NADPH oxidase 1 promotes hepatic steatosis in obese mice and is abrogated by augmented skeletal muscle mass.

Exercise as a lifestyle modification is a frontline therapy for nonalcoholic fatty liver disease (NAFLD), but how components of exercise attenuate steatosis is unclear. To uncouple the effect of increased muscle mass from weight loss in obesity, myostatin knockout mice were bred on a lean and obese db/db background. Myostatin deletion increases gastrocnemius (Gastrocn.) mass and reduces hepatic steatosis and hepatic sterol regulatory element binding protein 1 (Srebp1) expression in obese mice, with no impact on adiposity or body weight. Interestingly, hypermuscularity reduces hepatic NADPH oxidase 1 (Nox1) expression but not NADPH oxidase 4 (Nox4) in db/db mice. To evaluate a deterministic function of Nox1 on steatosis, Nox1 knockout mice were bred on a lean and db/db background. NOX1 deletion significantly attenuates hepatic oxidant stress, steatosis, and Srebp1 programming in obese mice to parallel hypermuscularity, with no improvement in adiposity, glucose control, or hypertriglyceridemia to suggest off-target effects. Directly assessing the role of NOX1 on SREBP1, insulin (Ins)-mediated SREBP1 expression was significantly increased in either NOX1, NADPH oxidase organizer 1 (NOXO1), and NADPH oxidase activator 1 (NOXA1) or NOX5-transfected HepG2 cells versus ?-galactosidase control virus, indicating superoxide is the key mechanistic agent for the actions of NOX1 on SREBP1. Metabolic Nox1 regulators were evaluated using physiological, genetic, and diet-induced animal models that modulated upstream glucose and insulin signaling, identifying hyperinsulinemia as the key metabolic derangement explaining Nox1-induced steatosis in obesity. GEO data revealed that hepatic NOX1 predicts steatosis in obese humans with biopsy-proven NAFLD. Taken together, these data suggest that hypermuscularity attenuates Srebp1 expression in db/db mice through a NOX1-dependent mechanism.NEW & NOTEWORTHY This study documents a novel mechanism by which changes in body composition, notably increased muscle mass, protect against fatty liver disease. This mechanism involves NADPH oxidase 1 (NOX1), an enzyme that increases superoxide and increases insulin signaling, leading to increased fat accumulation in the liver. NOX1 may represent a new early target for preventing fatty liver to stave off later liver diseases such as cirrhosis or liver cancer.

A Reappraisal of the Utility of L-012 to Measure Superoxide from Biologically Relevant Sources.

The detection of superoxide anion (O2●-) in biological tissues remains challenging. Barriers to convenient and reproducible measurements include expensive equipment, custom probes, and the need for high sensitivity and specificity. The luminol derivative, L-012, has been used to measure O2●- since 1993 with mixed results and concerns over specificity. The goal of this study was to better define the conditions for use and their specificity. We found that L-012 coupled with depolymerized orthovanadate, a relatively impermeable tyrosine phosphatase inhibitor, yielded a highly sensitive approach to detect extracellular O2●-. In O2●- producing HEK-NOX5 cells, orthovanadate increased L-012 luminescence 100-fold. The combination of L-012 and orthovanadate was highly sensitive, stable, scalable, completely reversed by superoxide dismutase, and selective for O2●- generating NOXes versus NOX4, which produces H2O2. Moreover, there was no signal from cells transfected with NOS3 (NO●) and NOS2(ONOO-). To exclude the effects of altered tyrosine phosphorylation, O2●- was detected using non-enzymatic synthesis with phenazine methosulfate and via novel coupling of L-012 with niobium oxalate, which was less active in inducing tyrosine phosphorylation. Overall, our data shows that L-012 coupled with orthovanadate or other periodic group 5 salts yields a reliable, sensitive, and specific approach to measuring extracellular O2●- in biological systems.

Galectin-3 Mediates Vascular Dysfunction in Obesity by Regulating NADPH Oxidase 1.

BACKGROUND: Obesity is associated with increased risk of cardiovascular disease, but underlying mechanisms remain elusive. Metabolic dysfunction, especially hyperglycemia, is thought to be a major contributor, but how glucose impacts vascular function is unclear. GAL3 (galectin-3) is a sugar-binding lectin upregulated by hyperglycemia, but its role as a causative mechanism of cardiovascular disease remains poorly understood. Therefore, the objective of this study was to determine the role of GAL3 in regulating microvascular endothelial vasodilation in obesity. METHODS: GAL3 was measured and found to be markedly increased in the plasma of overweight and obese patients, as well as in the microvascular endothelium of diabetic patients. To investigate causative mechanisms in cardiovascular disease, mice deficient in GAL3 were bred with obese db/db mice to generate lean, lean GAL3 knockout, obese, and obese GAL3 knockout genotypes. Endothelial cell-specific GAL3 knockout mice with novel AAV-induced obesity recapitulated whole-body knockout studies to confirm cell specificity. RESULTS: Deletion of GAL3 did not alter body mass, adiposity, or plasma indices of glycemia and lipidemia, but levels of plasma reactive oxygen species as assessed by plasma thiobarbituric acid reactive substances were normalized in obese GAL3 knockout mice. Obese mice exhibited profound endothelial dysfunction and hypertension, both of which were rescued by GAL3 deletion. Isolated microvascular endothelial cells from obese mice had increased expression of NOX1 (nicotinamide adenine dinucleotide phosphate oxidase 1), which we have previously shown to contribute to increased oxidative stress and endothelial dysfunction, which was normalized in microvascular endothelium from mice lacking GAL3. Cell-specific deletion confirmed that endothelial GAL3 regulates obesity-induced NOX1 overexpression and subsequent microvascular function. Furthermore, improvement of metabolic syndrome by increasing muscle mass, improving insulin signaling, or treating with metformin decreased microvascular GAL3, and thereby NOX1, expression levels. CONCLUSIONS: Deletion of GAL3 normalizes microvascular endothelial function in obese db/db mice, likely through a NOX1-mediated mechanism. Pathological levels of GAL3, and in turn NOX1, are amenable to improvements in metabolic status, presenting a potential therapeutic target to ameliorate pathological cardiovascular consequences of obesity.

Galectin-3 Mediates Vascular Dysfunction in Obesity by Regulating NADPH Oxidase 1.

Rationale: Obesity increases the risk of cardiovascular disease (CVD) through mechanisms that remain incompletely defined. Metabolic dysfunction, especially hyperglycemia, is thought to be a major contributor but how glucose impacts vascular function is unclear. Galectin-3 (GAL3) is a sugar binding lectin upregulated by hyperglycemia but its role as a causative mechanism of CVD remains poorly understood. Objective: To determine the role of GAL3 in regulating microvascular endothelial vasodilation in obesity. Methods and Results: GAL3 was markedly increased in the plasma of overweight and obese patients, as well as in the microvascular endothelium of diabetic patients. To investigate a role for GAL3 in CVD, mice deficient in GAL3 were bred with obese db/db mice to generate lean, lean GAL3 knockout (KO), obese, and obese GAL3 KO genotypes. GAL3 KO did not alter body mass, adiposity, glycemia or lipidemia, but normalized elevated markers of reactive oxygen species (TBARS) in plasma. Obese mice exhibited profound endothelial dysfunction and hypertension, both of which were rescued by GAL3 deletion. Isolated microvascular endothelial cells (EC) from obese mice had increased NOX1 expression, which we have previously shown to contribute to increased oxidative stress and endothelial dysfunction, and NOX1 levels were normalized in EC from obese mice lacking GAL3. EC-specific GAL3 knockout mice made obese using a novel AAV-approach recapitulated whole-body knockout studies, confirming that endothelial GAL3 drives obesity-induced NOX1 overexpression and endothelial dysfunction. Improved metabolism through increased muscle mass, enhanced insulin signaling, or metformin treatment, decreased microvascular GAL3 and NOX1. GAL3 increased NOX1 promoter activity and this was dependent on GAL3 oligomerization. Conclusions: Deletion of GAL3 normalizes microvascular endothelial function in obese db/db mice, likely through a NOX1-mediated mechanism. Pathological levels of GAL3 and in turn, NOX1, are amenable to improvements in metabolic status, presenting a potential therapeutic target to ameliorate pathological cardiovascular consequences of obesity.

The Long Noncoding RNA Cardiac Mesoderm Enhancer-Associated Noncoding RNA (Carmn) Is a Critical Regulator of Gastrointestinal Smooth Muscle Contractile Function and Motility.

BACKGROUND & AIMS: Visceral smooth muscle cells (SMCs) are an integral component of the gastrointestinal (GI) tract that regulate GI motility. SMC contraction is regulated by posttranslational signaling and the state of differentiation. Impaired SMC contraction is associated with significant morbidity and mortality, but the mechanisms regulating SMC-specific contractile gene expression, including the role of long noncoding RNAs (lncRNAs), remain largely unexplored. Herein, we reveal a critical role of Carmn (cardiac mesoderm enhancer-associated noncoding RNA), an SMC-specific lncRNA, in regulating visceral SMC phenotype and contractility of the GI tract. METHODS: Genotype-Tissue Expression and publicly available single-cell RNA sequencing (scRNA-seq) data sets from embryonic, adult human, and mouse GI tissues were interrogated to identify SMC-specific lncRNAs. The functional role of Carmn was investigated using novel green fluorescent protein (GFP) knock-in (KI) reporter/knock-out (KO) mice. Bulk RNA-seq and single nucleus RNA sequencing (snRNA-seq) of colonic muscularis were used to investigate underlying mechanisms. RESULTS: Unbiased in silico analyses and GFP expression patterns in Carmn GFP KI mice revealed that Carmn is highly expressed in GI SMCs in humans and mice. Premature lethality was observed in global Carmn KO and inducible SMC-specific KO mice due to GI pseudo-obstruction and severe distension of the GI tract, with dysmotility in cecum and colon segments. Histology, GI transit, and muscle myography analysis revealed severe dilation, significantly delayed GI transit, and impaired GI contractility in Carmn KO vs control mice. Bulk RNA-seq of GI muscularis revealed that loss of Carmn promotes SMC phenotypic switching, as evidenced by up-regulation of extracellular matrix genes and down-regulation of SMC contractile genes, including Mylk, a key regulator of SMC contraction. snRNA-seq further revealed SMC Carmn KO not only compromised myogenic motility by reducing contractile gene expression but also impaired neurogenic motility by disrupting cell-cell connectivity in the colonic muscularis. These findings may have translational significance, because silencing CARMN in human colonic SMCs significantly attenuated contractile gene expression, including MYLK, and decreased SMC contractility. Luciferase reporter assays showed that CARMN enhances the transactivation activity of the master regulator of SMC contractile phenotype, myocardin, thereby maintaining the GI SMC myogenic program. CONCLUSIONS: Our data suggest that Carmn is indispensable for maintaining GI SMC contractile function in mice and that loss of function of CARMN may contribute to human visceral myopathy. To our knowledge this is the first study showing an essential role of lncRNA in the regulation of visceral SMC phenotype.

The biological clock enhancer nobiletin ameliorates steatosis in genetically obese mice by restoring aberrant hepatic circadian rhythm.

Nonalcoholic fatty liver disease (NAFLD) is associated with disruption of homeostatic lipid metabolism, but underlying processes are poorly understood. One possible mechanism is impairment in hepatic circadian rhythm, which regulates key lipogenic mediators in the liver and whose circadian oscillation is diminished in obesity. Nobiletin enhances biological rhythms by activating RAR-related orphan receptor nuclear receptor, protecting against metabolic syndrome in a clock-dependent manner. The effect of nobiletin in NAFLD is unclear. In this study, we investigate the clock-enhancing effects of nobiletin in genetically obese (db/db) PER2::LUCIFERASE reporter mice with fatty liver. We report microarray expression data suggesting hepatic circadian signaling is impaired in db/db mice with profound hepatic steatosis. Circadian PER2 activity, as assessed by mRNA and luciferase assay, was significantly diminished in liver of db/db PER2::LUCIFERASE reporter mice. Continuous animal monitoring systems and constant dark studies suggest the primary circadian defect in db/db mice lies within peripheral hepatic oscillators and not behavioral rhythms or the master clock. In vitro, nobiletin restored PER2 amplitude in lipid-laden PER2::LUCIFERASE reporter macrophages. In vivo, nobiletin dramatically upregulated core clock gene expression, hepatic PER2 activity, and ameliorated steatosis in db/db PER2::LUCIFERASE reporter mice. Mechanistically, nobiletin reduced serum insulin levels, decreased hepatic Srebp1c, Acaca1, Tnfα, and Fgf21 expression, but did not improve Plin2, Plin5, or Cpt1, suggesting nobiletin attenuates steatosis in db/db mice via downregulation of hepatic lipid accumulation. These data suggest restoring endogenous rhythm with nobiletin resolves steatosis in obesity, proposing that hypothesis that targeting the biological clock may be an attractive therapeutic strategy for NAFLD.NEW & NOTEWORTHY NAFLD is the most common chronic liver disease, but underlying mechanisms are unclear. We show here that genetically obese (db/db) mice with fatty liver have impaired hepatic circadian rhythm. Hepatic Per2 expression and PER2 reporter activity are diminished in db/db PER2::LUCIFERASE mice. The biological clock-enhancer nobiletin restores hepatic PER2 in db/db PER2::LUCIFERASE mice, resolving steatosis via downregulation of Srebp1c. These studies suggest targeting the circadian clock may be beneficial strategy in NAFLD.

Protective role of Cav-1 in pneumolysin-induced endothelial barrier dysfunction.

Pneumolysin (PLY) is a bacterial pore forming toxin and primary virulence factor of Streptococcus pneumonia, a major cause of pneumonia. PLY binds cholesterol-rich domains of the endothelial cell (EC) plasma membrane resulting in pore assembly and increased intracellular (IC) Ca2+ levels that compromise endothelial barrier integrity. Caveolae are specialized plasmalemma microdomains of ECs enriched in cholesterol. We hypothesized that the abundance of cholesterol-rich domains in EC plasma membranes confers cellular susceptibility to PLY. Contrary to this hypothesis, we found increased PLY-induced IC Ca2+ following membrane cholesterol depletion. Caveolin-1 (Cav-1) is an essential structural protein of caveolae and its regulation by cholesterol levels suggested a possible role in EC barrier function. Indeed, Cav-1 and its scaffolding domain peptide protected the endothelial barrier from PLY-induced disruption. In loss of function experiments, Cav-1 was knocked-out using CRISPR-Cas9 or silenced in human lung microvascular ECs. Loss of Cav-1 significantly enhanced the ability of PLY to disrupt endothelial barrier integrity. Rescue experiments with re-expression of Cav-1 or its scaffolding domain peptide protected the EC barrier against PLY-induced barrier disruption. Dynamin-2 (DNM2) is known to regulate caveolar membrane endocytosis. Inhibition of endocytosis, with dynamin inhibitors or siDNM2 amplified PLY induced EC barrier dysfunction. These results suggest that Cav-1 protects the endothelial barrier against PLY by promoting endocytosis of damaged membrane, thus reducing calcium entry and PLY-dependent signaling.

Endothelin-1 response to whole-body vibration in obese and normal weight individuals.

Upregulation of endothelin-1 (ET-1) is the hallmark of various cardiovascular diseases (CVD). The purpose of the present study was to assess the ET-1 response to an acute bout of whole-body vibration (WBV) in humans and to determine the role of adiposity. Twenty-two participants volunteered for the study; they were grouped into overweight/obese [(OW/OB): n = 11, Age: 33 ± 4 years, Body mass index (BMI): 35 ± 10 kg/m2 ] or normal weight [(NW): n = 11, Age: 28 ± 7 years, BMI: 21 ± 2 kg/m2 ]. Participants engaged in 10 cycles of WBV exercise (1 cycle = 1 min WBV followed by 30 s of rest). Blood samples were analyzed for ET-1 pre-WBV (PRE), immediately post (POST), 1 h (1H), 3 h (3H), and 24 h (24H) post-WBV. There was a significant time main effect of WBV on circulating ET-1 (F = 12.5, p < 0.001); however, the ET-1 response was similar (F = 0.180, p = 0.677) between groups. Specifically, compared to PRE, a significant increase in ET-1 was observed at 1H (p = 0.017) and 3H (p = 0.025). In addition, concentrations of ET-1 were significantly lower at 24H compared to PRE (p = 0.019), 1H (p < 0.001), and 3H (p < 0.001). Maximal oxygen uptake during WBV was similar between the two groups. Acute WBV resulted in an initial rise in ET-1, followed by a significantly lower ET-1 at 24H in both groups. Findings support the utility of routine WBV exercise to elicit a decrease in ET-1 and improve CVD risk, similar to what has been reported with traditional modes of exercise.

Obesity Induces Disruption of Microvascular Endothelial Circadian Rhythm.

Obese individuals are at significantly elevated risk of developing cardiovascular disease (CVD). Additionally, obesity has been associated with disrupted circadian rhythm, manifesting in abnormal sleeping and feeding patterns. To date, the mechanisms linking obesity, circadian disruption, and CVD are incompletely understood, and insight into novel mechanistic pathways is desperately needed to improve therapeutic potential and decrease morbidity and mortality. The objective of this study was to investigate the roles of metabolic and circadian disruptions in obesity and assess their contributions in promoting vascular disease. Lean (db/+) and obese (db/db) mice were subjected to 12 weeks of constant darkness to differentiate diurnal and circadian rhythms, and were assessed for changes in metabolism, gene expression, and vascular function. Expression of endothelial nitric oxide synthase (eNOS), an essential enzyme for vascular health, was blunted in obesity and correlated with the oscillatory loss of the novel regulator cezanne (OTUD7B). Lean mice subjected to constant darkness displayed marked reduction in vasodilatory capacity, while endothelial dysfunction of obese mice was not further compounded by diurnal insult. Endothelial gene expression of essential circadian clock components was altered in obesity, but imperfectly phenocopied in lean mice housed in constant darkness, suggesting overlapping but separate mechanisms driving endothelial dysfunction in obesity and circadian disruption. Taken together, these data provide insight into the nature of endothelial circadian rhythm in obesity and suggest a distinct mechanism by which obesity causes a unique circadian defect in the vasculature.

Protein kinase N2 connects blood flow with NO production in a double AKT.

Shear stress is an important regulator of blood flow, and luminal endothelial cells (ECs) sense increases in frictional forces and respond with an appropriate release of vasoactive mediators. In this issue of the JCI, Jin et al. identified a mechanism by which ECs respond to shear stress with endothelial NOS (eNOS) activation and NO release. The authors showed that PKN2 was activated by fluid shear stress and contributed to eNOS activation via a double play - indirect phosphorylation at serine 1177 (S1177) via AKT and direct phosphorylation of the S1179 site. Phosphorylation of both sites individually increased eNOS activity, but together they had an additive effect. In sum, these findings reveal exciting details about how shear stress regulates eNOS and have important implications for blood flow and blood pressure.

Nf1 heterozygous mice recapitulate the anthropometric and metabolic features of human neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is a heritable cancer predisposition syndrome resulting from mutations in the NF1 tumor suppressor gene. Genotype-phenotype correlations for NF1 are rare due to the large number of NF1 mutations and role of modifier genes in manifestations of NF1; however, emerging reports suggest that persons with NF1 display a distinct anthropometric and metabolic phenotype featuring short stature, low body mass index, increased insulin sensitivity, and protection from diabetes. Nf1 heterozygous (Nf1+/-) mice accurately reflect the dominant inheritance of NF1 and are regularly employed as a model of NF1. Here, we sought to identify whether Nf1+/- mice recapitulate the anthropometric and metabolic features identified in persons with NF1. Littermate 16-20 week-old male wildtype (WT) and Nf1+/- C57B/6J mice underwent nuclear magnetic resonance (NMR), indirect calorimetry, and glucose/insulin/pyruvate tolerance testing. In some experiments, tissues were harvested for NMR and histologic characterization. Nf1+/- mice are leaner with significantly reduced visceral and subcutaneous fat mass, which corresponds with an increased density of small adipocytes and reduced leptin levels. Additionally, Nf1+/- mice are highly reliant on carbohydrates as an energy substrate and display increased glucose clearance and insulin sensitivity, but normal response to pyruvate suggesting enhanced glucose utilization and preserved gluconeogenesis. Finally, WT and Nf1+/- mice subjected to high glucose diet were protected from diet-induced obesity and hyperglycemia. Our data suggest that Nf1+/- mice closely recapitulate the anthropometric and metabolic phenotype identified in persons with NF1, which will impact the interpretation of previous and future translational studies of NF1.

Whole body vibration elicits differential immune and metabolic responses in obese and normal weight individuals.

Traditional aerobic exercise reduces the risk of developing chronic diseases by inducing immune, metabolic, and myokine responses. Following traditional exercise, both the magnitude and time-course of these beneficial responses are different between obese compared to normal weight individuals. Although obesity may affect the ability to engage in traditional exercise, whole body vibration (WBV) has emerged as a more tolerable form of exercise . The impact of WBV on immune, metabolic, and myokine responses as well as differences between normal weight and obese individuals, however, is unknown. Purpose: To determine if WBV elicits differential magnitudes and time-courses of immune, metabolic, and myokine responses between obese and normal weight individuals. Methods: 21 participants [Obese (OB): n = 11, Age: 33 ±â€¯4 y, percent body fat (%BF): 39.1 ±â€¯2.4% & Normal weight (NW) n = 10, Age: 28 ±â€¯8 y, %BF: 17.4 ±â€¯2.1%] engaged in 10 cycles of WBV exercise [1 cycle = 1 min of vibration followed by 30 s of rest]. Blood samples were collected pre-WBV (PRE), immediately (POST), 3 h (3H), and 24 h (24H) post-WBV and analyzed for leukocytes, insulin, glucose, and myokines (IL-6, decorin, myostatin). Results: The peak (3H) percent change in neutrophil counts (OB: 13.9 ±â€¯17.4 vs. NW: 47.2 ±â€¯6.2%Δ; p = 0.007) was different between groups. The percent change in neutrophil percentages was increased in NW (POST: -1.6 ±â€¯2.0 vs. 3H: 13.0 ±â€¯7.2%Δ, p = 0.019) but not OB (p > 0.05). HOMA ß-cell function was increased at 24H (PRE: 83.4 ±â€¯5.4 vs. 24H: 131.0 ±â€¯14.1%; p = 0.013) in NW and was not altered in OB (p > 0.05). PRE IL-6 was greater in OB compared to NW (OB: 2.7 ±â€¯0.6 vs. NW: 0.6 ±â€¯0.1 pg/mL; p = 0.011); however, the percent change from PRE to peak (3H) was greater in NW (OB: 148.1 ±â€¯47.9 vs. NW: 1277.9 ±â€¯597.6 %Δ; p = 0.035). Creatine kinase, decorin, and myostatin were not significantly altered in either group (p > 0.05). Conclusion: Taken together, these data suggest that acute whole body vibration elicits favorable immune, metabolic, and myokine responses and that these responses differ between obese and normal weight individuals.

Galectin-3 is expressed in vascular smooth muscle cells and promotes pulmonary hypertension through changes in proliferation, apoptosis, and fibrosis.

A defining characteristic of pulmonary hypertension (PH) is the extensive remodeling of pulmonary arteries (PAs), which results in progressive increases in vascular resistance and stiffness and eventual failure of the right ventricle. There is no cure for PH and identification of novel molecular mechanisms that underlie increased proliferation, reduced apoptosis, and excessive extracellular matrix production in pulmonary artery smooth muscle cells (PASMCs) is a vital objective. Galectin-3 (Gal-3) is a chimeric lectin and potent driver of many aspects of fibrosis, but its role in regulating PASMC behavior in PH remains poorly understood. Herein, we evaluated the importance of increased Gal-3 expression and signaling on PA vascular remodeling and cardiopulmonary function in experimental models of PH. Gal-3 expression was quantified by qRT-PCR, immunoblotting, and immunofluorescence imaging, and its functional role was assessed by specific Gal-3 inhibitors and CRISPR/Cas9-mediated knockout of Gal-3 in the rat. In rat models of PH, we observed increased Gal-3 expression in PASMCs, which stimulated migration and resistance to apoptosis, whereas silencing or genetic deletion reduced cellular migration and PA fibrosis and increased apoptosis. Gal-3 inhibitors attenuated and reversed PA remodeling and fibrosis, as well as hemodynamic indices in monocrotaline (MCT)-treated rats in vivo. These results were supported by genetic deletion of Gal-3 in both MCT and Sugen Hypoxia rat models. In conclusion, our results suggest that elevated Gal-3 levels contribute to inappropriate PA remodeling in PH by enhancing multiple profibrotic mechanisms. Therapeutic strategies targeting Gal-3 may be of benefit in the treatment of PH.

Increased Muscle Mass Protects Against Hypertension and Renal Injury in Obesity.

Background Obesity compromises cardiometabolic function and is associated with hypertension and chronic kidney disease. Exercise ameliorates these conditions, even without weight loss. Although the mechanisms of exercise's benefits remain unclear, augmented lean body mass is a suspected mechanism. Myostatin is a potent negative regulator of skeletal muscle mass that is upregulated in obesity and downregulated with exercise. The current study tested the hypothesis that deletion of myostatin would increase muscle mass and reduce blood pressure and kidney injury in obesity. Methods and Results Myostatin knockout mice were crossed to db/db mice, and metabolic and cardiovascular functions were examined. Deletion of myostatin increased skeletal muscle mass by ≈50% to 60% without concomitant weight loss or reduction in fat mass. Increased blood pressure in obesity was prevented by the deletion of myostatin, but did not confer additional benefit against salt loading. Kidney injury was evident because of increased albuminuria, which was abolished in obese mice lacking myostatin. Glycosuria, total urine volume, and whole kidney NOX-4 levels were increased in obesity and prevented by myostatin deletion, arguing that increased muscle mass provides a multipronged defense against renal dysfunction in obese mice. Conclusions These experimental observations suggest that loss of muscle mass is a novel risk factor in obesity-derived cardiovascular dysfunction. Interventions that increase muscle mass, either through exercise or pharmacologically, may help limit cardiovascular disease in obese individuals.

Hsp70 Suppresses Mitochondrial Reactive Oxygen Species and Preserves Pulmonary Microvascular Barrier Integrity Following Exposure to Bacterial Toxins.

Pneumonia is a leading cause of death in children and the elderly worldwide, accounting for 15% of all deaths of children under 5 years old. Streptococcus pneumoniae is a common and aggressive cause of pneumonia and can also contribute to meningitis and sepsis. Despite the widespread use of antibiotics, mortality rates for pneumonia remain unacceptably high in part due to the release of bacterial toxins. Pneumolysin (PLY) is a cholesterol-dependent toxin that is produced by Streptococcus, and it is both necessary and sufficient for the development of the extensive pulmonary permeability edema that underlies acute lung injury. The mechanisms by which PLY disrupts the pulmonary endothelial barrier are not fully understood. Previously, we found that reactive oxygen species (ROS) contribute to the barrier destructive effects of PLY and identified an unexpected but potent role of Hsp70 in suppressing ROS production. The ability of Hsp70 to influence PLY-induced barrier dysfunction is not yet described, and the goal of the current study was to identify whether Hsp70 upregulation is an effective strategy to protect the lung microvascular endothelial barrier from G+ bacterial toxins. Overexpression of Hsp70 via adenovirus-mediated gene transfer attenuated PLY-induced increases in permeability in human lung microvascular endothelial cells (HLMVEC) with no evidence of cytotoxicity. To adopt a more translational approach, we employed a pharmacological approach using geranylgeranylacetone (GGA) to acutely upregulate endogenous Hsp70 expression. Following acute treatment (6 h) with GGA, HLMVECs exposed to PLY displayed improved cell viability and enhanced endothelial barrier function as measured by both Electric Cell-substrate Impedance Sensing (ECIS) and transwell permeability assays compared to control treated cells. PLY promoted increased mitochondrial ROS, decreased mitochondrial oxygen consumption, and increased caspase 3 cleavage and cell death, which were collectively improved in cells pretreated with GGA. In mice, IP pretreatment with GGA 24 h prior to IT administration of PLY resulted in significantly less Evans Blue Dye extravasation compared to vehicle, indicating preserved endothelial barrier integrity and suggesting that the acute upregulation of Hsp70 may be an effective therapeutic approach in the treatment of lung injury associated with pneumonia.

A single high-fat meal provokes pathological erythrocyte remodeling and increases myeloperoxidase levels: implications for acute coronary syndrome.

High-fat meal (HFM) consumption can produce acute lipemia and trigger myocardial infarction in patients with atherosclerosis, but the mechanisms are poorly understood. Erythrocytes (red blood cells, RBCs) intimately interact with inflammatory cells and blood vessels and play a complex role in regulating vascular function. Chronic high-fat feeding in mice induces pathological RBC remodeling, suggesting a novel link between HFM, RBCs, and vascular dysfunction. However, whether acute HFM can induce RBC remodeling in humans is unknown. Ten healthy individuals were subjected to biochemical testing and assessment of endothelial-dependent flow-mediated dilation (FMD) before and after a single HFM or iso-caloric meal (ICM). Following the HFM, triglyceride, cholesterol, and free fatty acid levels were all significantly increased, in conjunction with impaired post-prandial FMD. Additionally, peripheral blood smears demonstrated microcytes, remodeled RBCs, and fatty monocytes. Increased intracellular ROS and nitration of protein band 3 was detected in RBCs following the HFM. The HFM elevated plasma and RBC-bound myeloperoxidase (MPO), which was associated with impaired FMD and oxidation of HDL. Monocytic cells exposed to lipid in vitro released MPO, while porcine coronary arteries exposed to fatty acids ex vivo took up MPO. We demonstrate in humans that a single HFM induces pathological RBC remodeling and concurrently elevates MPO, which can potentially enter the blood vessel wall to trigger oxidative stress and destabilize vulnerable plaques. These novel findings may have implications for the short-term risk of HFM consumption and alimentary lipemia in patients with atherosclerosis.

Deletion of the Duffy antigen receptor for chemokines (DARC) promotes insulin resistance and adipose tissue inflammation during high fat feeding.

OBJECTIVE: Inflammation in adipose tissues in obesity promotes insulin resistance and metabolic disease. The Duffy antigen receptor for chemokines (DARC) is a promiscuous non-signaling receptor expressed on erythrocytes and other cell types that modulates tissue inflammation by binding chemokines such as monocyte chemoattractant protein-1 (MCP-1) and by acting as a chemokine reservoir. DARC allelic variants are common in humans, but the role of DARC in modulating obesity-related metabolic disease is unknown. METHODS: We examined body weight gain, tissue adiposity, metabolic parameters and inflammatory marker expression in wild-type and DARC knockout mice fed a chow diet (CD) and high fat diet (HFD). RESULTS: Compared to wild-type mice, HFD-fed DARC knockout mice developed glucose intolerance and insulin resistance independent of increases in body weight or adiposity. Interestingly, insulin sensitivity was also diminished in lean male DARC knockout mice fed a chow diet. Insulin production was not reduced by DARC gene deletion, and plasma leptin levels were similar in HFD fed wild-type and DARC knockout mice. MCP-1 levels in plasma rose significantly in the HFD fed wild-type mice, but not in the DARC knockout mice. Conversely, adipose tissue MCP-1 levels were higher, and more macrophage crown-like structures were detected, in the HFD fed DARC knockout mice as compared with the wild-type mice, consistent with augmented adipose tissue inflammation that is not accurately reflected by plasma levels of DARC-bound MCP-1 in these mice. CONCLUSIONS: These findings suggest that DARC regulates metabolic function and adipose tissue inflammation, which may impact obesity-related disease in ethnic populations with high frequencies of DARC allelic variants.