. [ABSTRACT FROM AUTHOR]

lcoholic myopathy is characterized by the reduction in cross-sectional area (CSA) of muscle fibers and impaired anabolic signaling. The goal of the current study was to investigate the causes and compare the changes in CSA and fiber type composition with the modifications of anabolic and catabolic signaling pathways at the early stages of chronic alcohol consumption in women. Skeletal muscle samples from 5 female patients with alcohol abuse (AL; 43 ± 5 yr old; alcohol abuse duration 5,6 ± 0,6 yr) were compared with the muscle from the control group of 8 healthy women (C; 35 ± 4 yr old). The average daily dose of alcohol consumption was 110 ± 10 ml of pure ethanol. In women patients, a significant decrease in CSA of type I and II muscle fibers, titin and nebulin content, plasma IGF-1 level and total IRS-1, p-Akt and p-4E-BP1 in vastus lateralis was found in comparison with the control group. The p-AMPK level was found to be increased versus the control group. In women patients with chronic alcoholic myopathy 1) both fast and slow muscle fibers are subjected to atrophy; 2) impairments in IGF-I-dependent signaling and pathways controlling translation initiation (AMPK/mTOR/4E-BP1), but not translation elongation, are observed; 3) the level of calpain-1 and ubiquitinated proteins increases, unlike E3 ligases content. [ABSTRACT FROM AUTHOR]

TNF-α is a multifunctional cytokine participating in immune disorders, inflammation, and tumor development with regulatory effects on energy metabolism. Our work focused on the function of TNF-α in adipogenesis of primary porcine adipocytes. TNF-α could suppress the insulin receptor (IR) at the mRNA and protein levels. Microarray analysis of TNF-α-treated porcine adipocytes was used to screen out 29 differentially expressed microRNAs (miRNAs), 13 of which were remarkably upregulated and 16 were intensely downregulated. These 29 differentially expressed miRNAs were predicted to mainly participate in the insulin signaling pathway, adipocytokine signaling pathway, and type 2 diabetes mellitus pathway by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses. miR-146a-5p, reportedly involved in immunity and cancer relevant processes, was one of the most highly differentially expressed miRNAs after TNF-α treatment. Red Oil O staining and TG assay revealed that miR-146a-5p suppressed adipogenesis. A dual-luciferase reporter and siRNA assay verified that miR-146a-5p targeted IR and could inhibit its protein expression. miR-146a-5p was also validated to be involved in the insulin signaling pathway by reducing tyrosine phosphorylation of insulin receptor substrate-1. Our study provides the first evidence of miR-146a-5p targeting IR, which facilitates future studies related to obesity and diabetes using pig models.

In view of the high incidence of metabolic syndrome and the role of exercise in promoting metabolic disorders, the present study aimed to investigate the therapeutic effects of swimming intervention on metabolic syndrome. In the present study, a total of 100 patients with metabolic syndrome and 100 healthy individuals were included. Fasting blood was extracted from each participant, and the serum levels of interleukin (IL)-1, high sensitivity C-reactive protein (hs-CRP), tumor necrosis factor α (TNF-α) and IL-8 were measured by sandwich enzyme-linked immunosorbent assay. Patients were randomly divided into five groups (groups A-E). Patients in group A was treated with conventional drug treatment. Besides conventional treatment, patients in groups B-E were also subjected to swimming intervention for 15, 30, 45 and 60 min each time, respectively, four times a week for 3 months. Changes in the homeostatic model assessment of β-cell function and insulin resistance (HOMA-IR) score, and in the serum levels of IL-1, hs-CRP, TNF-α and IL-8 were recorded. Furthermore, muscle tissues were collected from patients, and the expression levels of insulin receptor substrate-1 (IRS-1), glucose transporter type 4 (GLUT4) and protein kinase B (Akt) in the tissues were detected by western blot assay. The results revealed that HOMA-IR and the serum levels of IL-1, hs-CRP, TNF-α and IL-8 were significantly higher in metabolic syndrome patients as compared with those in the normal controls, while swimming intervention reduced HOMA-IR and these serum levels to different extents. Swimming intervention also promoted IRS-1 and Akt phosphorylation, and increased GLUT4 expression level. Thus, it is concluded that swimming intervention may improve metabolic syndrome through multiple pathways. [ABSTRACT FROM AUTHOR]

Abstract Regulation of tyrosine phosphorylation on insulin receptor substrate-1 (IRS-1) is essential for insulin signaling. The protein tyrosine phosphatase (PTP) C1-Ten/Tensin2 has been implicated in the regulation of IRS-1, but the molecular basis of this dephosphorylation is not fully understood. Here, we demonstrate that the cellular phosphatase activity of C1-Ten/Tensin2 on IRS-1 is mediated by the binding of the C1-Ten/Tensin2 Src-homology 2 (SH2) domain to phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ). We show that the role of C1-Ten/Tensin2 is dependent on insulin-induced phosphoinositide 3-kinase activity. The C1-Ten/Tensin2 SH2 domain showed strong preference and high affinity for PtdIns(3,4,5)P 3. Using site-directed mutagenesis, we identified three basic residues in the C1-Ten/Tensin2 SH2 domain that were critical for PtdIns(3,4,5)P 3 binding but were not involved in phosphotyrosine binding and PTP activity. Using a PtdIns(3,4,5)P 3 binding-deficient mutant, we showed that the specific binding of the C1-Ten/Tensin2 SH2 domain to PtdIns(3,4,5)P 3 allowed C1-Ten/Tensin2 to function as a PTP in cells. Collectively, our findings suggest that the interaction between the C1-Ten/Tensin2 SH2 domain and PtdIns(3,4,5)P 3 produces a negative feedback loop of insulin signaling through IRS-1. Highlights • C1-Ten's function as a PTP for IRS-1 in insulin signaling is controlled by PI3K. • The C1-Ten SH2 domain binds to PtdIns(3,4,5)P 3 with high affinity. • The PtdIns(3,4,5)P 3 binding of C1-Ten is necessary for its function as PTP. [ABSTRACT FROM AUTHOR]

Introduction: Sedentary life style and high calorie foods lead to obesity, hence the aim of the study was to investigate the effects of different doses of exercise and caloric restriction on insulin receptor substrate-1 in obese male rats. Methods and Materials: 48, eight-week-old male Wistar rats (190 ± 16 g) fed high-fat palate for seventeen weeks. After obesity induction, the obesity group was randomly assigned to one of the six groups: EXE, CR, EXCR1, EXCR2, and EXCR3 as experimental groups and OC as the control group. Then 10% of food intake was reduced. Exercise intensity was adjusted about 70-75% vo2max, 28 m/min for 48 to 55 minute. Negative energy balance was applied through caloric restriction (CR) (6d/w), exercise training (EXE) (6d/w), and in three combinations: EXCR1 (2d/w exercise + 4d/w CR,) EXCR2 (3d/w exercise + 3d/w CR) and EXCR3 (4d/w exercise + 2d/w CR) for two month. One way ANOVA and Tukey post-hoc test was applied for data analysis by SPSS version 16 (p<0.05). Results: IRS-1 mRNA expression was significantly higher in CR in comparison to OC (p=0.024). Also, IRS-1 expression significantly increased in EXCR2 comparing to OC (p=0.033). Conclusion: 10% of negative energy balance in forms of caloric restriction per se or in conjunction with exercise can provoke mRNA expression of insulin substrate receptor-1 that may lead to insulin sensitivity improvement. [ABSTRACT FROM AUTHOR]

It is widely accepted that infusion of mesenchymal stem cells (MSCs) ameliorates hyperglycemia by alleviating insulin resistance in rats with type 2 diabetes mellitus (T2D). However, the detailed underlying mechanisms are not clearly defined. Mitsugumin 53 (MG53) is an E3 ligase that has recently been implicated in the aggravation of insulin resistance by promoting the ubiquitinoylation of insulin receptor substrate‑1 (IRS‑1) in skeletal muscles. It was therefore hypothesized that MG53 may be involved in MSC‑mediated therapeutic effects on insulin resistance. To test this hypothesis, in the present study, T2D rat models were induced by a high‑fat diet combined with streptozotocin administration and MSC infusion was performed four times (once every 2 weeks for 8 weeks). The therapeutic effects of MSC infusion on insulin resistance were evaluated and the effect on the expression of MG53 and insulin receptor signaling elements in skeletal muscle was also investigated by immunofluorescence staining and western blotting. The results demonstrated that MSC infusion ameliorated hyperglycemia and insulin resistance in T2D rats. Furthermore, MSC infusion inhibited MG53 elevation and reversed the decreases in glucose transporter type 4, insulin receptor, IRS‑1 and phosphorylated‑AKT levels in the skeletal muscle of T2D rats. These results indicated that MSC infusion has therapeutic effects in rats and that MG53 in skeletal muscle may be a promising novel therapeutic target protein for MSC‑mediated amelioration of insulin resistance in T2D. [ABSTRACT FROM AUTHOR]

Despite the consensus that neuro-inflammation and insulin resistance (IR) are two hallmarks of Alzheimer disease (AD), the molecular mechanisms responsible for the development of IR remain uncharacterized. MAPKs are signaling molecules that are implicated in the pathology of AD and have a role in IR development. Given that inflammatory mediators are shown to interfere with insulin signaling pathway in different cell types, the present work aimed to investigate whether neuro-inflammation induced memory loss is associated with hippocampal IR and whether insulin treatment protects against this IR. Subsequently, possible roles of MAPKs in this situation were investigated. Male Wistar rats were cannulated, and LPS (15 µg, day 0), insulin (3 mU) or saline (vehicle) were administered intra-cerebroventricularly (ICV) (days 1-6). Spatial memory performance was assessed during days 7-10 by Morris Water Maze test. Consequently, analysis of the amount of hippocampal phosphorylated forms of P38, JNK, ERK, IRS1 (ser307) and Akt (ser473) were done by Western blot. The outcomes indicated that while LPS induced memory loss and hippocampal IR (shown by elevated IRS1 and decreased Akt phosphorylation), insulin treatment nullified these effects. Molecular results also showed that LPS mediated IR and memory loss are associated with P38 but not JNK and ERK activation; this P38 activation was reversed by insulin treatment. These observations implied that one of the ways by which neuro-inflammation participates in AD is via induction of IR. It seems that this IR is mainly mediated by P38. Therefore, P38 could be considered as a molecular target for preventing IR development. [ABSTRACT FROM AUTHOR]

The present study aimed to investigate the effect of androgens on chronic heart failure (CHF) in a rat model. A total of 120 Sprague Dawley male rats were randomly divided into the following groups: (A) sham operation group, (B) castrated group, (C) heart failure (HF) group, (D) castrated + HF group, and (E) castrated + HF + testosterone (T) replacement therapy group. There were 20 rats in group A, and 25 rats in the other groups. Surgical castration was performed on groups B, D and E, and T replacement therapy was administered to group E. Groups C, D and E were treated with doxorubicin hydrochloride to prepare the CHF animal model. The insulin sensitivity index (ISI) was calculated from fasting blood glucose and fasting insulin levels. Echocardiography was performed. Venous blood was collected for plasma T level test. Myocardial tissue was used for apoptosis index analysis. The expression levels of myocardial insulin receptor (IR) and insulin receptor substrate-1 (IRS-1) were measured by reverse transcription semi-quantitative polymerase chain reaction. Compared with group A, the T level and ISI decreased, whereas the expression level of IR and IRS-1 were increased in the CHF group (P<0.05). Following castration, the T level and ISI were significantly decreased, and the expression of IR and IRS-1 were increased compared with the uncastrated CHF rats (P<0.01). Following androgen administration, the ISI increased, expression of IR and IRS-1 decreased, and the myocardial apoptosis index decreased (P<0.05). Taken together, these results demonstrated that androgen supplementation could improve insulin resistance and affect the expression of IR and IRS-1 in CHF, thereby reducing myocardial apoptosis and improving cardiac function. [ABSTRACT FROM AUTHOR]

A growing body of evidence demonstrates an association between Angiotensin II (Ang II) receptor blockers (ARBs) and enhanced glucose metabolism during ischemic heart disease. Despite these encouraging results, the mechanisms responsible for these effects during ischemia remain poorly understood. In this study we investigated the influence of losartan, an AT1 receptor blocker, and secreted Ang II (sAng II) on glucose uptake and insulin receptor substrate (IRS-1) levels during cardiomyocyte swelling. H9c2 cells were differentiated to cardiac muscle and the levels of myogenin, Myosin Light Chain (MLC), and membrane AT1 receptors were measured using flow cytometry. Intracellular Ang II (iAng II) was overexpressed in differentiated cardiomyocytes and swelling was induced after incubation with hypotonic solution for 40 min. Glucose uptake and IRS-1 levels were monitored by flow cytometry using 2-NBDG fluorescent glucose (10 μM) or an anti-IRS-1 monoclonal antibody in the presence or absence of losartan (10 −7 M). Secreted Angiotensin II was quantified from the medium using a specific Ang II-EIA kit. To evaluate the relationship between sAng II and losartan effects on glucose uptake, transfected cells were pretreated with the drug for 24 h and then exposed to hypotonic solution in the presence or absence of the secreted peptide. The results indicate that: (1) swelling of transfected cardiomyocytes decreased glucose uptake and induced the secretion of Ang II to the extracellular medium; (2) losartan antagonized the effects of swelling on glucose uptake and IRS-1 levels in transfected cardiomyocytes; (3) the effects of losartan on glucose uptake were observed during swelling only in the presence of sAng II in the culture medium. Our study demonstrates that both losartan and sAng II have essential roles in glucose metabolism during cardiomyocyte swelling. [ABSTRACT FROM AUTHOR]

Tangningtongluo (TNTL), a traditional Chinese medicine, has been widely used in clinics for decades in southwest China. Its pharmacological properties and underlying molecular mechanisms remain unclear. The main goal of ethnopharmacology is to identify novel bioactive compounds derived from plants for use in indigenous medical practice. This knowledge can be used to develop novel pharmaceuticals. In the present study, hyperglycemic C57BL/KsJ-db/db (db/db) mice were used to test the effect of TNTL on microvasculature of the retina and hypoglycemia. Metformin (Met) was selected as a positive control. 26-week-old mice were randomly assigned to receive either the anti-diabetic agent Met [140 mg/kg body weight (BW)], 1.8, 0.9 or 0.45 g/kg BW TNTL, or a placebo. The fasting blood glucose, serum insulin and glycated hemoglobin levels were measured. Histopathologic examination of the pancreas was performed to confirm the hypoglycemic effect. Fluorescein angiography was applied to detect diabetes-induced retinal angioma in the db/db mice. TNTL intake significantly decreased the fasting blood glucose level in a dose-dependent manner. Additionally, TNTL intervention resulted in a significant decrease in the insulin resistance index. Notably, TNTL treatment markedly reduced the speed of retinal degeneration and mildly reversed microvascular caliber degeneration. Western blot analysis indicated that upregulation of phosphorylated insulin receptor substrate-1 (p-IRS-1) by the administration of TNTL may be strongly involved in the improvement of insulin resistance. In conclusion, TNTL exerted a strong hypoglycemic effect and reversed retinal degeneration via upregulation of ISR-1. The present findings provide important scientific evidence supporting TNTL as an effective alternative approach for the management of Type 2 diabetes mellitus. [ABSTRACT FROM AUTHOR]

Apomorphine (APO) promotes intraneuronal amyloid-β (Aβ) degradation and improves memory function in an Alzheimer's disease (AD) model, 3xTg-AD mice. Since insulin resistance is increased in AD neurons, we investigated the effects of APO on brain insulin resistance in 3xTg-AD mice at early and late stages. After 1-month subcutaneous injection of Apokyn® to 3xTg-AD mice at 6 or 12 months of age, memory function was significantly improved in both age groups. Protein levels of insulin-degrading enzyme (IDE), which is linked to insulin signaling and degrades Aβ, significantly increased in the 3xTg-AD mice brain compared with non-transgenic mice, and were further increased by APO. Protein levels of two types of serine-phosphorylated insulin receptor substrate-1 (IRS-1), pS616 and pS636/639, significantly decreased following APO treatment in the 13-month-old 3xTg-AD mice brain, suggesting improved brain insulin resistance. Immunostaining of the IDE, pS616 and pS636/639 IRS-1 demonstrated similar changes due to APO treatment. Thus, brain insulin resistance is considered an important therapeutic target in AD, and APO may provide improved neuronal insulin resistance. [ABSTRACT FROM AUTHOR]

Thyroid-stimulating hormone (TSH) receptor is expressed in extrathyroidal tissues such as hepatocytes, adipocytes, and skeletal muscle, which suggests a possible novel role of TSH in various metabolic processes in extrathyroidal tissues independent of thyroid hormones. We investigated whether TSH has any effects on glucose tolerance and insulin sensitivity in the skeletal muscle using diet-induced obesity (DIO) mouse models and rodent skeletal muscle cells. TSH improved glucose tolerance in DIO mice and this was associated with an improvement of skeletal muscle insulin sensitivity resulting from the increased expression of insulin receptor substrate (IRS)-1 protein and mRNA therein. TSH significantly increased both basal and insulin-stimulated glucose transport in rat L6 myotubes and increased the expression of IRS-1 protein and mRNA in these cells as well. TSH also stimulated Irs1 promoter activation; this stimulation was abolished by protein kinase A (PKA) inhibition using H89 or by mutation of the cAMP-response element site located at −1155 to −875 bp of the Irs1 promoter region, supporting a novel role of TSH activated-cAMP/PKA/CREB signaling in the regulation of Irs1 expression. In conclusion, TSH improves insulin sensitivity in skeletal muscle by increasing Irs1 gene expression. This regulatory effect is mediated by a PKA-CREB-dependent pathway. [ABSTRACT FROM AUTHOR]

Objective To observe the expression changes of insulin receptor substrate-1/2 ( IRS-1/2) and phosphatidy inositol 3 kinase (PI3K) in gestational diabetes mellitus (GDM) adipocyte cells after anthropogenic Omentin-1, visceral adipose tissue-derived serine protease inhibitor (Vaspin) transfection. Methods Recoveried, extended and differentiated GDM preadipocyte cells were prepared, and then we built Omentin-1, Vaspin overexpression carrier, and transfected the extended adipose cells with different overexpression levels (1.0, 2.5 and 5.0μg).Meanwhile, no transfection group was taken as the contrast. Using Q-PCR to detect the mRNA expression of Omentin-1, Vaspin, IRS-1/2 and PI3K (P85a), using Western blotting to detect protein expression of Omentin-1, Vaspin, IRS-1/2 and PI3K ( P85a) as well as IRS-1/2 phosphorylation levels, using [3H]-2-deoxidation-D-glucose uptake assay to detect the rates of glucose uptake in different transfection groups. Results With the increasing Omentin-1 expression, the mRNA and protein expression of IRS-1 and PI3K(P85a) were increased, but those of IRS-2 had no significant changes, IRS-1 phosphorylation increased obviously, IRS-2 phosphorylation had no significant changes, and the glucose uptake rate increased significantly. With the increasing Vaspin expression, all of the mRNA, protein and phosphorylation in IRS-1/2 and PI3K(P85a) had no significant changes, so did the glucose uptake rates. Conclusion In GDM adipocyte cells after anthropogenic Omentin-1 transfection, IRS-1, PI3K( P85a) and glucose uptake rates were increased, but no changes in those of GDM adipocyte cells after anthropogenic Vaspin transfection. [ABSTRACT FROM AUTHOR]

Insulin receptor substrate-1 (IRS-1) is a signaling adaptor protein that interfaces with many pathways activated in lung cancer. It has been assumed that IRS-1 promotes tumor growth through its ability to activate PI3K signaling downstream of the insulin-like growth factor receptor. Surprisingly, tumors with reduced IRS-1 staining in a human lung adenocarcinoma tissue microarray displayed a significant survival disadvantage, especially within the Kirsten rat sarcoma viral oncogene homolog (KRAS) mutant subgroup. Accordingly, adenoviral Cre recombinase (AdCre)-treated LSL-Kras/Irs-1fl/fl (Kras/Irs-1-/-) mice displayed increased tumor burden and mortality comparedwith controls. Mechanistically, IRS-1 deficiency promotes Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling via the IL-22 receptor, resulting in enhanced tumor-promoting inflammation. Treatment of Kras/Irs-1+/+ and Kras/Irs-1-/- mice with JAK inhibitors significantly reduced tumor burden, most notably in the IRS-1-deficient group. [ABSTRACT FROM AUTHOR]

The mechanisms by which exendin-4 and selenium exert their antidiabetic actions are still unclear. Here, we investigated the effects of exendin-4 or selenium administration on the expression of glucagon-like peptide-1 receptor (GLP-1R), insulin receptor substrate-1 (IRS-1), and preproinsulin in the pancreas of diabetic rats. Diabetes was induced by streptozotocin administration. Diabetic rats were injected intraperitoneally with 0.03 μg exendin-4/kg body weight/daily or treated with 5 ppm selenium in drinking water for a period of 4 weeks. GLP-1R and IRS-1 levels were decreased while the level of preproinsulin messenger RNA (mRNA) was increased in the pancreas of diabetic untreated rats, as compared to that in control rats. Treatment of diabetic rats with exendin-4 increased protein and mRNA levels of GLP-1R, and IRS-1, and the mRNA level of preproinsulin in the pancreas, as compared to their levels in diabetic untreated rats. Selenium treatment of diabetic rats increased the pancreatic mRNA levels of GLP-1R, IRS-1, and preproinsulin. Exendin-4 or selenium treatment of diabetic rats also increased the numbers of pancreatic islets and GLP-1R molecules in the pancreas. Therefore, exendin-4 and selenium may exert their antidiabetic effects by increasing GLP-1R, IRS-1, and preproinsulin expression in the pancreas and by increasing the number of pancreatic islets. [ABSTRACT FROM AUTHOR]

Studies were conducted to evaluate the flux of various carbon sources to lipogenesis during brown adipocyte differentiation. 13C labeling and isotopomer spectral analysis quantified the contribution of metabolites to de novo lipogenesis in wild-type (WT) and insulin receptor substrate-1 knockout (KO) brown adipocytes. Both glucose (Glc) and glutamine (Gln) provided substantial fractions of the lipogenic acetyl CoA for both WT and KO cells in standard media, together contributing 60%. Adding acetoacetate (AcAc; 10 mM) to the medium resulted in a large flux of AcAc to lipid, representing 70% of the lipogenic acetyl CoA and decreasing the contribution of Glc plus Gln to 30%. For WT cells, the fractional synthesis of new fatty acids during 4 days of differentiation was 80% of the total. Similarly, 80% of the lipidic glycerol was derived from Glc in the medium; Gln was not a precursor for glycerol. When Gln was removed from the medium, the contribution of Glc to fatty acid synthesis doubled, replacing most of the contribution of Gln and maintaining total lipogenesis. Conversely, removal of Glc dramatically decreased lipogenesis.These results indicate that Glc's distinct role in lipid synthesis during differentiation cannot be replaced by other carbon sources, consistent with the role of Glc supplying NADPH and/or glycerol for triglyceride synthesis.

Background/aim: Pancreatic cancer is known to have one of the worst prognoses of all cancers, and its tumor cells are highly resistant to chemotherapeutic drugs. Pancreatic cancer cells coexist with stromal cells; however, their involvement in anticancer drug resistance remains poorly understood. Thus, in this study, we analyzed drug sensitivity using an in vitro co-culture system containing pancreatic cancer cells and stromal cells treated with a compound library., Materials and Methods: We examined the viability of the pancreatic cancer cell lines BxPC-3, Capan-1, and Panc-1 against compounds in an in vitro co-culture model containing pancreatic stromal cells (PSCs) and analyzed the protein expression for drug resistance by western blotting., Results: We found that co-cultured pancreatic cancer cells were resistant to vandetanib, which is an inhibitor of multi-tyrosine kinases. The key factor involved in drug resistance in these pancreatic cancer cells was oncostatin M, which was secreted by stromal cells. The addition of oncostatin M increased the vandetanib resistance of pancreatic cancer cells, while it inhibited the suppression of insulin receptor substrate-1 (IRS1) and the phosphorylation of extracellular signal-regulated kinase (ERK) by vandetanib., Conclusion: Oncostatin M secreted by stromal cells derived from the pancreas activates the IRS1-ERK axis, causing resistance to vandetanib., (Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: The mechanisms leading to the high on-treatment platelet reactivity in diabetes patients are not fully elucidated. The genetic factors may be associated with the diminished antiplatelet efficacy of dual antiplatelet therapy. We investigated the possible association between insulin receptor substrate-1 (IRS-1) polymorphisms and high platelet reactivity in coronary artery disease (CAD) patients with type 2 diabetes mellitus (T2DM). Methods: A total of 674 CAD patients with T2DM were enrolled in this study. Platelet aggregation and platelet activation were assessed with light transmission aggregometry and flow cytometry analysis, respectively. Participants were divided into high platelet reactivity (HPR) group and non-HPR group according to their maximal platelet aggregation. Genotypes were identified by polymerase chain reaction and direct sequencing of genomic DNA. The association between IRS-1 genetic variants and platelet function was assessed. Results: There were 233 participants in the HPR group and 441 participants in the non-HPR group. G allele frequencies of rs13431554 were 27.7% for the HPR group and 18.6% for the non-HPR group (p < 0.001). Adenosine diphosphate and arachidonic acid induced platelet aggregation were significantly higher in G allele carriers compared with non-carriers (56.8 ± 16.2 vs 52.0 ± 17.9%, p < 0.01, 28.9 ± 18.6 vs 25.2 ± 17.8%, p < 0.01, respectively). We observed that P-selectin expression and PAC-1 binding were higher in G allele carriers compared with non-carriers (40.8 ± 12.4 vs 36.2 ± 13.8, p = 0.01; 43.7 ± 15.9 vs 38.7 ± 19.9, p = 0.03, respectively). Conclusion: The G allele of rs13431554 in the IRS-1 gene was associated with a hyperreactive platelet phenotype in the CAD patients with T2DM. [ABSTRACT FROM AUTHOR]

Aim To explore the association between the Arg972 insulin receptor substrate ( IRS)-1 polymorphism (rs1801278) and the risk and disease activity/severity of rheumatoid arthritis ( RA). Method We genotyped the Arg972 IRS-1 polymorphism in 871 pairs of age-, sex-, body mass index-, residence area- and current smoking status-matched RA patients and controls. We assessed RA severity using the disease activity score of 28 joints ( DAS28). Results The AA (homozygous Arg972 IRS-1) and GA (heterozygous Arg972 IRS-1) genotypes were significantly associated with high risk of RA with or without adjustment for comorbidities ( P < 0.001). The A allele was significantly associated with high risk of RA ( P < 0.001). The AA genotype was significantly associated with high/severe RA activity ( P < 0.001), while the GG genotype (wild type IRS-1) had protective effects. Conclusion The Arg972 IRS-1 polymorphism is associated with increased risk and disease activity/severity of RA, and therefore may be a potential prognostic factor for RA. This study adds novel insights into the pathogenesis of RA. [ABSTRACT FROM AUTHOR]

Objective To investigate the molecular mechanism of sterol regulatory element binding protein-1c (SREBP- 1c) suppressing insulin receptor substrate-1(IRS-1) in skeletal muscle cells. Methods Luciferase plasmid for rat IRS- 1 promoter, expression plasmid of SREBP-1c and pRL-TK renilla plasmid were cotransfected into L6 cells using Lipofectamine 2000 (Invitrogen). After transfection for 36 h, luciferase activity was measured using the dual-luciferase reporter assay system following the manufacturer' s instructions. L6 myotubes were infected with adenoviral vectors expressing SREBP-1c. Adenovirus expressing green fluorescent protein (GFP) was used as control. The cells were harvested for nucleus protein after infection for 48 h. Electrophoretic mobility shift assay (EMSA) was performed using a Light Shift Chemiluminescent EMSA Kit. Chromatin immunoprecipitation assay was performed by CHIP Kit. The interaction between the transcription factor SREBP-1c and the promoter region of IRS-1 was assessed by above experiments. Differences among the groups were determined using two-way ANOVA. Results The luciferase deletion studies suggested the region from -450 to -210 bp on the IRS-1 promoter was a potential target region for SREBP-1c. Sequence analysis showed that the target region of the rat IRS-1 promoter gene contained a potential binding site (GCCTCCCGAG), which was located between -302 and -292 bp. PCR site- directed mutagenesis (TGTTAAATTA) was generated and analyzed using a luciferase assay. The transcriptional activity of wild-type IRS-1 promoter was dramatically down-regulated by SREBP-1c, whereas SREBP-1c had no effect on the IRS-1 promoter bearing mutation of SREBP-1c binding site(7.03±1.28 vs 19.09±2.45,3.55±1.68 vs 3.96±1.09,F=114.437,0.251,all P>0.05). The EMSA results showed that the labeled wild- type probe successfully formed a complex with nuclear proteins. But the unlabelled mutant probe did not interfere the complex. The results from CHIP assay showed that SREBP-1c could bind directly to the IRS- 1 promoter region. The quantity of SREBP- 1c protein binding to the IRS- 1 promoter was two times of control under PA conditions in L6 cells or five times of control when SREBP-1c was over-expressed (2.15±0.03 vs 1.07±0.24,5.48±1.28 vs 0.86±0.19,t=6.877,5.495,all P<0.05). Conclusion SREBP- 1c could directly bind to the atypical SRE sequence in the promoter region of IRS- 1, suppressing IRS- 1 expression and the subsequent insulin signaling pathway. [ABSTRACT FROM AUTHOR]

Both endothelin-1 (ET-1) and cAMP are implicated for inducing insulin resistance. Since we have shown previously that there is a crosstalk between ET-1 and cAMP signaling pathways in regulating glucose uptake in 3T3-L1 adipocytes, we extended our investigation in this study on whether they may have a synergistic effect on inducing insulin resistance. Our results showed that it was indeed the case. Insulin-stimulated glucose uptake, phosphorylation of PKB, IRS-1-associated PI3K, and IRS-1 tyrosine phosphorylation were all inhibited by ET-1 and 8-bromo cAMP in a synergistic manner. IRS-1 protein levels were similarly decreased by ET-1 and 8-bromo cAMP, attributable to suppressed mRNA expression. In addition, after correction for the loss in IRS-1 protein, the inhibition of insulin-stimulated IRS-1 tyrosine phosphorylation or IRS-1-associated PI3K was mainly caused by cAMP. Moreover, whereas IRS-2 protein levels were increased by cAMP or ET-1 plus cAMP, insulin-stimulated IRS-2-associated PI3K activities were abolished by both treatments. Furthermore, ET-1 and β-adrenergic agonists had similar synergistic inhibition on insulin-stimulated glucose uptake. In conclusion, we have shown that ET-1 and cAMP may synergistically induce insulin resistance in adipocytes via inhibiting IRS-1 expression as well as insulin-stimulated IRS-1/IRS-2 activities. [ABSTRACT FROM AUTHOR]

Excessive fluoride ion (F) intake promotes systemic metabolic alterations, such as a decrease in insulin secretion, hyperglycemia, and insulin resistance, similar to those observed in diabetes. Cardiovascular disease is responsible for a large degree of morbidity and mortality in individuals with diabetes, and dyslipidemia is considered to be one of the most important risk factors for the development of cardiovascular disease. This study aimed to evaluate the chronic effect of NaF on insulin signal transduction in liver and in muscle, and to determine the plasma concentrations of triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and very low-density lipoprotein cholesterol (VLDL-C). Seven-week-old castrated male Wistar rats were randomly distributed into a control group, which received 76.4 mg/ L NaCl in their drinking water, and a fluoride group, which received 54.9 mg/L NaF in their drinking water and F in their food pellets (estimated total F intake: 4.0 mg/kg body weight (bw)/day). After 42 days, the insulin receptor substrate-1 (IRS-1) serine phosphorylation status in the liver and muscle tissues and plasma concentrations of triglycerides, total cholesterol, HDL-C, LDL-C, VLDL-C and F were evaluated. The chronic treatment with F promoted an increase in the plasma concentrations of TG, TC, VLDL-C and F but no alteration in the plasma concentrations of HDL-C and LDLC or in the IRS-1 serine phosphorylation status in liver or muscle tissue. These results demonstrate that chronic treatment with NaF (4.0 mg/kg bw/day) promotes dyslipidemia in castrated rats but does not interfere with the IRS-1 serine phosphorylation status, one of the inhibitory pathways of the insulin signal in muscle and liver tissues. [ABSTRACT FROM AUTHOR]

The presence of Arg972 insulin receptor substrate-1 (IRS-1) is associated with impaired insulin/IRS-1 signaling to activate phosphatidylinositol-3 kinase (PI3K). Tumor necrosis factor-α (TNF-α), an inflammatory cytokine with a central role in the pathogenesis of rheumatoid arthritis (RA), induces apoptosis in osteoblasts, which are the principal cell type responsible for bone loss in RA. In our previous study, an association between Arg972 IRS-1 and a high risk and severity of RA was identified. In the present study, the effects of Arg972 IRS-1 and IRS-1 on TNF-α-induced apoptosis in human osteoblasts were examined. Normal and RA osteoblasts were stably transfected with Arg972 IRS-1 and IRS-1. In addition, cells were stably transduced with IRS-1-shRNA to knock down IRS-1. Following stimulation with 10 nM insulin for 30 min, the stable overexpression of Arg972 IRS-1 and knock down of IRS-1 significantly decreased IRS-1-associated PI3K activity and Akt activation/phosphorylation at serine 473 (ser473) and enhanced TNF-α-induced apoptosis in normal and in RA osteoblasts. By contrast, the stable overexpression of IRS-1 significantly increased the levels of IRS-1-associated PI3K activity and Akt phosphorylation (ser473) and inhibited TNF-α-induced apoptosis, which was eliminated by pretreatment with 50 μn BJM120, a selective PI3K inhibitor, for 30 min. In conclusion, the present study provided the first evidence, to the best of our knowledge, that insulin stimulation of Arg972 IRS-1 and IRS-1 enhanced and inhibited TNF-α-induced apoptosis, respectively in normal and RA osteoblasts by a PI3K-dependent mechanism. These findings suggest that insulin/IRS-1 signaling is important in the pathogenesis of RA. [ABSTRACT FROM AUTHOR]

Background: Insulin resistance is characterized by deficient responses to insulin in its target tissues. In the present study, we examined the effects of L-Citrulline (L-Cit) on insulin sensitivity and signaling cascades in rat hepatoma H4IIE cells and SHRSP.Z-Leprfa/IzmDmcr rats. Methods: H4IIE cells were pretreated in the presence or absence of 250 LJM L-Cit in serum-free medium and then incubated in the presence or absence of 0.1 nM insulin. Rats were allocated into 2 groups; a control group (not treated) and L-Cit group (2 g/kg/day, L-Cit) and treated for 8 weeks. Results: L-Cit enhanced the insulin-induced phosphorylation of Akt in H4IIE cells. Moreover, the inhibited expression of Dex/cAMP-induced PEPCK mRNA by insulin was enhanced by the L-Cit treatment. The phosphorylation of tyrosine, which is upstream of Akt, in insulin receptor substrate-1 (IRS-1) was increased by the L-Cit treatment. The L-Cit-induced enhancement in insulin signaling was not related to the binding affinity of insulin to the insulin receptor orto the expression of the insulin receptor, but to a decrease in the phosphorylation of serine 1101 in IRS-1. These results were also confirmed in animal experiments. In the livers of L-Cit-treated rats, PI3K/Akt signaling was improved by decreases in the phosphorylation of serine 1101. Conclusions: We herein demonstrated for the first time the beneficial effects of L-Cit on improved insulin resistance associated with enhanced insulin sensitivity. These results may have clinical applications for insulin resistance and the treatment of type-2 diabetes. [ABSTRACT FROM AUTHOR]

Here, we demonstrate that insulin receptor (IR) tyrosine kinase catalyzes Tyr-19 and Tyr-27 phosphorylation of caveolin-2 (cav-2), leading to stimulation of signaling proteins downstream of IR, and that the catalysis is dependent on fatty acylation status of cav-2, promoting its interaction with IR. Cav-2 is myristoylated at Gly-2 and palmitoylated at Cys-109, Cys-122, and Cys-145. The fatty acylation deficient mutants are unable to localize in the plasma membrane and not phosphorylated by IR tyrosine kinase. IR interacts with the C-terminal domain of cav-2 containing the cysteines for palmitoylation. IR mutants, Y999F and K1057A, but not W1220S, fail interaction with cav-2. Insulin receptor substrate-1 (IRS-1) is recruited to interact with the IR-catalyzed phospho-tyrosine cav-2, which facilitates IRS-1 association with and activation by IR to initiate IRS-1-mediated downstream signaling. Cav-2 fatty acylation and tyrosine phosphorylation are necessary for the IRS-1-dependent PI3K-Akt and ERK activations responsible for glucose uptake and cell survival and proliferation. In conclusion, fatty acylated cav-2 is a new substrate of IR tyrosine kinase, and the fatty acylation and phosphorylation of cav-2 present novel mechanisms by which insulin signaling is activated. [ABSTRACT FROM AUTHOR]

The current study aims to investigate the therapeutic potential of bone marrow-derived mesenchymal stem cells (MSCs) as a solo therapy in ameliorating both skin lesions and liver injury induced by cutaneous severe burn injury (SBI) in rats. In anesthetized male adult Wistar albino rats, 30 % total burn surface area and established hepatic injury was achieved via direct contact of each experimental animal's dorsum with heated metal rod (100 °C) for 10 s. On the next day following burn, human MSCs or mouse MSCs was administered locally around the burn site and intraperitonially (0.5 × 106 cells/rat for each route) and outcomes were investigated at 4 and 14 days following burn induction. Both types of MSCs significantly improved skin and liver histology, decreased liver enzymes, and ameliorated oxidative stress in hepatocytes of SBI-rats. Further, SBI-induced rises in hepatic apoptotic marker (caspase-3, Bax) and serum inflammatory markers (TNF-α, IL-1β, and IL-6) were reduced following either human or mouse MSC administration. In addition, MSCs augmented insulin receptor substrate-1, phosphorylated protein kinase-B (phospho-Akt), while alleviating serum glucose levels in SBI-rats. These previous effects persisted even at the 14-day time point. Following single administration, bone marrow-derived MSCs is capable of counteracting SBI-induced skin lesions as well as related hepatic complications, specifically via mitigating postburn hyperglycemia and hyperinflammation. [Display omitted] • Severe burn predisposes to hepatic injury. • Altered metabolism with insulin resistance is an outcome in severe burn injury. • Mesenchymal stem cells alleviated hepatic injury associated with severe burn. • Mesenchymal stem cells attenuated hepatic oxidative stress and apoptosis in severe burn. • Mesenchymal stem cells mitigated insulin resistance post severe burn. [ABSTRACT FROM AUTHOR]

Background: There is evidence that several messenger RNAs (mRNAs) are bifunctional RNAs, i.e. RNA transcript carrying both protein-coding capacity and activity as functional non-coding RNA via 5' and 3' untranslated regions (UTRs). Results: In this study, we identified a novel bifunctional RNA that is transcribed from insulin receptor substrate-1 (Irs-1) gene with full-length 5'UTR sequence (FL-Irs-1 mRNA). FL-Irs-1 mRNA was highly expressed only in skeletal muscle tissue. In cultured skeletal muscle C2C12 cells, the FL-Irs-1 transcript functioned as a bifunctional mRNA. The FL-Irs-1 transcript produced IRS-1 protein during differentiation of myoblasts into myotubes; however, this transcript functioned as a regulatory RNA in proliferating myoblasts. The FL-Irs-1 5'UTR contains a partial complementary sequence to Rb mRNA, which is a critical factor for myogenic differentiation. The overexpression of the 5'UTR markedly reduced Rb mRNA expression, and this reduction was fully dependent on the complementary element and was not compensated by IRS-1 protein. Conversely, knockdown of FL-Irs-1 mRNA increased Rb mRNA expression and enhanced myoblast differentiation into myotubes. Conclusions: Our findings suggest that the FL-Irs-1 transcript regulates myogenic differentiation as a regulatory RNA in myoblasts. [ABSTRACT FROM AUTHOR]