Edwin R. Miranda

Nox2 contributes to hyperinsulinemia-induced redox imbalance and impaired vascular function

Insulin resistance promotes vascular endothelial dysfunction and subsequent development of cardiovascular disease. Previously we found that skeletal muscle arteriolar flow-induced dilation (FID) was reduced following a hyperinsulinemic clamp in healthy adults. Therefore, we hypothesized that hyperinsulinemia, a hallmark of insulin resistance, contributes to microvascular endothelial cell dysfunction via inducing oxidative stress that is mediated by NADPH oxidase (Nox) system. We examined the effect of insulin, at levels that are comparable with human hyperinsulinemia on 1) FID of isolated arterioles from human skeletal muscle tissue in the presence and absence of Nox inhibitors and 2) human adipose microvascular endothelial cell (HAMECs) expression of nitric oxide (NO), endothelial NO synthase (eNOS), and Nox-mediated oxidative stress. In six lean healthy participants (mean age 25.5±1.6 y, BMI 21.8±0.9), reactive oxygen species (ROS) were increased while NO and arteriolar FID were reduced following 60 min of ex vivo insulin incubation. These changes were reversed after co-incubation with the Nox isoform 2 (Nox2) inhibitor, VAS2870. In HAMECs, insulin-induced time-dependent increases in Nox2 expression and P47phox phosphorylation were echoed by elevations of superoxide production. In contrast, phosphorylation of eNOS and expression of superoxide dismutase (SOD2 and SOD3) isoforms showed a biphasic response with an increased expression at earlier time points followed by a steep reduction phase. Insulin induced eNOS uncoupling that was synchronized with a drop of NO and a surge of ROS production. These effects were reversed by Tempol (SOD mimetic), Tetrahydrobiopterin (BH4; eNOS cofactor), and VAS2870. Finally, insulin induced nitrotyrosine formation which was reversed by inhibiting NO or superoxide generation. In conclusions, hyperinsulinemia may reduce FID via inducing Nox2-mediated superoxide production in microvascular endothelial cells which reduce the availability of NO and enhances peroxynitrite formation. Therefore, the Nox2 pathway should be considered as a target for the prevention of oxidative stress-associated endothelial dysfunction during hyperinsulinemia.

Circulating soluble RAGE isoforms are attenuated in obese, impaired-glucose-tolerant individuals and are associated with the development of type 2 diabetes

The soluble receptor for advanced glycation end products (sRAGE) may be protective against inflammation associated with obesity and type 2 diabetes (T2DM). The aim of this study was to determine the distribution of sRAGE isoforms and whether sRAGE isoforms are associated with risk of T2DM development in subjects spanning the glucose tolerance continuum. In this retrospective analysis, circulating total sRAGE and endogenous secretory RAGE (esRAGE) were quantified via ELISA, and cleaved RAGE (cRAGE) was calculated in 274 individuals stratified by glucose tolerance status (GTS) and obesity. Group differences were probed by ANOVA, and multivariate ordinal logistic regression was used to test the association between sRAGE isoform concentrations and the proportional odds of developing diabetes, vs. normal glucose tolerance (NGT) or impaired glucose tolerance (IGT). When stratified by GTS, total sRAGE, cRAGE, and esRAGE were all lower with IGT and T2DM, while the ratio of cRAGE to esRAGE (cRAGE:esRAGE) was only lower (P < 0.01) with T2DM compared with NGT. When stratified by GTS and obesity, cRAGE:esRAGE was higher with obesity and lower with IGT (P < 0.0001) compared with lean, NGT. In ordinal logistic regression models, greater total sRAGE (odds ratio, 0.91; P < 0.01) and cRAGE (odds ratio, 0.84; P < 0.01) were associated with lower proportional odds of developing T2DM. Reduced values of sRAGE isoforms observed with both obesity and IGT are independently associated with greater proportional odds of developing T2DM. The mechanisms by which each respective isoform contributes to obesity and insulin resistance may reveal novel treatment strategies for diabetes.

Dicarbonyl stress and glyoxalase enzyme system regulation in human skeletal muscle

Skeletal muscle insulin resistance is a hallmark of Type 2 diabetes (T2DM) and may be exacerbated by protein modifications by methylglyoxal (MG), known as dicarbonyl stress. The glyoxalase enzyme system composed of glyoxalase 1/2 (GLO1/GLO2) is the natural defense against dicarbonyl stress, yet its protein expression, activity, and regulation remain largely unexplored in skeletal muscle. Therefore, this study investigated dicarbonyl stress and the glyoxalase enzyme system in the skeletal muscle of subjects with T2DM (age: 56 ± 5 yr.; BMI: 32 ± 2 kg/m2) compared with lean healthy control subjects (LHC; age: 27 ± 1 yr.; BMI: 22 ± 1 kg/m2). Skeletal muscle biopsies obtained from the vastus lateralis at basal and insulin-stimulated states of the hyperinsulinemic (40 mU·m-2·min-1)-euglycemic (5 mM) clamp were analyzed for proteins related to dicarbonyl stress and glyoxalase biology. At baseline, T2DM had increased carbonyl stress and lower GLO1 protein expression (-78.8%), which inversely correlated with BMI, percent body fat, and HOMA-IR, while positively correlating with clamp-derived glucose disposal rates. T2DM also had lower NRF2 protein expression (-31.6%), which is a positive regulator of GLO1, while Keap1 protein expression, a negative regulator of GLO1, was elevated (207%). Additionally, insulin stimulation during the clamp had a differential effect on NRF2, Keap1, and MG-modified protein expression. These data suggest that dicarbonyl stress and the glyoxalase enzyme system are dysregulated in T2DM skeletal muscle and may underlie skeletal muscle insulin resistance. Whether these phenotypic differences contribute to the development of T2DM warrants further investigation.

Metabolic Derangements Contribute to Reduced sRAGE Isoforms in Subjects with Alzheimer’s Disease

Although there is evidence for metabolic dysfunction and chronic inflammation in Alzheimers disease (AD), circulating levels of soluble receptor for advanced glycation end products (sRAGE) and the receptor for advanced glycation end products (RAGE) ligand S100B have not been characterized. sRAGE is an important mediator in disease as it can act as a ligand decoy for RAGE and attenuate downstream inflammatory signaling. Cognitively healthy elderly and AD participants with and without type 2 diabetes (n = 135) were stratified according to the clinical dementia rating (CDR; 0 = normal cognition (NC); ≥0.5 = AD). Total serum sRAGE, endogenous secretory RAGE (esRAGE), and S100B were assayed via ELISAs, and cleaved RAGE (cRAGE) and the cRAGE : esRAGE ratio were calculated. cRAGE : esRAGE was lower in AD compared to NC (p < 0.05). Metabolic substratifications were used to investigate the factors that influence sRAGE pathology in AD. Stratification by BMI classification, median fat mass, median HOMA-IR, median insulin, and median amylin were all metabolic or anthropometric factors which significantly interacted with sRAGE profiles within AD subjects. There were no significant differences in serum S100B between groups. These characterizations of sRAGE contribute evidence to the link between impaired metabolism and cognitive decline due to AD.

ID: 83: PRODUCTION OF SOLUBLE RECEPTOR FOR ADVANCED GLYCATION END-PRODUCTS FOLLOWING ACUTE AEROBIC EXERCISE IS GENDER SPECIFIC

The Receptor for Advanced Glycation End Products (RAGE) is a transmembrane receptor that initiates a self-propagating inflammatory cascade and has been implicated in the onset of complications involved with aging, diabetes and neuroinflammation. Soluble RAGE (sRAGE) inhibits this inflammatory signaling by competitively binding to RAGE ligands without stimulating downstream effectors. Evidence from our lab demonstrates chronic aerobic exercise increases the cleaved isoform of sRAGE (sRAGEc). However, the effects of acute aerobic exercise on sRAGEc production have not been comprehensively examined. Furthermore, recent data suggests that estrogen may play a role in exacerbating RAGE signaling and perturbing sRAGE production in diabetic women. Therefore, the primary objective of this study was to investigate changes in plasma sRAGE with acute aerobic exercise in both lean healthy (LH) and obese insulin resistant (OB-IR) individuals. A secondary objective of the study was to compare exercise responses between men and women. 8 LH participants (4 M, 4 F) and 14 OB-IR participants (4 M, 10 F) were recruited for the study. VO2max was determined via treadmill test and participants returned to the lab on a separate day following an overnight fast and exercised at 65% VO2max for 30 minutes. Blood samples were collected before and following exercise after participants rested in seated position for 30 minutes. Quantification of plasma sRAGE and endogeonous secretory RAGE were determined via ELISA and sRAGEc was calculated by subtraction. Between-group comparisons were made via independent T Test and the effect of gender was analyzed via two-way ANOVA. At baseline the OB-IR group was older (41±3 y vs. 26±1 y, p<.001), more obese (BMI 35.1±0.9 vs. 22.2±0.9 kg . m−2, p<.001) and less aerobically fit (VO2max 27.8±1.8 vs. 50.2±2.9 mL/kg−1. min−1, p<.001) compared the LH group. There was no main effect of group (OB-IR vs. LHC) on change in sRAGE or sRAGEc in response to exercise (ΔsRAGE 20.3±53.2 vs. 13.8±34.4 pg/mL, p=.93), (ΔsRAGEc 28.7±47.1 vs. 14.4±34.8 pg/mL, p=.33). However there was an effect of gender on the response to acute exercise. Males in both groups saw a significantly greater increase in plasma sRAGE (131.49±46.46 vs. −46.94±39.23 pg/mL, p<.05) and plasma sRAGEc (127.73±47.04 vs. −36.08±34.13 pg/mL, p<.05) compared to females. This study is the first to show that young healthy women and obese/insulin resistant women have an impaired ability to increase sRAGE plasma levels with acute aerobic exercise. Recent data has suggested that estrogen can exacerbate RAGE signaling as well as inhibit sRAGE production although the precise mechanism for this interaction is unclear and warrants further investigation.