Thyroid-Stimulating Hormone Inhibits Insulin Receptor Substrate-1 Expression and Tyrosyl Phosphorylation in 3T3-L1 Adipocytes by Increasing NF-κB DNA-Binding Activity.

Background: Abundant evidence indicates that thyroid-stimulating hormone (TSH) levels are associated with insulin resistance in adipocytes. However, the potential mechanism of the association remains uncertain. The objective of this study was to determine the potential role of TSH in the suppression of insulin receptor substrate-1 (IRS-1) expression and IRS-1 tyrosyl phosphorylation, which might contribute to insulin resistance. Methods: Mouse 3T3-L1 preadipocytes were differentiated into adipocytes. After treatment with 0.01, 0.1, and 1.0 mIU/ml bovine TSH, the TNF-α concentration in the medium was determined by enzyme-linked immunosorbent assay (ELISA). Nuclear factor-kappa B (NF-κB) DNA-binding activity was quantified by electrophoretic mobility shift assay (EMSA). IRS-1 levels in adipocytes were quantified by Western blotting, and tyrosine phosphorylation was measured by immunoprecipitation. Results: TSH induced TNF-α secretion in a dose-dependent manner. There was a significant positive correlation between NF-κB DNA-binding activity and TNF-α secretion. This effect and correlation were weakened by BAY 11-7082 (a nuclear NF-κB inhibitor) and H89 (an inhibitor of cyclic adenosine monophosphate- (cAMP-) dependent protein kinase A (PKA)). Treatment of cultured adipocytes with TSH inhibited insulin-stimulated IRS-1 tyrosyl phosphorylation but promoted TSH-dependent secretion of TNF-α and activation of NF-κB DNA-binding activity. The effects of TSH were significantly inhibited by BAY 11-7082 and H89 and were completely blocked by the TNF-α antagonist WP9QY. Conclusion: TSH inhibited IRS-1 protein expression and tyrosyl phosphorylation in 3T3-L1 adipocytes by stimulating TNF-α production via promotion of NF-κB DNA-binding activity. TSH might play a pivotal role in the development of insulin resistance.

IRS4 promotes the progression of non-small cell lung cancer and confers resistance to EGFR-TKI through the activation of PI3K/Akt and Ras-MAPK pathways.

IRS4 is a member of the insulin receptor substrate (IRS) protein family. It acts as a cytoplasmic adaptor protein, integrating and transmitting signals from receptor protein tyrosine kinases to the intracellular environment. IRS4 can induce mammary tumorigenesis and is usually overexpressed in non-small cell lung cancer (NSCLC). However, little is known about the role of IRS4 in the development and progression of lung cancer. In this study, we show that IRS4 knockout suppresses the proliferation, colony formation, migration, and invasion of A549 lung cancer cells, as well as tumor growth in a nude mouse xenograft model. In contrast, stable expression of IRS4 showed the opposite effects. As expected, IRS4 was found to activate the PI3K/Akt and Ras-MAPK pathways, and we also showed that IRS4 depletion significantly enhanced the sensitivity of EGFR tyrosine kinase inhibitor (EGFR-TKI)-resistant cells to gefitinib. Taken together, these results show that IRS4 promotes NSCLC progression and may represent a potential therapeutic target for EGFR-TKI-resistant NSCLC.

SOX21-AS1 modulates neuronal injury of MMP+-treated SH-SY5Y cells via targeting miR-7-5p and inhibiting IRS2.

Parkinson's disease (PD), caused by the decreased number of dopaminergic neurons in the substantia nigra, is identified as the second most familiar age-dependent neurodegenerative disease to the public. Long non-coding RNAs (lncRNAs) have been reported to participate in the development of PD. In our research, the expression of lncRNA SRY-box transcription factor 21 antisense divergent transcript 1 (SOX21-AS1) was up-regulated in 1-methyl-4-phenylpyridinium (MMP+)-treated SH-SY5Y cells. In addition, SOX21-AS1 depletion weakened the cell injury induced by MMP+. Moreover, SOX21-AS1 knockdown decreased Reactive Oxygen Species (ROS) generation and levels of TNF-α, IL-1ß and IL-6, but increased SOD activity. However, SOX21-AS1 up-regulation led to opposite results. Further, SOX21-AS1 could bind with miR-7-5p, whose overexpression relieved MMP+-induced cell injury. Additionally, insulin receptor substrate 2 (IRS2) served as the target gene of miR-7-5p, and its expression was positively modulated by SOX21-AS1. Similarly, IRS2 knockdown also had alleviative effects on cell injury stimulated by MMP+ treatment. In sum up, our study demonstrated a new regulatory network consisted of SOX21-AS1, miR-7-5p and IRS2 in SH-SY5Y cells, supplying with a better comprehension about the pathogenic mechanism of PD.

Effects of supplementing different chromium histidinate complexes on glucose and lipid metabolism and related protein expressions in rats fed a high-fat diet.

BACKGROUND: The objective of this study was to investigate the effects of different chromium histidinate (CrHis) complexes added to the diet of rats fed a high-fat diet (HFD) on body weight changes, glucose and lipid metabolism parameters, and changes in biomarkers such as PPAR-γ, IRS-1, GLUTs, and NF-κB proteins. METHODS: Forty-two Sprague-Dawley rats were divided equally into six groups and fed either a control, an HFD, or an HFD supplemented with either CrHis1, CrHis2, CrHis3, or a combination of the CrHis complexes as CrHisM. RESULTS: Feeding an HFD to rats increased body weights, HOMA-IR values, fasting serum glucose, insulin, leptin, free fatty acid, total cholesterol, low-density lipoprotein cholesterol, and MDA concentrations as well as AST activities, and decreased serum and brain serotonin concentrations compared with rats fed a control diet (P < 0.0001). The levels of the PPAR-γ, IRS-1, and GLUTs in the liver and brain decreased, while NF-κB level increased, with feeding an HFD (P < 0.05). Although all the CrHis supplements reversed the negative effects of feeding an HFD (P < 0.05), the CrHis1 complex was most effective in changing the protein levels, while CrHisM was most effective in influencing certain parameters such as body weight and serum metabolites. CONCLUSION: The results of the present work suggest that the CrHis1 complex is most potent for alleviating the negative effects of feeding an HFD. The efficacy of CrHisM is likely due to the presence of the CrHis1 complex.

NT157, an IGF1R-IRS1/2 inhibitor, exhibits antineoplastic effects in pre-clinical models of chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is successfully treated with BCR-ABL1 tyrosine kinase inhibitors, but a significant percentage of patients develop resistance. Insulin receptor substrate 1 (IRS1) has been shown to constitutively associate with BCR-ABL1, and IRS1-specific silencing leads to antineoplastic effects in CML cell lines. Here, we characterized the efficacy of NT157, a pharmacological inhibitor of IGF1R-IRS1/2, in CML cells and observed significantly reduced cell viability and proliferation, accompanied by induction of apoptosis. In human K562 cells and in murine Ba/F3 cells, engineered to express either wild-type BCR-ABL1 or the imatinib-resistant BCR-ABL1T315I mutant, NT157 inhibited BCR-ABL1, IGF1R, IRS1/2, PI3K/AKT/mTOR, and STAT3/5 signaling, increased CDKN1A, FOS and JUN tumor suppressor gene expression, and reduced MYC and BCL2 oncogenes. NT157 significantly reduced colony formation of human primary CML cells with minimal effect on normal hematopoietic cells. Exposure of primary CML cells harboring BCR-ABL1T315I to NT157 resulted in increased apoptosis, reduced cell proliferation and decreased phospho-CRKL levels. In conclusion, NT157 has antineoplastic effects on BCR-ABL1 leukemogenesis, independent of T315I mutational status.

Berberine promotes peri-implant osteogenesis in diabetic rats by ROS-mediated IRS-1 pathway.

Accompanying with diabetes mellitus-induced osteoporosis (DM-OS), diabetic patients show poor peri-implant osteogenesis after implantation for dentition defect. Berberine (BBR), a candidate oral hypoglycemic agent, is a promising agent for treating DM-OS. In this study, BBR was applied on DM rats and high-glucose-cultured bone mesenchymal stem cells (BMSCs) to investigate its therapeutic mechanism on DM-OS, thus laying a theoretical basis for the future application of BBR in implant restoration. Phenotypes were assessed in the DM rats after 4 w of gavage with BBR. Furthermore, BMSCs were cultured with high glucose and BBR. Cell Counting Kit-8, 2',7'-dichlorofluorescin diacetate (H2 DCF-DA), quantitative real-time PCR (qRT-PCR), and western blot were performed to estimate the cell proliferation, oxidative stress, and osteogenic differentiation. Moreover, the DM rats treated with BBR and insulin receptor substrate-1 anti-sense oligonucleotide (IRS-1-ASO) underwent a 4-w implant-healing period and then micro computed tomography (Micro-CT) and histology were performed to verify the mechanism. Results showed that the 4-w administration of BBR markedly improved the glucose metabolism and bone metabolism in the DM rats. in vitro experiments revealed that BBR alleviated high-glucose-inhibited osteogenesis of the BMSCs by upregulating reactive oxygen species (ROS)-mediated IRS-1 signaling. Besides, injection of IRS-1-ASO abolished the BBR promotion of implant osseointegration in the DM rats. In conclusion, targeting ROS-mediated IRS-1 signaling, BBR acted as an efficient agent to advance osseointegration in DM, which indicated that BBR use is a good strategy for future implants restoration in diabetic patients.

Saturated fatty acids induce insulin resistance in podocytes through inhibition of IRS1 via activation of both IKKß and mTORC1.

Diabetic nephropathy (DN), a microvascular complication of diabetes, is the leading cause of end-stage renal disease worldwide. Multiple studies have shown that podocyte dysfunction is a central event in the progression of the disease. Beside chronic hyperglycemia, dyslipidemia can induce insulin resistance and dysfunction in podocytes. However, the exact mechanisms of free fatty acid (FFA)-induced podocyte insulin unresponsiveness are poorly understood. We used a type 2 diabetic mouse model (db/db) and mouse podocytes exposed to palmitic acid for 24 h followed by an insulin stimulation. Renal function and pathology were evaluated at 25 weeks of age to confirm the DN development. Our results demonstrate that saturated FFA activated the serine/threonine kinases IκB kinase (IKK)ß/IκBα and mTORC1/S6K1, but not protein kinase C and c-jun N-terminal kinase, in podocytes and glomeruli of db/db mice. Activation of both kinases promoted serine 307 phosphorylation of IRS1, a residue known to provoke IRS1 inhibition. Using IKK, mTORC1 and ceramide production inhibitors, we were able to blunt IRS1 serine 307 phosphorylation and restore insulin stimulation of Akt. In conclusion, our results indicate that FFA and diabetes contribute to insulin resistance through the activation of IKKß and S6K1 leading to podocyte dysfunction and DN.

NT157 has antineoplastic effects and inhibits IRS1/2 and STAT3/5 in JAK2V617F-positive myeloproliferative neoplasm cells.

Recent data indicate that IGF1R/IRS signaling is a potential therapeutic target in BCR-ABL1-negative myeloproliferative neoplasms (MPN); in this pathway, IRS2 is involved in the malignant transformation induced by JAK2V617F, and upregulation of IGF1R signaling induces the MPN phenotype. NT157, a synthetic compound designed as an IGF1R-IRS1/2 inhibitor, has been shown to induce antineoplastic effects in solid tumors. Herein, we aimed to characterize the molecular and cellular effects of NT157 in JAK2V617F-positive MPN cell lines (HEL and SET2) and primary patient hematopoietic cells. In JAK2V617F cell lines, NT157 decreased cell viability, clonogenicity, and cell proliferation, resulting in increases in apoptosis and cell cycle arrest in the G2/M phase (p < 0.05). NT157 treatment inhibited IRS1/2, JAK2/STAT, and NFκB signaling, and it activated the AP-1 complex, downregulated four oncogenes (CCND1, MYB, WT1, and NFKB1), and upregulated three apoptotic-related genes (CDKN1A, FOS, and JUN) (p < 0.05). NT157 induced genotoxic stress in a JAK2/STAT-independent manner. NT157 inhibited erythropoietin-independent colony formation in cells from polycythemia vera patients (p < 0.05). These findings further elucidate the mechanism of NT157 action in a MPN context and suggest that targeting IRS1/2 proteins may represent a promising therapeutic strategy for MPN.

MiRNA-200a-3p suppresses the proliferation, migration and invasion of non-small cell lung cancer through targeting IRS2.

OBJECTIVE: To uncover the biological role of microRNA-200a-3p (miRNA-200a-3p) in the progression of non-small cell lung cancer (NSCLC) and the underlying mechanism. PATIENTS AND METHODS: The expression levels of miRNA-200a-3p and IRS2 in NSCLC tissues and cell lines were examined through quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The correlation between the miRNA-200a-3p level and pathological characteristics of NSCLC patients was analyzed. The prognostic value of miRNA-200a-3p in NSCLC was assessed through the Kaplan-Meier method. The potential interaction between miRNA-200a-3p and IRS2 was explored through Dual-Luciferase Reporter Gene Assay and Spearman correlation test. The regulatory effects of miRNA-200a-3p/IRS2 on the proliferative, migratory, and invasive abilities of NSCLC were evaluated by Cell Counting Kit-8 (CCK-8) and the transwell assay. The protein levels of the epithelial-mesenchymal transition (EMT)-related genes in NSCLC cells influenced by miRNA-200a-3p were detected by Western blot. RESULTS: MiRNA-200a-3p was downregulated in NSCLC tissues and cell lines. The expression level of miRNA-200a-3p was related to tumor size, TNM staging, and lymphatic metastasis of NSCLC. The low level of miRNA-200a-3p predicted worse prognosis in NSCLC patients. The overexpression of miRNA-200a-3p inhibited A549 cells from proliferating, migrating, and invading. The protein levels of E-cadherin were upregulated, while N-cadherin and Vimentin were downregulated in A549 cells overexpressing miRNA-200a-3p. The Dual-Luciferase Reporter Gene Assay verified the binding between miRNA-200a-3p and IRS2. The level of IRS2 was negatively regulated by miRNA-200a-3p. Moreover, the overexpression of IRS2 could reverse the regulatory role of miRNA-200a-3p in the cellular behaviors of A549 cells. CONCLUSIONS: MiRNA-200a-3p suppresses the proliferative, migratory, and invasive abilities of NSCLC by targeting IRS2, thus alleviating the progression of NSCLC.

IGF1R/IRS1 targeting has cytotoxic activity and inhibits PI3K/AKT/mTOR and MAPK signaling in acute lymphoblastic leukemia cells.

The IGF1R/IRS1 signaling is activated in acute lymphoblastic leukemia (ALL) and can be targeted by the pharmacological inhibitors NT157 (IGF1R-IRS1/2 inhibitor) and OSI-906 (IGF1R/IR inhibitor). Here we investigate the cellular and molecular effects of NT157 and OSI-906 in ALL cells. NT157 and OSI-906 treatment reduced viability, proliferation and cell cycle progression in ALL cell lines. Similarly, in primary samples of patients with ALL, both OSI-906 and NT157 reduced viability, but only NT157 induced apoptosis. NT157 and OSI-906 did not show cytotoxicity in primary samples from healthy donor. NT157 and OSI-906 significantly decreased Jurkat cell migration, but did not modulate Namalwa migration. Consistent with the more potent effect of NT157 on cells, NT157 significantly modulated expression of 25 genes related to the MAPK signaling pathway in Jurkat cells, including oncogenes and tumor suppressor genes. Both compounds inhibited mTOR and p70S6K activity, but only NT157 inhibited AKT and 4-EBP1 activation. In summary, in ALL cells, NT157 has cytotoxic activity, whereas OSI-906 is cytostatic. NT157 has a stronger effect on ALL cells, and thus the direct inhibition of IRS1 may be a potential therapeutic target in ALL.

MicroRNA-128 confers protection against cardiac microvascular endothelial cell injury in coronary heart disease via negative regulation of IRS1.

OBJECTIVE: Cardiac microvascular endothelial cells (CMECs) play a critical role in the physiological regulation of coronary blood flow and its dysfunction is associated with myocardium ischemic injury. This study was performed to clarify the effect of microRNA-128 (miR-128) on the CMEC injury in coronary heart disease (CHD) by binding to insulin receptor substrate 1 (IRS1). METHODS: The rat CMECs were cultured by explant culture method and identified by CD31 immunofluorescence assay. CMECs were treated with homocysteine (Hcy), which underwent stress of CHD, followed by treatment of miR-128 mimics/inhibitors or IRS1 siRNA. Expression of miR-128, IRS1, and vascular endothelial growth factor (VEGF) was determined. The viability, apoptosis, migration ability, and tube formation ability of CMECs were evaluated. The superoxide dismutase (SOD), malondialdehyde (MDA), and reactive oxygen species (ROS) of CMECs were evaluated, respectively. RESULTS: In rat CMECs, miR-128 was poorly expressed and IRS1 was highly expressed. Notably, miR-128 targeted and negatively regulated IRS1. Additionally, the treatment with Hcy in CMECs led to reduced viability, migration ability, tube formation, VEGF expression, SOD activity as well as increased cell apoptosis, MDA and ROS levels. The experimental results demonstrated that miR-128 mimics and IRS1 siRNA in rat CMECs promoted viability, migration ability, tube formation, VEGF expression, SOD activity, while repressing cell apoptosis, MDA and ROS levels. MiR-128 inhibitors could reverse the tendencies. CONCLUSION: Collectively, our study provides evidence that miR-128 targeted and negatively regulated IRS1 expression, whereby the functional injury of CMECs induced by Hcy was ameliorated. Furthermore, protection of miR-128 was stimulated by reducing oxidative stress.

miR-431 inhibits adipogenic differentiation of human bone marrow-derived mesenchymal stem cells via targeting insulin receptor substance 2.

BACKGROUND: An understanding of the mechanism underlying adipogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) will provide new therapeutic approaches for many diseases, including osteoporosis. This study aimed to investigate the role of miR-431 in adipogenic differentiation of hMSCs. METHODS: hMSCs were induced for adipogenic differentiation and miR-431 was detected by polymerase chain reaction (PCR). hMSCs were transfected by miR-431 or small interfering RNA (siRNA) for insulin receptor substance 2 (IRS2). The expression of IRS2 was detected by PCR and Western blot analysis. The targeting of the 3'-untranslated region (UTR) of IRS2 by miR-431 was examined by luciferase assay. RESULTS: miR-431 expression was decreased during adipogenesis of hMSCs. Overexpression of miR-431 inhibited adipogenic differentiation, accompanied by the downregulation of CCAAT/enhancer binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ), two key regulators of adipogenesis. Moreover, miR-431 decreased both protein and mRNA levels of IRS2. The expression of IRS2 was increased during adipogenic differentiation of hMSCs in conjunction with decreased levels of miR-431, and knockdown of IRS2 in hMSCs inhibited adipogenic differentiation. Luciferase assay confirmed that miR-431 targeted the 3'-UTR of IRS2 in hMSCs. CONCLUSIONS: This is the first study to show that miR-431 inhibits adipogenic differentiation of hMSCs via targeting IRS2.

Therapeutic Targeting of TFE3/IRS-1/PI3K/mTOR Axis in Translocation Renal Cell Carcinoma.

PURPOSE: Translocation renal cell carcinoma (tRCC) represents a rare subtype of kidney cancer associated with various TFE3, TFEB, or MITF gene fusions that are not responsive to standard treatments for RCC. Therefore, the identification of new therapeutic targets represents an unmet need for this disease. EXPERIMENTAL DESIGN: We have established and characterized a tRCC patient-derived xenograft, RP-R07, as a novel preclinical model for drug development by using next-generation sequencing and bioinformatics analysis. We then assessed the therapeutic potential of inhibiting the identified pathway using in vitro and in vivo models. RESULTS: The presence of a SFPQ-TFE3 fusion [t(X;1) (p11.2; p34)] with chromosomal break-points was identified by RNA-seq and validated by RT-PCR. TFE3 chromatin immunoprecipitation followed by deep sequencing analysis indicated a strong enrichment for the PI3K/AKT/mTOR pathway. Consistently, miRNA microarray analysis also identified PI3K/AKT/mTOR as a highly enriched pathway in RP-R07. Upregulation of PI3/AKT/mTOR pathway in additional TFE3-tRCC models was confirmed by significantly higher expression of phospho-S6 (P < 0.0001) and phospho-4EBP1 (P < 0.0001) in established tRCC cell lines compared with clear cell RCC cells. Simultaneous vertical targeting of both PI3K/AKT and mTOR axis provided a greater antiproliferative effect both in vitro (P < 0.0001) and in vivo (P < 0.01) compared with single-node inhibition. Knockdown of TFE3 in RP-R07 resulted in decreased expression of IRS-1 and inhibited cell proliferation. CONCLUSIONS: These results identify TFE3/IRS-1/PI3K/AKT/mTOR as a potential dysregulated pathway in TFE3-tRCC, and suggest a therapeutic potential of vertical inhibition of this axis by using a dual PI3K/mTOR inhibitor for patients with TFE3-tRCC.

An MG53-IRS1-interaction disruptor ameliorates insulin resistance.

Mitsugumin 53 (MG53) is an E3 ligase that induces insulin receptor substrate-1 (IRS-1) ubiquitination and degradation in skeletal muscle. We previously demonstrated that the pharmaceutical disruption of the MG53-IRS-1 interaction improves insulin sensitivity by abrogating IRS-1 ubiquitination and increasing IRS-1 levels in C2C12 myotubes. Here, we developed a novel MG53-IRS-1 interaction disruptor (MID-00935) that ameliorates insulin resistance in diet-induced obese (DIO) mice. MID-00935 disrupted the molecular interaction of MG53 and IRS-1, abrogated MG53-induced IRS-1 ubiquitination and degradation and improved insulin signaling in C2C12 myotubes. Oral administration of MID-00935 increased insulin-induced IRS-1, Akt, and Erk phosphorylation via increasing IRS-1 levels in the skeletal muscle of DIO mice. In DIO mice, MID-00935 treatment lowered fasting blood glucose levels and improved glucose disposal in glucose and insulin tolerance tests. These results suggest that MID-00935 may be a potential muscle-targeting drug candidate for treating insulin resistance.

Microcystis bloom containing microcystin-LR induces type 2 diabetes mellitus.

Epidemiological data from Lake Taihu showed significantly higher incidences of type 2 diabetes mellitus (T2DM) than in other areas of China. This may be related to the occurrence of a Microcystis bloom in Lake Taihu in the summer and autumn every year. The objective of this study is to investigate whether the contaminated water from the Microcystis bloom and the derivative pollutant microcystin-LR (MC-LR) can explain the higher incidences of T2DM. Healthy male mice were fed with water from different regions of Lake Taihu, and were either acutely or chronically exposed to MC-LR through oral administration or intraperitoneal injection. Serum lipid profiles were determined, and the effects on T2DM-related gene expression and insulin receptor signaling pathway were investigated. Intraperitoneal glucose tolerance (IPGTT) and insulin resistance (IRT) tests were implemented, and the functions of pancreatic islet and ß-cell were also evaluated. The results showed that both water sampled from the region with a Microcysis bloom and those containing MC-LR altered the serum glucide and lipid profiles in mice after exposure. The exposure to a Microcysis bloom water affected the expression T2DM-related genes: up-regulated the mRNA levels of FASn, ACACA, G6pc, LPL, and Insig2, and down-regulated the mRNA level of PEPCK and Gsk-3ß. Both acute and chronic exposure of MC-LR, even at very low concentrations (1 µg/L), impaired the insulin receptor signalling pathway and induced hyperinsulinemia and insulin resistance in mice. In this study, the most important intracellular target of MC-LR was found to be hetapocellular mitochondria. Thus, exposure to Microcystis bloom water containing microcystin-LR can induce the incidence of T2DM, by impairing the function of mitochondria by microcystin-LR. The study suggests a review of the risk assessment concerning 1 µg/L MC-LR as the reference dose in surface water.

Liraglutide ameliorates palmitate-induced insulin resistance through inhibiting the IRS-1 serine phosphorylation in mouse skeletal muscle cells.

OBJECTIVE: A reduction in insulin-stimulated glucose uptake in skeletal muscles is a characteristic of insulin resistance and type 2 diabetes mellitus (T2DM). The glucagon-like peptide (GLP)-1 agonist liraglutide can reduce blood glucose levels in individuals with T2DM. However, its effect on insulin-induced glucose metabolism in the skeletal muscle of insulin resistance is unknown. We investigated the effects and action mechanisms of liraglutide on insulin resistance (IR) in the skeletal muscle cells treatment with palmitic acid (PA). METHODS: The cell-surface GLUT4myc levels were determined by an antibody-coupled colorimetric assay. The phosphorylation levels of Akt, PI3K(p85α), AS160, IRS1, IKK, and JNK were determined by western blotting. The quantifications of mRNA levels of TNFα, IL-1ß, and IL-6 were determined by real-time PCR. Analysis of variance was used for data analysis. RESULTS: PA elevated not only phosphorylation of JNK, IRS1 serines, and IKKα/ß, but also the expression of IL-6, TNFα and IL-1ß in C2C12-GLUT4myc cells. PA can reduce phosphorylation of IRS1 tyrosine. These effects of PA were reversed by liraglutide. In addition, liraglutide can reverse PA-decreased insulin-stimulated cell-surface GLUT4 levels, Akt, PI3K(p85α), and AS160 phosphorylation. CONCLUSIONS: Liraglutide can enhance insulin-induced GLUT4 translocation by inhibiting IRS1 serine phosphorylation in PA-treated muscle cells.

Upregulation of IRS1 Enhances IGF1 Response in Y537S and D538G ESR1 Mutant Breast Cancer Cells.

Increased evidence suggests that somatic mutations in the ligand-binding domain of estrogen receptor [ER (ERα/ESR1)] are critical mediators of endocrine-resistant breast cancer progression. Insulinlike growth factor-1 (IGF1) is an essential regulator of breast development and tumorigenesis and also has a role in endocrine resistance. A recent study showed enhanced crosstalk between IGF1 and ERα in ESR1 mutant cells, but detailed mechanisms are incompletely understood. Using genome-edited MCF-7 and T47D cell lines harboring Y537S and D538G ESR1 mutations, we characterized altered IGF1 signaling. RNA sequencing revealed upregulation of multiple genes in the IGF1 pathway, including insulin receptor substrate-1 (IRS1), consistent in both Y537S and D538G ESR1 mutant cell line models. Higher IRS1 expression was confirmed by quantitative reverse transcription polymerase chain reaction and immunoblotting. ESR1 mutant cells also showed increased levels of IGF-regulated genes, reflected by activation of an IGF signature. IGF1 showed increased sensitivity and potency in growth stimulation of ESR1 mutant cells. Analysis of downstream signaling revealed the phosphoinositide 3-kinase (PI3K)-Akt axis as a major pathway mediating the enhanced IGF1 response in ESR1 mutant cells. Decreasing IRS1 expression by small interfering RNA diminished the increased sensitivity to IGF1. Combination treatment with inhibitors against IGF1 receptor (IGF1R; OSI-906) and ER (fulvestrant) showed synergistic growth inhibition in ESR1 mutant cells, particularly at lower effective concentrations. Our study supports a critical role of enhanced IGF1 signaling in ESR1 mutant cell lines, pointing toward a potential for cotargeting IGF1R and ERα in endocrine-resistant breast tumors with mutant ESR1.

Novel all-hydrocarbon stapled p110α[E545K] peptides as blockers of the oncogenic p110α[E545K]-IRS1 interaction.

To follow up on our recent discovery of the 18-amino acid all-hydrocarbon [i, i + 4]-stapled p110α[E545K] peptide 1 that was shown to potently block the intracellular p110α[E545K]-IRS1 interaction (a protein-protein interaction uniquely present in cancer cells expressing p110α[E545K]) and the growth of the xenograft tumors formed by cancers harboring this mutation, in the current study we prepared and examined six derivatives of 1, i.e. stapled peptides 2-A, 2-B, 3-A, 3-B, 4-A, 4-B. We found that 2-A, 2-B, 4-A, and 4-B had higher % α-helicity than 1; moreover, the enhanced % α-helicity also led to an enhanced proteolytic stability. When compared with 1, the structurally simplified 14-amino acid 4-A and 4-B were found to more potently deactivate the AKT phosphorylation at Ser473 in the p110α[E545K]-expressing colon cancer cells, whose activation was previously demonstrated by us to be specifically derived from the p110α[E545K]-IRS1 interaction. The preliminary findings from the current study have laid a foundation for future more extensive studies on the stapled p110α[E545K] peptides newly identified in the current study.

Involvement of Insulin Signaling Disturbances in Bisphenol A-Induced Alzheimer's Disease-like Neurotoxicity.

Bisphenol A (BPA), a member of the environmental endocrine disruptors (EDCs), has recently received increased attention because of its effects on brain insulin resistance. Available data have indicated that brain insulin resistance may contribute to neurodegenerative diseases. However, the associated mechanisms that underlie BPA-induced brain-related outcomes remain largely unknown. In the present study, we identified significant insulin signaling disturbances in the SH-SY5Y cell line that were mediated by BPA, including the inhibition of physiological p-IR Tyr1355 tyrosine, p-IRS1 tyrosine 896, p-AKT serine 473 and p-GSK3α/ß serine 21/9 phosphorylation, as well as the enhancement of IRS1 Ser307 phosphorylation; these effects were clearly attenuated by insulin and rosiglitazone. Intriguingly, Alzheimer's disease (AD)-associated pathological proteins, such as BACE-1, APP, ß-CTF, α-CTF, Aß 1-42 and phosphorylated tau proteins (S199, S396, T205, S214 and S404), were substantially increased after BPA exposure, and these effects were abrogated by insulin and rosiglitazone treatment; these findings underscore the specific roles of insulin signaling in BPA-mediated AD-like neurotoxicity. Thus, an understanding of the regulation of insulin signaling may provide novel insights into potential therapeutic targets for BPA-mediated AD-like neurotoxicity.

Downregulation of miR-493 promoted melanoma proliferation by suppressing IRS4 expression.

Accumulating evidence indicated that aberrantly expressed microRNAs play critical roles in the initiation and progression of human cancers. However, the underlying functions of miR-493 in human melanoma remains unknown. Here, our study found that miR-493 expression was downregulated in human melanoma tissues and cells. Overexpression of miR-493 suppressed cell proliferation and cell cycle in human melanoma cell line A375. IRS4 was defined as a target for downregulation by miR-493 and was confirmed by luciferase assay. We also found that knockdown of IRS4 counteracted the proliferation promotion by miR-493 inhibitor. In summary, these results demonstrated that miR-493 acts as a tumor suppressor and inhibits cell proliferation and cell cycle in human melanoma by directly targeting IRS4.