Monomethylarsonous Acid, But Not Inorganic Arsenic, Is a Mitochondrial-Specific Intoxicant in Vascular Smooth Muscle Cells

Diabetes-Metabolic Syndrome 250 Monomethylarsonous Acid, But Not Inorganic Arsenic, Is a Mitochondrial-Specific Intoxicant in Vascular Smooth Muscle Cells Clare Pace1, Tania Das Banerjee1, Barrett Welch2, Roxana Khalili3, Ruben Dagda1, and Jeff Angermann1 1 University of Nevada, Reno, USA, 2Oregon State University, USA, 3University of California, Irvine, USA Arsenic exposure is associated with vascular disease, yet the cellular mechanisms of toxicity are largely unknown. The present study investigates mitochondrial dysfunction in relation to the vascular toxicity of arsenic. The relative effects of inorganic arsenic (iAs) and the metabolite monomethylarsonous acid (MMA) on mitochondrial function in vascular aortic smooth muscle cells (VSMC) were assessed through cell proliferation, cell death, oxygen consumption rates, glycolysis, mitochondrial morphology, ATP generation, and expression of several mitochondriallylocalized proteins. Survival curves and determination of mitochondrial function following a 24-hr. exposure indicate that MMA is significantly more toxic than iAs as determined by trypan blue and MTS assays. A 3 hr. exposure of MMA, but not iAs, caused a significant decrease in basal and maximal respiration and a concomitant increase in compensatory glycolysis as measured by an XF24Extracellular Flux Analyzer. Significant decreases in mitochondrial ATP generation occurred following 6 hr. treatment with all species and were significantly more pronounced for MMA compared to iAs. MMA treatment, but not of iAs, caused a significant increase in hydrogen peroxide levels. Exposure of VSMCs to high doses of MMA fragmented mitochondria and caused a decrease in the percent of mitochondria occupied by cytosol. In contrast, treating VSMCs with a high dose of MMA for 6 hrs. or a low dose for 24 hrs. enhanced mitochondrial levels as assessed by single cell analysis of mitochondrial content or by immunoblotting for the total levels of several mitochondrial proteins. Overall our data demonstrate that MMA, but not iAs, is a mitochondrial-specific intoxicant in VSMCs. Bidirectional changes in mitochondrial morphology and content, and elevated glycolysis induced by MMA are likely compensatory manifestations secondary to mitochondrial dysfunction and ROS elevation. Therefore, subsequent work will explore additional mechanisms of MMA-mediated mitochondrial dysfunction including identifying other mitochondrial sources of ROS induced by MMA, and analyzing the effects of MMA on substrate utilization by mitochondrial complexes. Funding: NIH-P20 GM103554 doi: 10.1016/j.freeradbiomed.2014.10.227

251 Location, Location, Location - The Location of Free Radical Generation Is a Key Determinant in Their Actions on ȕ-Cells Katarzyna Broniowska1, Bryndon Oleson1, Clayton Mathews2, and John Corbett1 1 Medical College of Wisconsin, USA, 2University of Florida, USA Inflammatory cytokines impair pancreatic ȕ-cell function by stimulating the expression of inducible nitric oxide synthase (iNOS) and production of nitric oxide (NO). In addition to NO, excessive formation of reactive oxygen species, such as superoxide and H2O2 KDV EHHQ VKRZQ WR FDXVH ȕ-cell damage. Here, we determined the forms and functions of reactive species

SURGXFHG LQ ȕ-cells in response to cytokines. Cytokine treatment of isolated islets or an insulinoma cell line results in the production of nitric oxide but does not stimulate the formation of peroxynitrite (assessed using the coumarin-7-boronate). Exposure of ȕ-cells to the NADPH oxidase activator phorbol 12-myristate 13-acetate fails to stimulate superoxide generation. Addition of either NO donor, menadione (to produce superoxide), or H2O2 alone results LQ LPSDLUHG PLWRFKRQGULDO R[LGDWLRQ DQG FDXVHV ȕ-cell death. In contrast, peroxynitrite does not impair oxidative metabolism or GHFUHDVHȕ-cell viability. Surprisingly, when NO-tUHDWHGȕ-cells are forced to produce superoxide (via treatment with menadione), SHUR[\QLWULWH LV JHQHUDWHG EXW R[LGDWLYH PHWDEROLVP DQG ȕ-cell viability are preserved. In the cells exposed to NO, when superoxide is generated in the extracellular medium (via xanthine R[LGDVHV\VWHP SHUR[\QLWULWHLVIRUPHGEXW$73OHYHOVDQGȕ-cell viability remain compromised. These findings demonstrate that NO is the toxic species produced in response to cytokines and that the location of radical generation and the site of radical reactions are key determinants that regulate the effect of reactive VSHFLHVRQȕ-cell function and viability. Extrapolating these results to islet inflammation during the development of diabetes, these findings would predict that radical generatiRQLQȕ-cells might be a key determinant in impairment of function. doi: 10.1016/j.freeradbiomed.2014.10.228

252 Adjustment of a Western Diet-Induced Obesity Mouse Model for Immunometabolic Studies Maria Cecilia Della-Vedova1,2, Marcos D Muñoz1,3, Martin Rinaldi Tosi1, Silvina Garcia1, Sandra E Gomez Mejiba1,3, And Dario C Ramirez1,2 1 Lab. Exp. Med. & Ther., IMIBIO-SL, Nat. Bureau of Sci. & Tech. Res. (CONICET), Argentina, 2Dept. of Biochemstry, School of Chemistry, Biochemistry & Pharmacy-UNSL, Argentina, 3Dept. of Nutrition, School of Health Sciences-UNSL, Argentina The increase in the consumption of chicken fat and fructose, which are cheaper ingredients for foods, coincides with an increase in the prevalence of obesity. Due to the world known difficulties Argentinean scientists to import commercially available diets herein we aimed at developing a mouse model of dietinduced obesity that uses local ingredients and that also highlights the main features of human obesity, including adiposity and insulin resistance (IR). To accomplish our aim we fed male 6 week-old B6 mice for 16 weeks with a rodent chow diet containing either 4 (LFD) or 22% (HFD) p/p chicken fat and water containing or not 10% p/v fructose (F). This experimental design resulted in 4 experimental groups: high fat diet plus water supplemented with 10% fructose (HFD+F); HFD only, low fat diet only (LFD) and LFD+F. The consumption of food and drink as well as body weight gain were measured throughout the experiment. At the end of the feeding period adiposity index, fasten glucemia and IR (glucose tolerance test) were measured. The results (mean valuesrSEM) showed that compared to mouse from other groups those from the HFD+F group: i) gained more weight (HFD+F:29.41±1.07 vs HFD:27.4r0.93; LFD+F:25.5r0.70; LFD:24.77r0.60); ii) have heavier epididymal fat-pads thus larger adiposity index ([epididymal fat(g)/body weight] x100 (HFD+F:4.38±1.36 vs HFD:3.34±0.27; LFD+F:2.12±0.17; LFD:1.43±0.10); iii) like the LFD+F this group drank more water (ml/mouse/day, HFD+F:8.7±0.46 and LFD+F:7.43±0.47 vs HFD:4.94±0.56 and LFD:4.52±0.70); iv) ate less (mg/mouse/day, HFD+F:2.16±0.07 vs HFD:2.55±0.03; LFD+F:2.35±0.06; LFD:2.95±0.07); v) have more fasten glucemia (HFD+F:271r50 vs HFD:233r50; LFD+F:212±44; LFD:183r45); and vi) are more

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