69 New Chalcones and Stilbenes as Modulators of hSIRT1 Activity Leonardo Santos1, Veronica Silva1, Gabriel Sagrer1, Gustavo Seoane1, and Ana Denicola1 1University of the Republic, Uruguay Sirtuins catalyze the NAD-dependent deacetylation of ε-acetyl-Lys residues of proteins. There are seven human sirtuins (SIRT1-7) and SIRT1, the nuclear isoform, has been implicated in several age-related diseases like cardiovascular, neurodegenerative and metabolic disorders (metabolic syndrome). Increasing SIRT1 activity either by transgenic overexpression or pharmacological activation with resveratrol has shown beneficial effects in rodent models of obesity-associated disorders, indicating that SIRT1 may represent a potential therapeutic target. A widely used assay for SIRT1 activity is a commercial kit that employs 7-amino-4-methylcoumarin (AMC) quenched at the C-terminal end of a tetrapeptide-containing acetyl-lysine substrate. On deacetylation, the peptide becomes a trypsin substrate, thus, upon trypsin cleavage the fluorophore AMC is released. The assay has been questioned; it is an end-point assay, not continous, but still useful as high-throughput screening We tested the ability of several chalcones and stilbenes synthesized in our laboratory to modulate the activity of hSIRT1 using this fluorescent assay, taking special attention to appropriate controls (dependence on enzyme concentration; no inner-filter effect; blank the intrinsic absorption; or fluorescence of the polyphenol assayed). We found polyphenolic structures which activated SIRT1 in vitro, on a level similar to that of resveratrol. Further studies including other activity assays as well as enzyme-activator interactions are underway. doi: 70 H2O2 Production and Glutathionylation of Ryanodine Receptor in Skeletal Muscle from Insulin Resistance Mice Alejandra Espinosa1, Julio Miranda1, Gina Sánchez2, Mario Bustamante2, José Luis Bucarey3, Cristián Campos1, Marcelo Ezquier4, Cecilia Hidalgo2, and Enrique Jaimovic2 1Universidad de Chile, Chile, 2Universidad de Chile, Chile, 3Universidad de Valparaíso, Chile, 4Universidad del Desarrollo, Chile Insulin resistance (IR) is defined as a reduced ability of insulin to stimulate glucose utilization and is characterized by higher plasma concentration of insulin. C57BL/6 mice fed chronically with a high fat diet (HFD) are an IR model. Recently we had shown that H2O2 participates as a signal evoked by insulin in myotubes. H2O2 alters the redox state shifting the ratio of reduced glutathione to oxidized glutathione (GSH/GSSG). Ryanodine receptor (RyR), a calcium channel located in sarcoplasmatic reticulum, is a target of redox modification as glutathionylation. We established a colony of insulin resistance mice and evaluated both, the IR skeletal muscle H2O2 production and the GSH/GSSG ratio and glutathionylation of RyR in isolated triads. C57BL/6J mice were fed control or HFD, ad libitum (D12492, Research Diets). IR was determined by HOMA-IR. Isolated muscle fibers in culture were transfected with a plasmid that encodes for HyPer protein, a specific probe for intracellular H2O2. Ratio GSH/GSSG was measured using Glutathione Detection Kit (Abcam). RyR glutathionylation was detected using antibodies against GSH-protein on a purified preparation of RyR. An IR mice colony was obtained after 3 months of treatment with HFD. HOMA-IR was 2.13±0.54 against the HFD mice result of 4.48±1.58. IR mice can generate higher amounts of H2O2 compared to control. The ratio GSH/GSSG decreased in insulin resistance mice. The ratio anti-GSH/anti-RyR was 2.8 fold in IR

mice respect to control. Our results suggest that IR mice can generate higher levels of H2O2 upon stimulation with insulin and alter the redox state of the skeletal muscle fiber. This could be involved in intracellular calcium release from RyR under pathological condition. FONDECYT 11090301, FONDAP 15010006. doi:

71 Inhibition of α­amylase and α­glucosidase Activity by Bioflavonoids: Implications for Carbohydrate Metabolism and type 2 Diabetes Meltem Yilmazer-Musa1, Anneke M Tucker1, and Balz Frei1 1Linus Pauling Institute, Oregon State University While it is often assumed that dietary flavonoids exert biological effects through antioxidant mechanisms, growing evidence suggests that they modulate carbohydrate metabolism at several levels independently of antioxidant activity. One of these mechanisms is to inhibit digestive enzymes and delay glucose absorption in the small intestine, thereby being potentially useful as therapeutic agents in metabolic syndrome and type 2 diabetes. In this study we evaluated the effect of dietary supplements containing bioflavonoids on two digestive enzymes, α-amylase and α-glucosidase, required for hydrolysis of dietary amylose and amylopectin (starch) into maltotriose, maltose, and glucose. Grape seed, cocoa, green tea, white tea, cinnamon, curcumin, and resveratrol (Japanese knotweed) extracts and Teavigo® (green tea extract containing ≥94% epigallocatechin gallate [EGCG]) were tested at various concentrations, as well as their constituent bioflavonoids: catechin (C), epicatechin (EC), epigallocatechin (EGC), EGCG, gallocatechin gallate (GCG), epicatechin gallate (ECG), and resveratrol. In all tests, acarbose, a pharmacological inhibitor of α-amylase and α-glucosidase used to treat type 2 diabetes and prediabetes, was included as positive control. For each compound, the concentration required for 50% inhibition of enzyme activity (IC50) was determined. Resveratrol extract more strongly inhibited α-amylase (IC50=4.87±0.50 µg/ml) than acarbose (IC50=7.17±0.24 µg/ml). Grape seed, cinnamon, and curcumin extracts exhibited similar α-amylase inhibitory activity (IC50=9.44±0.70; 9.74±0.49; and 11.86±2.67 µg/ml, respectively) to acarbose. All other extracts and individual bioflavonoids tested, even at the highest concentration (1 mM), only partially (20-56%) inhibited α-amylase activity. Interestingly, all extracts showed stronger α-glucosidase inhibitory activity (IC50=0.25-42.9 µg/ml) than acarbose (IC50=101.2±3.5 µg/ml). Also, EGCG, GCG, ECG, and resveratrol very effectively (IC50<4 µg/ml) inhibited α-glucosidase, while C, EC, and EGC were ineffective. We are currently investigating whether selected extracts and bioflavonoids can delay carbohydrate absorption and lower postprandial glycemia and insulinemia in human volunteers consuming a defined, high-carbohydrate meal.  This work was supported by a grant from USANA Health Sciences Inc., Salt Lake City, UT. doi:

72 Hyperglycemic Carbonyl Stress in Vascular Endothelial Cells and Protection by Heavy Metal Chelators Travis Owen Gurney1, Sean Sliman1, Rishi Patel1, Sainath Kotha1, and Narasimham Parinandi1 1Ohio State University During diabetes, vascular endothelium is susceptible to damage caused by elevated levels of the glucose-derived oxoaldehyde, glyoxal, through carbonyl stress and the formation of advanced

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