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.

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.

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.

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.

Genetic Deletion of NADPH Oxidase 1 Rescues Microvascular Function in Mice With Metabolic Disease.

RATIONALE: Early vascular changes in metabolic disease that precipitate the development of cardiovascular complications are largely driven by reactive oxygen species accumulation, yet the extent to which excess reactive oxygen species derive from specific NADPH oxidase isoforms remains ill defined. OBJECTIVE: Identify the role of Nox1 in the development of microvascular dysfunction in metabolic disease. METHODS AND RESULTS: Four genotypes were generated by breeding Nox1 knockout mice with db/db mice: lean (HdbWnox1), lean Nox1 knockout (HdbKnox1), obese (KdbWnox1), and obese KK (KdbKnox1). The degree of adiposity, insulin resistance, and dyslipidemia in KW mice was not influenced by Nox1 deletion as determined by nuclear magnetic resonance spectroscopy, glucose tolerance tests, and plasma analyses. Endothelium-dependent responses to acetylcholine in pressurized mesenteric arteries were reduced in KW versus HW (P<0.01), whereas deletion of Nox1 in KW mice normalized dilation. Vasodilator responses after inhibition of NO synthase blunted acetylcholine responses in KK and lean controls, but had no impact in KW, attributing recovered dilatory capacity in KK to normalization of NO. Acetylcholine responses were improved (P<0.05) with Tempol, and histochemistry revealed oxidative stress in KW animals, whereas Tempol had no impact and reactive oxygen species staining was negligible in KK. Blunted dilatory responses to an NO donor and loss of myogenic tone in KW animals were also rescued with Nox1 deletion. CONCLUSIONS: Nox1 deletion reduces oxidant load and restores microvascular health in db/db mice without influencing the degree of metabolic dysfunction. Therefore, targeted Nox1 inhibition may be effective in the prevention of vascular complications.

Ptp1b deletion in pro-opiomelanocortin neurons increases energy expenditure and impairs endothelial function via TNF-α dependent mechanisms.

Protein tyrosine phosphatase 1b (Ptp1b) is a negative regulator of leptin and insulin-signalling pathways. Its targeted deletion in proopiomelanocortin (POMC) neurons protects mice from obesity and diabetes by increasing energy expenditure. Inflammation accompanies increased energy expenditure. Therefore, the present study aimed to determine whether POMC-Ptp1b deletion increases energy expenditure via an inflammatory process, which would impair endothelial function. We characterized the metabolic and cardiovascular phenotypes of Ptp1b+/+ and POMC-Ptp1b-/- mice. Clamp studies revealed that POMC-Ptp1b deletion reduced body fat and increased energy expenditure as evidenced by a decrease in feed efficiency and an increase in oxygen consumption and respiratory exchange ratio. POMC-Ptp1b deletion induced a 2.5-fold increase in plasma tumour necrosis factor α (TNF-α) levels and elevated body temperature. Vascular studies revealed an endothelial dysfunction in POMC-Ptp1b-/- mice. Nitric oxide synthase inhibition [N-nitro-L-arginine methyl ester (L-NAME)] reduced relaxation to a similar extent in Ptp1b+/+ and POMC-Ptp1b-/- mice. POMC-Ptp1b deletion decreased ROS-scavenging enzymes [superoxide dismutases (SODs)] whereas it increased ROS-generating enzymes [NADPH oxidases (NOXs)] and cyclooxygenase-2 (COX-1) expression, in aorta. ROS scavenging or NADPH oxidase inhibition only partially improved relaxation whereas COX-2 inhibition and thromboxane-A2 (TXA2) antagonism fully restored relaxation in POMC-Ptp1b-/- mice Chronic treatment with the soluble TNF-α receptor etanercept decreased body temperature, restored endothelial function and reestablished aortic COX-2, NOXs and SOD expression to their baseline levels in POMC-Ptp1b-/- mice. However, etanercept promoted body weight gain and decreased energy expenditure in POMC-Ptp1b-/- mice. POMC-Ptp1b deletion increases plasma TNF-α levels, which contribute to body weight regulation via increased energy expenditure and impair endothelial function via COX-2 and ROS-dependent mechanisms.

Berardinelli-Seip congenital lipodystrophy 2 regulates adipocyte lipolysis, browning, and energy balance in adult animals.

Mutations in BSCL2/SEIPIN cause Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2), but the mechanisms whereby Bscl2 regulates adipose tissue function are unclear. Here, we generated adipose tissue (mature) Bscl2 knockout (Ad-mKO) mice, in which Bscl2 was specifically ablated in adipocytes of adult animals, to investigate the impact of acquired Bscl2 deletion on adipose tissue function and energy balance. Ad-mKO mice displayed reduced adiposity and were protected against high fat diet-induced obesity, but not insulin resistance or hepatic steatosis. Gene expression profiling and biochemical assays revealed increased lipolysis and fatty acid oxidation in white adipose tissue (WAT) and brown adipose tissue , as well as browning of WAT, owing to induction of cAMP/protein kinase A signaling upon Bscl2 deletion. Interestingly, Bscl2 deletion reduced food intake and downregulated adipose ß3-adrenergic receptor (ADRB3) expression. Impaired ADRB3 signaling partially offsets upregulated browning-induced energy expenditure and thermogenesis in Ad-mKO mice housed at ambient temperature. However, this counter-regulatory response was abrogated under thermoneutral conditions, resulting in even greater body mass loss in Ad-mKO mice. These findings suggest that Bscl2 regulates adipocyte lipolysis and ß-adrenergic signaling to produce complex effects on adipose tissues and whole-body energy balance.

Obesity alters the peripheral circadian clock in the aorta and microcirculation.

OBJECTIVE: Perturbation of daily rhythm increases cardiovascular risk. The aim of this study was to determine whether obesity alters circadian gene expression and microvascular function in lean mice and obese (db/db) mice. METHODS: Mice were subjected to normal LD or DD to alter circadian rhythm. Metabolic parameters and microvascular vasoreactivity were evaluated. Array studies were conducted in the am and pm cycles to assess the rhythmicity of the entire genomics. Rhythmic expression of specific clock genes (Bmal1, Clock, Npas2, Per1, Per2, and Cry1), clock output genes (dbp), and vascular relaxation-related genes (eNOS, GTPCH1) were assessed. RESULTS: Obesity was associated with metabolic dysfunction and impaired endothelial dilation in the microvasculature. Circadian rhythm of gene expression was suppressed 80% in both macro- and microcirculations of obese mice. Circadian disruption with DD increased fasting serum glucose and HbA1c in obese but not lean mice. Endothelium-dependent dilation was attenuated in obese mice and in lean mice subjected to DD. Rhythmic expression of per1 and dbp was depressed in obesity. Expression of eNOS expression was suppressed and GTPCH1 lost rhythmic expression both in obesity and by constant darkness. CONCLUSION: These results suggest that obesity reduces circadian gene expression in concert with impaired endothelial function. The causal relationship remains to be determined.

12-week Dolutegravir treatment marginally reduces energy expenditure but does not increase body weight or alter vascular function in a murine model of Human Immunodeficiency Virus infection.

Combination antiretroviral therapy (cART) has markedly increased life expectancy in people with HIV (PWH) but has also resulted in an increased prevalence of cardiometabolic disorders, whose etiopathology remains ill-defined. Notably, the respective contribution of cART and HIV-derived proteins to obesity and vascular alterations remain poorly understood. Therefore, we investigated the individual and combined effects of HIV-proteins and of the integrase strand transfer inhibitor Dolutegravir (DTG) on body composition and vascular reactivity. Male wildtype (WT) and HIV transgenic (Tg26) mice, received DTG or vehicle for 12 weeks. Viral proteins expression in Tg26 mice lowered fat mass, increased heat production, and induced a 2-fold increase in brown adipose tissue (BAT) uncoupling protein 1 (UCP1) expression. DTG increased the expression of markers of adipogenesis in adipocytes in culture, but also reduced heat production and BAT UCP1 and UCP3 expression in Tg26 mice. DTG increased food intake, fat percentage and protected from lean mass reduction in Tg26 mice only. However, DTG did not increase body weight in either WT or Tg26 mice. Viral protein expression reduced acetylcholine (endothelium)-mediated relaxation by 14% in mesenteric arteries preconstricted with phenylephrine. However, DTG did not impair nor improve endothelium-dependent relaxation. Together, these data indicate that DTG's effects on food intake, adipogenesis and energy expenditure are insufficient to increase body weight, even in the presence of HIV-proteins, suggesting that body weight gain in PWH involves additional factors likely including other cART components and pre-existing comorbidities. Moreover, these data rule out DTG as a source of vascular disorders in PWH.

Perivascular adipose tissue promotes vascular dysfunction in murine lupus.

Introduction: Patients with systemic lupus erythematosus (SLE) are at elevated risk for Q10 cardiovascular disease (CVD) due to accelerated atherosclerosis. Compared to heathy control subjects, lupus patients have higher volumes and densities of thoracic aortic perivascular adipose tissue (PVAT), which independently associates with vascular calcification, a marker of subclinical atherosclerosis. However, the biological and functional role of PVAT in SLE has not been directly investigated. Methods: Using mouse models of lupus, we studied the phenotype and function of PVAT, and the mechanisms linking PVAT and vascular dysfunction in lupus disease. Results and discussion: Lupus mice were hypermetabolic and exhibited partial lipodystrophy, with sparing of thoracic aortic PVAT. Using wire myography, we found that mice with active lupus exhibited impaired endothelium-dependent relaxation of thoracic aorta, which was further exacerbated in the presence of thoracic aortic PVAT. Interestingly, PVAT from lupus mice exhibited phenotypic switching, as evidenced by "whitening" and hypertrophy of perivascular adipocytes along with immune cell infiltration, in association with adventitial hyperplasia. In addition, expression of UCP1, a brown/beige adipose marker, was dramatically decreased, while CD45-positive leukocyte infiltration was increased, in PVAT from lupus mice. Furthermore, PVAT from lupus mice exhibited a marked decrease in adipogenic gene expression, concomitant with increased pro-inflammatory adipocytokine and leukocyte marker expression. Taken together, these results suggest that dysfunctional, inflamed PVAT may contribute to vascular disease in lupus.

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.

Pin1 prolyl isomerase regulates endothelial nitric oxide synthase.

OBJECTIVE: The Pin1 prolyl isomerase acts in concert with proline-directed protein kinases to regulate function of protein substrates through isomerization of peptide bonds that link phosphoserine or phosphothreonine to proline. We sought to determine whether Pin1 interacts with endothelial nitric oxide synthase (eNOS) in endothelial cells in a manner that depends on proline-directed phosphorylation of the eNOS enzyme and whether this interaction influences basal or agonist-stimulated eNOS activity. METHODS AND RESULTS: Inhibitors of the extracellular-regulated kinase (ERK) 1/2 MAP kinases inhibit proline-directed phosphorylation of eNOS at serine 116 (Ser116) in bovine aortic endothelial cells (BAECs). Moreover, eNOS and Pin1 can be coimmunoprecipitated from BAECs only when Ser116 is phosphorylated. In addition, phosphomimetic Ser116Asp eNOS, but not wild-type eNOS, can be coimmunoprecipitated with Pin1 coexpressed in COS-7 cells. Inhibition of Pin1 in BAECs by juglone or by dominant negative Pin1 increases basal and agonist-stimulated NO release from the cells, whereas overexpression of wild-type Pin1 in BAECs suppresses basal and agonist-stimulated NO production. Overexpression of wild-type Pin1 in intact aortae also reduces agonist-induced relaxation of aortic rings. CONCLUSIONS: Our results demonstrate a novel form of eNOS regulation in endothelial cells and blood vessels through Ser116 phosphorylation-dependent interaction of eNOS with Pin1.

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.

Deletion of protein tyrosine phosphatase 1b improves peripheral insulin resistance and vascular function in obese, leptin-resistant mice via reduced oxidant tone.

RATIONALE: Obesity is a risk factor for cardiovascular dysfunction, yet the underlying factors driving this impaired function remain poorly understood. Insulin resistance is a common pathology in obese patients and has been shown to impair vascular function. Whether insulin resistance or obesity, itself, is causal remains unclear. OBJECTIVE: The present study tested the hypothesis that insulin resistance is the underlying mediator for impaired NO-mediated dilation in obesity by genetic deletion of the insulin-desensitizing enzyme protein tyrosine phosphatase (PTP)1B in db/db mice. METHODS AND RESULTS: The db/db mouse is morbidly obese, insulin-resistant, and has tissue-specific elevation in PTP1B expression compared to lean controls. In db/db mice, PTP1B deletion improved glucose clearance, dyslipidemia, and insulin receptor signaling in muscle and fat. Hepatic insulin signaling in db/db mice was not improved by deletion of PTP1B, indicating specific amelioration of peripheral insulin resistance. Additionally, obese mice demonstrate an impaired endothelium dependent and independent vasodilation to acetylcholine and sodium nitroprusside, respectively. This impairment, which correlated with increased superoxide in the db/db mice, was corrected by superoxide scavenging. Increased superoxide production was associated with increased expression of NAD(P)H oxidase 1 and its molecular regulators, Noxo1 and Noxa1. CONCLUSIONS: Deletion of PTP1B improved both endothelium dependent and independent NO-mediated dilation and reduced superoxide generation in db/db mice. PTP1B deletion did not affect any vascular function in lean mice. Taken together, these data reveal a role for peripheral insulin resistance as the mediator of vascular dysfunction in obesity.

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.

Protein tyrosine phosphatase 1B, a major regulator of leptin-mediated control of cardiovascular function.

BACKGROUND: Obesity causes hypertension and sympathoactivation, a process proposed to be mediated by leptin. Protein tyrosine phosphatase 1B (PTP1B), a major new pharmaceutical target in the treatment of obesity and type II diabetes mellitus, constrains the metabolic actions of leptin, but the extent to which PTP1B regulates its cardiovascular effects is unclear. This study examined the hypothesis that PTP1B is a negative regulator of the cardiovascular effects of leptin. METHODS AND RESULTS: PTP1B knockout mice had lower body fat but higher mean arterial pressure (116+/-5 versus 105+/-5 mm Hg, P<0.05) than controls. Leptin infusion produced a greater anorexic effect in PTP1B knockout mice and a marked increase in mean arterial pressure (135+/-5 mm Hg) in PTP1B knockout mice only. The decrease in mean arterial pressure in response to ganglionic blockade was higher in PTP1B knockout mice (-38+/-3% versus -29+/-3%, P<0.05), which suggests increased sympathetic tone. PTP1B deletion blunted mean arterial pressure responses to phenylephrine injection (55+/-10% versus 93+/-7%, P<0.05). Phenylephrine-induced aortic contraction was reduced in PTP1B knockout mice (57.7+/-9% versus 96.3+/-12% of KCl, P<0.05), consistent with desensitization to chronically elevated sympathetic tone. Furthermore, PTP1B deletion significantly reduced gene expression of 3 alpha(1)-adrenergic receptor subtypes, consistent with blunted constriction to phenylephrine. CONCLUSIONS: These data indicate that PTP1B is a key regulator of the cardiovascular effects of leptin and that reduced vascular adrenergic reactivity provides a compensatory limit to the effects of leptin on mean arterial pressure.

Preventing increased blood pressure in the obese Zucker rat improves severity of stroke.

Obesity is a risk factor for stroke, but the determinants of increased stroke risk in obesity are unknown. We have previously reported that obese Zucker rats (OZRs) have a worse stroke outcome and display evidence of remodeling of the middle cerebral artery (MCA), in parallel with hypertension, compared with lean controls. This study tested the hypothesis that hypertension is an essential determinant of cerebral vascular remodeling and increased stroke damage in OZRs. Blood pressure was measured by telemetry in lean and obese rats with and without hydrochlorthiazide (HCT; 2 mg.kg(-1).day(-1)) from 8 to 15 wk of age. A separate group of rats was also chronically fed a low-sodium (LS) diet. Vessel structure was assessed in isolated, pressurized MCAs. Cerebral ischemia was induced for 60 min using an intralumenal suture technique, followed by 24 h of reperfusion. HCT treatment effectively prevented the increase in blood pressure in obese rats; however, the LS diet did not lower pressure. Importantly, infarct size was normalized by HCT after ischemia-reperfusion injury. Additionally, HCT improved the changes in MCA structure observed in untreated OZRs. There were no benefits of the LS diet on stroke injury or vessel structure. These results indicate that increased pressure is essential for driving the changes in infarct size in OZRs.

Acculturation, childhood trauma and the cortisol awakening response in Mexican-American adults.

Exposure to chronic and traumatic stress has been associated with the dysregulation of crucial stress response systems. Acculturation has been associated with unique forms of chronic psychosocial stress. The purpose of this study was to examine the effects of exposure to early traumatic stress and acculturation on dysregulation of the cortisol awakening response (CAR) in Mexican-American adults. Salivary cortisol samples were collected at awakening and 30, 45, and 60 min thereafter, on two consecutive weekdays from 59 healthy Mexican-American adult males (26) and females (33), ages 18-38 years. Participants were assessed for level of acculturation and exposure to early trauma. Data were analyzed using a mixed effects regression model with repeated measures at four time points. Mixed effects regression results indicated a significant Early Trauma x Time interaction (p=.0029) and a significant Acculturation x Time interaction (p=.0015), after controlling for age and sex. Subsequent analyses of the interaction of Trauma x Acculturation x Time showed that more than minimal exposure to either risk factor was associated with attenuation of the awakening cortisol response (p=.0002). Higher levels of acculturation with greater Anglo-orientation were associated with attenuation of the CAR in Mexican-American adults. Both moderate and higher levels of exposure to early trauma were associated with an attenuated CAR. However, greater exposure to both risk factors was only incrementally worse than exposure to either one.

Insulin resistance impairs endothelial function but not adrenergic reactivity or vascular structure in fructose-fed rats.

Obesity and diabetes are major risk factors for the development of vascular disease in the lower limbs. Previous studies have demonstrated reduced nitric oxide (NO)-mediated vasodilation, increased adrenergic constriction, and inward, atrophic remodeling in the limb circulation of obese Zucker rats, but the component of the "metabolic syndrome" driving these changes is unclear. Because insulin resistance precedes the state of frank diabetes, the current study hypothesized that insulin resistance independent of obesity induced by fructose feeding would impair microvascular function in the skeletal muscle circulation in lean Zucker rats (LZR). A 66% fructose diet impaired glucose tolerance and induced moderate insulin resistance with no changes in whole-body hemodynamics of anesthetized rats (FF-LZR), compared to control LZR. NO-mediated vasodilation of isolated gracilis arteries, assessed in vitro with acetylcholine and sodium nitroprusside, was reduced approximately 20% in FF-LZR vs. LZR. NO-independent cGMP-mediated vasodilation was unimpaired. Pretreatment of isolated vessels with the superoxide scavenger, tempol, improved responses to both vasodilators. Reactivity to adrenergic stimulation was unaltered in FF-LZR vs. LZR, although constriction to endothelin was increased. Structural and passive mechanical characteristics of isolated gracilis arteries were similar in both LZR and FF-LZR. Taken together, these findings indicate that moderate insulin resistance is sufficient to impair endothelial function in an oxidant-dependent manner in the rat hindlimb circulation. Other aspects of skeletal muscle vascular function documented in obese models, specifically adrenergic tone and inward remodeling, must reflect either severe insulin resistance or other aspects of obesity. The factors accounting for nonendothelial vasculopathies remain unknown.