Despite increasing evidence suggesting a role for NADPH oxidases (Nox) in bone pathophysiology, whether Nox enzymes contribute to obesity-mediated bone remodeling remains to be clearly elucidated. Nox2 is one of the predominant Nox enzymes expressed in the bone marrow microenvironment and is a major source of ROS generation during inflammatory processes. It is also well recognized that a high-fat diet (HFD) induces obesity, which negatively impacts bone remodeling. In this work, we investigated the effect of Nox2 loss of function on obesity-mediated alteration of bone remodeling using wild-type (WT) and Nox2-knockout (KO) mice fed with a standard lab chow diet (SD) as a control or a HFD as an obesity model. Bone mineral density (BMD) of mice was assessed at the beginning and after 3 months of feeding with SD or HFD. Our results show that HFD increased bone mineral density to a greater extent in KO mice than in WT mice without affecting the total body weight and fat mass. HFD also significantly increased the number of adipocytes in the bone marrow microenvironment of WT mice as compared to KO mice. The bone levels of proinflammatory cytokines and proosteoclastogenic factors were also significantly elevated in WT-HFD mice as compared to KO-HFD mice. Furthermore, the in vitro differentiation of bone marrow cells into osteoclasts was significantly increased when using bone marrow cells from WT-HFD mice as compared to KO-HFD mice. Our data collectively suggest that Nox2 is implicated in HFD-induced deleterious bone remodeling by enhancing bone marrow adipogenesis and osteoclastogenesis.

Inflammation and oxidative stress through the production of reactive oxygen species (ROS) are consistently associated with metabolic syndrome/type 2 diabetes. Although the role of Nox2, a major ROS-generating enzyme, is well described in host defense and inflammation, little is known about its potential role in insulin resistance in skeletal muscle. Insulin resistance induced by a high fat diet was mitigated in Nox2-null mice compared with wild-type mice after 3 or 9 months on the diet. High fat feeding increased Nox2 expression, superoxide production, and impaired insulin signaling in skeletal muscle tissue of wild-type mice but not in Nox2-null mice. Exposure of C2C12 cultured myotubes to either high glucose concentration, palmitate, or H2O2 decreases insulin-induced Akt phosphorylation and glucose uptake. Pretreatment with catalase abrogated these effects, indicating a key role for H2O2 in mediating insulin resistance. Down-regulation of Nox2 in C2C12 cells by shRNA prevented insulin resistance induced by high glucose or palmitate but not H2O2. These data indicate that increased production of ROS in insulin resistance induced by high glucose in skeletal muscle cells is a consequence of Nox2 activation. This is the first report to show that Nox2 is a key mediator of insulin resistance in skeletal muscle.

Background The identity of the ROS-producing enzyme(s) in human spermatozoa remains uncertain. Results NOX5 NADPH oxidase, but not NOX1/2/4, is expressed in human spermatozoa and produces superoxide. Inhibition of NOX5 activity reduces spermatozoa motility. Conc lusion NOX5 is the main source of superoxide and is implicated in human spermatozoa motility. Signif icance NOX5 might control the numerous ROSdependent (patho)physiological processes in human spermatozoa.

Physiological and pathological processes in spermatozoa involve the production of reactive oxygen species (ROS), but the identity of the ROS-producing enzyme system(s) remains a matter of speculation. We provide the first evidence that NOX5 NADPH oxidase is expressed and functions in human spermatozoa. Immunofluorescence microscopy detected NOX5 protein in both the flagella/neck region and the acrosome. Functionally, spermatozoa exposed to calcium ionophore, phorbol ester, or H(2)O(2) exhibited superoxide anion production, which was blocked by addition of superoxide dismutase, a Ca(2+) chelator, or inhibitors of either flavoprotein oxidases (diphenylene iododonium) or NOX enzymes (GKT136901). Consistent with our previous overexpression studies, we found that H(2)O(2)-induced superoxide production by primary sperm cells was mediated by the non-receptor tyrosine kinase c-Abl. Moreover, the H(V)1 proton channel, which was recently implicated in spermatozoa motility, was required for optimal superoxide production by spermatozoa. Immunoprecipitation experiments suggested an interaction among NOX5, c-Abl, and H(V)1. H(2)O(2) treatment increased the proportion of motile sperm in a NOX5-dependent manner. Statistical analyses showed a pH-dependent correlation between superoxide production and enhanced sperm motility. Collectively, our findings show that NOX5 is a major source of ROS in human spermatozoa and indicate a role for NOX5-dependent ROS generation in human spermatozoa motility.

Insulin resistance due in part to impaired glucose uptake in skeletal muscle is consistently associated with oxidative stress. Skeletal muscle cells express NADPH oxidase type 2 (NOX2), a potent generator of reactive oxygen species (ROS). Our central hypothesis is that NOX2 is a key factor in the development of insulin resistance. In vivo, our preliminary data show that insulin resistance induced by a high‐fat diet (HFD) is associated with increased ROS production, NOX2 expression, and leukocyte infiltration in skeletal muscle. Furthermore, superoxide production in skeletal muscle, fasting blood glucose and insulin levels, and the insulin resistance index were all reduced in HFD‐fed NOX2‐null vs. wild‐type mice. These results suggest that NOX2 is implicated in the development of insulin resistance. A full understanding of NOX2 regulation is likely to help for the prevention or treatment of type 2 diabetes patients.

Abstract The San Antonio Nathan Shock Center Conferences have attracted international speakers and participants since 1995. This annual conference, held in Bandera, Texas, USA, addresses a different topic in the biology of aging each year. The venue's intimate setting, relatively remote location, and common areas are ideal for a small conference (80–100 participants), where copious informal intellectual interchange supplements that of the formal sessions. The 2011 meeting, part of an annual series sponsored by the University of Texas Health Science Center San Antonio, TX, USA, and the Nathan Shock Center of Excellence in the Biology of Aging, addressed the causes of age-associated inflammation and its effect on age-associated diseases.

Clinical evidence indicates that fat is inversely proportional to bone mass in elderly obese women. However, it remains unclear whether obesity accelerates bone loss. In this report we present evidence that increased visceral fat leads to inflammation and subsequent bone loss in 12-month-old C57BL/6J mice that were fed 10% corn oil (CO)-based diet and a control lab chow (LC) for 6 months. As expected from our previous work, CO-fed mice demonstrated increased visceral fat and enhanced total body fat mass compared to LC. The adipocyte-specific PPARγ and bone marrow (BM) adiposity were increased in CO-fed mice. In correlation with those modifications, inflammatory cytokines (IL-1β, IL-6, TNF-α) were significantly elevated in CO-fed mice compared to LC-fed mice. This inflammatory BM microenvironment resulted in increased superoxide production in osteoclasts and undifferentiated BM cells. In CO-fed mice, the increased number of osteoclasts per trabecular bone length and the increased osteoclastogenesis assessed ex-vivo suggest that CO diet induces bone resorption. Additionally, the up-regulation of osteoclast-specific cathepsin k and RANKL expression and down-regulation of osteoblast-specific RUNX2/Cbfa1 supports this bone resorption in CO-fed mice. Also, CO-fed mice exhibited lower trabecular bone volume in the distal femoral metaphysis and had reduced OPG expression. Collectively, our results suggest that increased bone resorption in mice fed a CO-enriched diet is possibly due to increased inflammation mediated by the accumulation of adipocytes in the BM microenvironment. This inflammation may consequently increase osteoclastogenesis, while reducing osteoblast development in CO-fed mice.

In this study, we examined the effect of CLA isomers in preventing age-associated muscle loss and the mechanisms underlying this effect, using 12-months-old C57BL/6 mice fed 10% corn oil (CO) or a diet supplemented with 0.5% c9t11-CLA, t10c12-CLA, or c9t11-CLA+t10c12-CLA (CLA-mix) for 6months. Both t10c12-CLA and CLA-mix groups showed significantly higher muscle mass, as compared to CO and c9t11-CLA groups, measured by dual-energy X-ray absorptiometry and muscle wet weight. Enhanced mitochondrial ATP production, with higher membrane potential, and elevated muscle antioxidant enzymes (catalase and glutathione peroxidase) production, accompanied by slight increase in H2O2 production was noted in t10c12-CLA and CLA-mix groups, as compared to that of CO and c9t11-CLA groups. Oxidative stress, as measured by serum malondialdehyde and inflammation, as measured by LPS-treated splenocyte IL-6 and TNF-α, were significantly less in CLA isomers groups. Thus, CLA may be a novel dietary supplement that will prevent sarcopenia by maintaining redox balance during aging.

The importance of H2O2 as a cellular signaling molecule has been demonstrated in a number of cell types and pathways. Here we explore a positive feedback mechanism of H2O2-mediated regulation of the phagocyte respiratory burst NADPH oxidase (NOX2). H2O2 induced a dose-dependent stimulation of superoxide production in human neutrophils, as well as in K562 leukemia cells overexpressing NOX2 system components. Stimulation was abrogated by the addition of catalase, the extracellular Ca2+ chelator BAPTA, the T-type Ca2+ channel inhibitor mibefradil, the PKCδ inhibitor rottlerin, or the c-Abl nonreceptor tyrosine kinase inhibitor imatinib mesylate or by overexpression of a dominant-negative form of c-Abl. H2O2 induced phosphorylation of tyrosine 311 on PKCδ and this activating phosphorylation was blocked by treatment with rottlerin, imatinib mesylate, or BAPTA. Rac GTPase activation in response to H2O2 was abrogated by BAPTA, imatinib mesylate, or rottlerin. In conclusion, H2O2 stimulates NOX2-mediated superoxide generation in neutrophils and K562/NOX2 cells via a signaling pathway involving Ca2+ influx and c-Abl tyrosine kinase acting upstream of PKCδ. This positive feedback regulatory pathway has important implications for amplifying the innate immune response and contributing to oxidative stress in inflammatory disorders.

We investigated the mechanism of H2O2 activation of the Ca2+-regulated NADPH oxidase NOX5. H2O2 induced a transient, dose-dependent increase in superoxide production in K562 cells expressing NOX5. Confocal studies demonstrated that the initial calcium influx generated by H2O2 is amplified by a feedback mechanism involving NOX5-dependent superoxide production and H2O2. H2O2 NOX5 activation was inhibited by extracellular Ca2+ chelators, a pharmacological inhibitor of c-Abl, and overexpression of kinase-dead c-Abl. Transfected kinase-active GFP–c-Abl colocalized with vesicular sites of superoxide production in a Ca2+-dependent manner. In contrast to H2O2, the Ca2+ ionophore ionomycin induced NOX5 activity independent of c-Abl. Immunoprecipitation of cell lysates revealed that active GFP–c-Abl formed oligomers with endogenous c-Abl and that phosphorylation of both proteins was increased by H2O2 treatment. Furthermore, H2O2-induced NOX5 activity correlated with increased localization of c-Abl to the membrane fraction, and NOX5 proteins could be coimmunoprecipitated with GFP–Abl proteins. Our data demonstrate for the first time that NOX5 is activated by c-Abl through a Ca2+-mediated, redox-dependent signaling pathway and suggest a functional association between NOX5 NADPH oxidase and c-Abl.

We investigated the role of the single SH3 domain of NOXA1 in NOX1 NADPH oxidase function using wild-type and mutated NOXA1 and the products of two variant NOXA1 transcripts isolated from CaCo2 cells by reverse transcription polymerase chain reaction. The first variant, NOXA1(trunc), contained a number of point mutations, including A51T, T261A, and a nonsense mutation at position 274. On transfection into K562 cells stably expressing NOX1 and NOXO1, both NOXA1(trunc) and an equivalent truncated wild-type NOXA1(1-273) were expressed as approximately 29-kDa truncated NOXA1 proteins lacking both PB1 and SH3 domains, yet both were as active as wild-type NOXA1 in phorbol-stimulated superoxide generation. Kinetic analysis demonstrated that truncated NOXA1 activated the NOX1 system at an accelerated rate compared with NOXA1. Deletion studies showed that the slower kinetics of wild-type NOXA1 depended primarily on its SH3 domain, suggesting SH3-dependent delay in forming the active NOX1/NOXO1/NOXA1 complex. The second variant, NOXA1(inhib), encoded a protein lacking the activation domain due to absence of exons 5 and 6 but including a heptapeptide (EPDVPLA) SH3 domain insertion resulting from alternative splicing in exon 14. NOXA1(inhib) failed to support superoxide-generating activity and exhibited transdominant inhibition of NOXA1. Insertion of the heptapeptide into the corresponding site in wild-type NOXA1 inhibited its activity by approximately 90%, rendered it a transdominant inhibitor of wild-type NOXA1, and abrogated binding of its SH3 domain to NOXO1 and p47(phox). These studies demonstrate that, in reconstituted NOX1/NOXO1/NOXA1 systems, the NOXA1 SH3 domain is not required for function but, when present, can critically modulate the activity of the enzyme system.

BACKGROUND Statins may improve left ventricular remodeling after myocardial infarction. We tested whether statins inhibit cardiomyocyte apoptosis through glycogen synthase kinase 3beta (GSK3beta) inactivation and evaluated activation of downstream transcription factors. METHODS/RESULTS Mevastatin and pravastatin activated serine/threonine kinase Akt in neonatal cardiomyocytes dose and time dependently with maximal activation at 15 min/10 microM. Caspase-3 activity was induced 2.73 +/- 0.29-fold by 6 h of hypoxia followed by 18 h of reoxygenation. Pravastatin added at the beginning of the reoxygenation period reduced caspase-3 activation to 1.26 +/- 0.06-fold compared to control cells (P < 0.001). Similar results were obtained for mevastatin (decreased to 1.98 +/- 0.45-fold, P < 0.05). TUNEL staining of neonatal cardiomyocytes after 24 h reoxygenation and 4',6'-diamidino-2-phenylindole staining of adult rat cardiomyocytes after 6 h H(2)O(2) showed reduced cardiomyocyte apoptosis in the presence of statin. Analysis of signaling pathways downstream of Akt revealed phosphorylation of GSK3beta. Transcription factor cAMP-responsive element binding (CREB) protein showed weak phosphorylation at serine 133; transcription factor NF-kappaB was not significantly activated after statin treatment as evaluated by EMSA. The GSK3beta target protein beta-catenin was stabilized at 3 h after statin treatment both in neonatal as well as adult rat cardiomyocytes. Transfection with constitutive active GSK3betaS9A sensitized neonatal cardiomyocytes to hypoxia/reoxygenation-induced apoptosis as measured by annexin V/propidium iodide staining. Furthermore, myocardial protein extracts of mice revealed GSK3beta inactivation after administration of pravastatin intraperitoneally. CONCLUSIONS Statins inhibit cardiomyocyte apoptosis in association with GSK3beta inactivation. Inactivation of GSK3beta leads to stabilization of beta-catenin in cardiomyocytes.

In vivo, left ventricular remodeling after myocardial infarction involves hypertrophy generally attributed to increased cardiac workload. We hypothesized that hypoxia/reoxygenation directly induces cardiomyocyte hypertrophy and studied several participating kinases and transcription factors in isolated cardiomyocytes. Hypoxia for 6 h followed by 42 h reoxygenation induced cardiomyocyte hypertrophy assessed by 3H leucine incorporation and immunohistochemistry. Inhibition of reactive oxygen species (ROS), serine/threonine kinase AKT, and ERK abolished reoxygenation-induced hypertrophy. In addition, a beta2-adrenergic receptor (beta2-AR) antagonist, as well as Gi inhibitor pertussis toxin, blocked reoxygenation-induced hypertrophy. Hypoxia for 6 h increased transcription factors CREB, NF-kappaB, and GATA DNA binding activities. However, only CREB DNA-binding was sustained during reoxygenation. Inhibition of PI3-kinase, ERK, and PKA abrogated reoxygenation-induced CREB DNA-binding without affecting CREB serine-133 phosphorylation. These same pathways were found to regulate hypoxia/reoxygenation-induced GSK3beta kinase activity and CREB serine-129 de-phosphorylation. GSK3beta mutants resistant to phosphorylation blocked the stimulation of CRE-dependent transcription induced by hypoxia/reoxygenation. Transfection of cardiomyocytes with a dominant-negative mutant of CREB abrogated hypoxia/reoxygenation-induced hypertrophy. We suggest that hypoxia/reoxygenation induces cardiomyocyte hypertrophy through CREB activation. Inactivation of GSK3beta by hypoxia/reoxygenation, possibly integrating PI3-kinase and ERK pathways downstream of beta2-AR and ROS, is a prerequisite for CRE-dependent transcription. Transient hypoxia may contribute to cardiac hypertrophy in ischemic heart disease independent of cardiac workload.