Tobacco Smoke Exposure Impairs Brain Insulin/IGF Signaling: Potential Co-Factor Role in Neurodegeneration

Background: Human studies suggest tobacco smoking is a risk factor for cognitive impairment and neurodegeneration, including Alzheimer’s disease (AD). However, experimental data linking tobacco smoke exposures to underlying mediators of neurodegeneration, including impairments in brain insulin and insulin-like growth factor (IGF) signaling in AD are lacking. Objective: This study tests the hypothesis that cigarette smoke (CS) exposures can impair brain insulin/IGF signaling and alter expression of AD-associated proteins. Methods: Adult male A/J mice were exposed to air for 8 weeks (A8), CS for 4 or 8 weeks (CS4, CS8), or CS8 followed by 2 weeks recovery (CS8+R). Gene expression was measured by qRT-PCR analysis and proteins were measured by multiplex bead-based or direct binding duplex ELISAs. Results: CS exposure effects on insulin/IGF and insulin receptor substrate (IRS) proteins and phosphorylated proteins were striking compared with the mRNA. The main consequences of CS4 or CS8 exposures were to significantly reduce insulin R, IGF-1R, IRS-1, and tyrosine phosphorylated insulin R and IGF-1R proteins. Paradoxically, these effects were even greater in the CS8+R group. In addition, relative levels of S312-IRS-1, which inhibits downstream signaling, were increased in the CS4, CS8, and CS8+R groups. Correspondingly, CS and CS8+R exposures inhibited expression of proteins and phosphoproteins required for signaling through Akt, PRAS40, and/or p70S6K, increased AβPP-Aβ, and reduced ASPH protein, which is a target of insulin/IGF-1 signaling. Conclusion: Secondhand CS exposures caused molecular and biochemical abnormalities in brain that overlap with the findings in AD, and many of these effects were sustained or worsened despite short-term CS withdrawal.

Phosphorylated and O-GlcNAc Modified IRS-1 (Ser1101) and -2 (Ser1149) Contribute to Human Diabetes type II

Background: The prevalence of the chronic metabolic disorder Type 2 diabetes mellitus (T2DM) is increasing steadily, and has even turned into an epidemic in some countries. T2DM results from defective responses to insulin and obesity is a major factor behind insulin resistance in T2DM. Insulin receptor substrate (IRS) proteins are adaptor proteins in the insulin receptor signalling pathway. The insulin signalling is controlled through tyrosine phosphorylation of IRS-1 and IRS2, and dysregulation of IRS proteins signalling may lead to glucose intolerance and eventually insulin resistance. Objective: In this work, we suggest that both glycosylation (O-GlcNAc modification) and phosphorylation of IRS-1 and -2 are involved in the pathogenesis of T2DM. Methods: Phosphorylation and O-GlcNAc modifications (Ser1101 in IRS-1 and Ser1149 in IRS-2) proteins were determined experimentally by sandwich ELISA with specific antibodies and with bioinformatics tools. Results: When IRS-1 (on Ser1101) and IRS-2 (Ser1149) become glycosylated following an increase in UDP-GlcNAc pools, it may contribute to insulin resistance. Whereas when the same (IRS-1 on Ser1101 and IRS-2 on Ser1149) are phosphorylated, the insulin signalling is inhibited. Discussion: In this work OGlcNAc-modified proteins were specifically detected using O-GlcNAc-specific antibodies, suggesting that elevated levels of O-GlcNAc-modified proteins are found, independently of their possible involvement in AGEs. Conclusion: This study suggests a mechanism, which is controlled by posttranslational modifications, and may contribute to the pathogenesis of type II diabetes.

Potential protection against type 2 diabetes in obesity through lower CD36 expression and improved exocytosis in β-cells

<div>Obesity is a risk factor for type 2 diabetes (T2D), however not all obese individuals develop the disease. In this study, we aimed to investigate the cause of differential insulin secretion capacity of pancreatic islets from T2D and non-T2D (ND) especially obese donors (BMI ≥30 kg/m2). Islets from obese T2D donors had reduced insulin secretion, decreased B-cell exocytosis and higher expression of fatty acid translocase CD36. We tested the hypothesis that CD36 is a key molecule in the reduced insulin secretion capacity. Indeed, CD36 overexpression led to decreased insulin secretion, impaired exocytosis and reduced granule docking. This was accompanied with reduced expression of the exocytotic proteins, SNAP25, STXBP1 and VAMP2, likely because CD36 induced down-regulation of the IRS proteins, suppressed insulin signaling PI3K-AKT pathway and increased nuclear localization of the transcription factor FoxO1. CD36 antibody treatment of the human B-cell line, EndoC-BH1, increased IRS1 and exocytotic protein levels, improved granule docking and enhanced insulin secretion. Our results demonstrate that B-cells from obese T2D donors have dysfunctional exocytosis likely due to an abnormal lipid handling represented by differential CD36 expression. Hence, CD36 could be a key molecule to limit B-cell function in T2D associated with obesity. <br></div><div> </div>

Potential protection against type 2 diabetes in obesity through lower CD36 expression and improved exocytosis in β-cells

<div>Obesity is a risk factor for type 2 diabetes (T2D), however not all obese individuals develop the disease. In this study, we aimed to investigate the cause of differential insulin secretion capacity of pancreatic islets from T2D and non-T2D (ND) especially obese donors (BMI ≥30 kg/m2). Islets from obese T2D donors had reduced insulin secretion, decreased B-cell exocytosis and higher expression of fatty acid translocase CD36. We tested the hypothesis that CD36 is a key molecule in the reduced insulin secretion capacity. Indeed, CD36 overexpression led to decreased insulin secretion, impaired exocytosis and reduced granule docking. This was accompanied with reduced expression of the exocytotic proteins, SNAP25, STXBP1 and VAMP2, likely because CD36 induced down-regulation of the IRS proteins, suppressed insulin signaling PI3K-AKT pathway and increased nuclear localization of the transcription factor FoxO1. CD36 antibody treatment of the human B-cell line, EndoC-BH1, increased IRS1 and exocytotic protein levels, improved granule docking and enhanced insulin secretion. Our results demonstrate that B-cells from obese T2D donors have dysfunctional exocytosis likely due to an abnormal lipid handling represented by differential CD36 expression. Hence, CD36 could be a key molecule to limit B-cell function in T2D associated with obesity. <br></div><div> </div>

Pharmacological IGF1R/IRS Inhibitor, NT157, Effectively Induces Apoptosis and CDKN1A Expression in Acute Lymphoblastic Leukemia Cells

Background: Acute lymphoblastic leukemia (ALL) is an aggressive cancer of immature progenitors that shows aberrant activation of signaling pathways. The IGF1/IGF1R signaling pathway is initiated through binding of the ligand (IGF1) to its transmembrane receptor (IGF1R), and subsequent activation of IRS1 and IRS2 proteins activating PI3K/Akt/mTOR and MAPK pathways, which are important signaling pathways reported to contributes to pathogenesis of ALL. However, the therapeutic potential of IGF1R/IRS signaling has not been previously studied in ALL cells. Aims: We herein aimed to investigate the effects of the pharmacological IGF1R/IR and IRS1/2 inhibition in ALL cells. Materials and Methods: T-ALL Jurkat and MOLT4, and B-ALL Namalwa and Raji cell lines were used. Peripheral blood or bone marrow mononuclear cells from ALL patients (T-ALL [n=2] and B-ALL [n=2]) at the time of diagnosis or relapse were used for functional studies and compared with health donors (n=2). Cell lines were treated or not with the IRS1/2 pharmacological inhibitor NT157, at 0.2, 0.4, 0.8, 1.6 and 3.2 µM, or with the IGF1R/IR inhibitor OSI-906, at 1, 5, 10 and 20 µM for 24, 48 and 72 hours. After drug exposure, cell lines were evaluated for cell viability (MTT assay), apoptosis (annexin V/PI and cleavage caspase 3), clonogenicity (colony forming assay), and protein expression/activation (Western blot) and PCR-array for MAPK signaling. Primary ALL cells were culture with IL7 (100 ng/mL), IL3 (30 ng/mL), SCF (30 ng/mL) and FLT3L (100 ng/mL) in the presence or not of NT157 or OSI-906 for 72 hours and them submitted to cell viability and apoptosis assays. Statistical analyzes were performed by the ANOVA or Student t test. P value <0.05 was considered statistically significant. Results: In ALL cell lines, NT157 treatment above 0.4 µM at 48 hours and 72 hours decreased cell viability in a dose and time-dependent manner (all p<0.05). Using a nonlinear regression analysis, IC50 cytotoxicity for Jurkat, MOLT4, Namalwa and Raji at 72 hours was 0.3, 0.9, 1.8, and 1.9 µM, respectively. NT157 significantly induced apoptosis in a dose and time-dependent manner (all p<0.05). The highest percentage of apoptotic cells were observed upon NT157 1.6 µM at 72 hours for all cell lines (Control vs. NT157 1.6 µM: 6% vs. 87% for Jurkat, 16% vs. 95% for MOLT4, 4% vs. 41% for Namalwa, and 25% vs. 50% for Raji cells). Western blot analysis revealed increased cleaved-caspase 3 levels in all cell lines. Jurkat and Raji cells were tested for clonogenicity; colonies were counted after 8 days. The number of colonies reduced by 14%, 14% and 23% for Jurkat and by 32%, 44% and 77% for Raji cells at the dose of 0.4, 0.8, and 1.6 µM, respectively. Western blot analysis revealed that NT157 treatment induced IRS1 down-regulation in a dose-dependent manner after 24 hours of treatment. In Jurkat cells, PCR-array analysis reveals that NT157 modulates 25 genes, including downregulation of MYC (proliferation-related genes) and upregulation of CDKN1A, CDKN1C and CDKN2A (cell cycle arrest-related genes), and JUN and FOS (apoptosis-related genes), validation experiments confirmed an upregulation of CDKN1A, JUN and FOS in all ALL cell lines upon NT157 treatment. Notable, IRS1/2 pharmacological inhibition by NT157 reduced cell viability of primary ALL cells and was a potent apoptosis inductor. Upon NT157 1.6 µM, cell viability for T-ALL and B-ALL primary cells was inhibited by 55±5% and 15±8%, respectively. The apoptosis rates for untreated cells and 1.6 µM NT157 was 12±8% and 59±27% for T-ALL primary cells and 19±2% and 37±1% and B-ALL primary cells, respectively. NT157 treatment did not presented citotoxicity in peripheral blood mononuclear cells (PBMC) from healthy donors. On the other hand, in ALL cell lines tested, OSI-906 treatment reduced cell viability, but did not induce apoptosis; higher doses of OSI-906 were necessary to induce cytotoxicity. OSI-906 did not modulate viability and apoptosis of primary ALL cells and normal PBMC. Conclusion: Our results revealed that pharmacological inhibition of IRS1/2, by NT157, exerts a cytotoxic effect in ALL cell lines and primary ALL cells, while IGF1R/IR inhibition, by OSI -906, has a predominant cytostatic effect only at higher doses. These data indicate that direct inhibition of IRS proteins by NT157 induces better antileukemic effects compared to OSI -906, and targeting IRS proteins may be an effective anti-ALL approach. Disclosures No relevant conflicts of interest to declare.