Nox4 mediates skeletal muscle metabolic responses to exercise.

OBJECTIVE: The immediate signals that couple exercise to metabolic adaptations are incompletely understood. Nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) produces reactive oxygen species (ROS) and plays a significant role in metabolic and vascular adaptation during stress conditions. Our objective was to determine the role of Nox4 in exercise-induced skeletal muscle metabolism. METHODS: Mice were subjected to acute exercise to assess their immediate responses. mRNA and protein expression responses to Nox4 and hydrogen peroxide (H2O2) were measured by qPCR and immunoblotting. Functional metabolic flux was measured via ex vivo fatty acid and glucose oxidation assays using 14C-labeled palmitate and glucose, respectively. A chronic exercise regimen was also utilized and the time to exhaustion along with key markers of exercise adaptation (skeletal muscle citrate synthase and beta-hydroxyacyl-coA-dehydrogenase activity) were measured. Endothelial-specific Nox4-deficient mice were then subjected to the same acute exercise regimen and their subsequent substrate oxidation was measured. RESULTS: We identified key exercise-responsive metabolic genes that depend on H2O2 and Nox4 using catalase and Nox4-deficient mice. Nox4 was required for the expression of uncoupling protein 3 (Ucp3), hexokinase 2 (Hk2), and pyruvate dehydrogenase kinase 4 (Pdk4), but not the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc-1α). Global Nox4 deletion resulted in decreased UCP3 protein expression and impaired glucose and fatty acid oxidization in response to acute exercise. Furthermore, Nox4-deficient mice demonstrated impaired adaptation to chronic exercise as measured by the time to exhaustion and activity of skeletal muscle citrate synthase and beta-hydroxyacyl-coA-dehydrogenase. Importantly, mice deficient in endothelial-Nox4 similarly demonstrated attenuated glucose and fatty acid oxidation following acute exercise. CONCLUSIONS: We report that H2O2 and Nox4 promote immediate responses to exercise in skeletal muscle. Glucose and fatty acid oxidation were blunted in the Nox4-deficient mice post-exercise, potentially through regulation of UCP3 expression. Our data demonstrate that endothelial-Nox4 is required for glucose and fatty acid oxidation, suggesting inter-tissue cross-talk between the endothelium and skeletal muscle in response to exercise.

Prospective study of gestational diabetes and fatty liver scores 9 to 16 years after pregnancy.

BACKGROUND: Women with gestational diabetes mellitus (GDM) may be at an increased risk of liver complications because chronic hyperglycemia is a risk factor for liver fat accumulation and potential liver dysfunction. Large prospective studies examining liver fat accumulation following a GDM pregnancy are lacking. METHODS: The Diabetes & Women's Health Study (2012-2014) examined the association between GDM and subsequent fatty liver scores among 607 women with and 619 women without GDM in the Danish National Birth Cohort. Nine to 16 years postpartum, a clinical examination was performed, with measurement of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and γ-glutamyl transferase, from which fatty liver scoring indices were calculated to assess liver fat score, fatty liver index, hepatic steatosis index, and liver fat percentage. Relative risks (RR) with 95% confidence intervals (CI) for elevated liver scoring indices by GDM status were assessed adjusting for major risk factors, including prepregnancy body mass index. RESULTS: Women with prior GDM had higher adjusted ALT and AST levels than women without GDM (by 6.7% [95% CI 1.7-12.0] and 4.8% [95% CI 0.6-9.1], respectively). Women with GDM also had adjusted increased risks for elevated liver fat score (RR 2.34; 95% CI 1.68-3.27), fatty liver index (RR 1.59; 95% CI 1.27-1.99), and hepatic steatosis index (RR 1.44; 95% CI 1.21-1.71). CONCLUSIONS: Women with GDM during pregnancy were at an increased risk for fatty liver 9 to 16 years postpartum. Gestational diabetes mellitus may serve as another risk indicator for the early identification and prevention of liver fat accumulation.

Longitudinal Maternal Vitamin D Status during Pregnancy Is Associated with Neonatal Anthropometric Measures.

Findings on maternal 25-hydroxyvitamin D (25[OH]D) and neonatal anthropometry are inconsistent, and may at least be partly due to variations in gestational week (GW) of 25(OH)D measurement and the lack of longitudinal 25(OH)D measurements across gestation. The aim of the current study was to examine the associations of longitudinal measures of maternal 25(OH)D and neonatal anthropometry at birth. This study included 321 mother⁻offspring pairs enrolled in the Eunice Kennedy Shriver National Institute of Child Health and Human Development Fetal Growth Studies⁻Singletons. This study was a prospective cohort design without supplementation and without data on dietary supplementation. Nevertheless, measurement of plasma 25(OH)D reflects vitamin D from different sources, including supplementation. Maternal concentrations of total 25(OH)D were measured at 10⁻14, 15⁻26, 23⁻31, and 33⁻39 GW and categorized as <50 nmol/L, 50⁻75 nmol/L, and >75 nmol/L. Generalized linear models were used to examine associations of 25(OH)D at each time-point with neonate birthweight z-score, length, and sum of skinfolds at birth. At 10⁻14 GW, 16.8% and 49.2% of women had 25(OH)D <50 nmol/L and between 50⁻75 nmol/L, respectively. The association of maternal 25(OH)D with neonatal anthropometry differed by GW and women's prepregnancy BMI (normal (<25.0 kg/m²), overweight/obese (25.0⁻44.9 kg/m²)). All analyses were stratified by prepregnancy BMI status. Among women with an overweight/obese BMI, 25(OH)D <50 nmol/L at 10⁻14 GW was associated with lower birthweight z-score (0.56; 95% CI: -0.99, -0.13) and length (-1.56 cm; 95% CI: -3.07, -0.06), and at 23⁻31 GW was associated with shorter length (-2.77 cm; 95% CI: -13.38, -4.98) and lower sum of skinfolds (-9.18 mm; 95% CI: -13.38, -4.98). Among women with a normal BMI, 25(OH)D <50 nmol/L at 10⁻14 GW was associated with lower sum of skinfolds (-2.64 mm; 95% CI: -5.03, -0.24), at 23⁻31 GW was associated with larger birthweight z-scores (0.64; 95% CI: 0.03, 1.25), and at 33-39 GW with both higher birthweight z-score (1.22; 95% CI: 0.71, 1.73) and longer length (1.94 cm; 95% CI: 0.37, 3.52). Maternal 25(OH)D status during pregnancy was associated with neonatal anthropometric measures, and the associations were specific to GW of 25(OH)D measurement and prepregnancy BMI.

Isolation of Mouse Stromal Vascular Cells for Monolayer Culture.

Positive energy balance contributes to adipose tissue expansion and dysfunction, which accounts largely for obesity and related metabolic disorders. Thermogenic fat can dissipate energy, activation or induction of which may promote energy balance and address the pressing health issues. Recent studies have shown that stromal vascular fraction (SVF) from white adipose tissue (WAT) can develop both white and brown-like adipocyte phenotypes, thus serving as a unique model to study adipogenesis and thermogenesis. Here, we describe a protocol for effective isolation of mouse SVF from WAT, induction of differentiation, and detection of adipogenesis. Success tips for isolation and culture of SVF are also discussed.