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Consumption of diets high in fat, sugar, and salt (Western diet, WD) is associated with accelerated arterial stiffening, a major independent risk factor for cardiovascular disease (CVD). Women with obesity are more prone to develop arterial stiffening leading to more frequent and severe CVD compared with men. As tissue transglutaminase (TG2) has been implicated in vascular stiffening, our goal herein was to determine the efficacy of cystamine, a nonspecific TG2 inhibitor, at reducing vascular stiffness in female mice chronically fed a WD. Three experimental groups of female mice were created. One was fed regular chow diet (CD) for 43 wk starting at 4 wk of age. The second was fed a WD for the same 43 wk, whereas a third cohort was fed WD, but also received cystamine (216 mg/kg/day) in the drinking water during the last 8 wk on the diet (WD + C). All vascular stiffness parameters assessed, including aortic pulse wave velocity and the incremental modulus of elasticity of isolated femoral and mesenteric arteries, were significantly increased in WD- versus CD-fed mice, and reduced in WD + C versus WD-fed mice. These changes coincided with respectively augmented and diminished vascular wall collagen and F-actin content, with no associated effect in blood pressure. In cultured human vascular smooth muscle cells, cystamine reduced TG2 activity, F-actin:G-actin ratio, collagen compaction capacity, and cellular stiffness. We conclude that cystamine treatment represents an effective approach to reduce vascular stiffness in female mice in the setting of WD consumption, likely because of its TG2 inhibitory capacity. NEW & NOTEWORTHY This study evaluates the novel role of transglutaminase 2 (TG2) inhibition to directly treat vascular stiffness. Our data demonstrate that cystamine, a nonspecific TG2 inhibitor, improves vascular stiffness induced by a diet rich in fat, fructose, and salt. This research suggests that TG2 inhibition might bear therapeutic potential to reduce the disproportionate burden of cardiovascular disease in females in conditions of chronic overnutrition.

Widespread consumption of diets high in fat and fructose (Western diet, WD) has led to increased prevalence of obesity and diastolic dysfunction (DD). DD is a prominent feature of heart failure with preserved ejection fraction (HFpEF). However, the underlying mechanisms of DD are poorly understood, and treatment options are still limited. We have previously shown that deletion of the cell-specific mineralocorticoid receptor in endothelial cells (ECMR) abrogates DD induced by WD feeding in female mice. However, the specific role of ECMR activation in the pathogenesis of DD in male mice has not been clarified. Therefore, we fed 4-wk-old ECMR knockout (ECMRKO) male mice and littermates (LM) with either a WD or chow diet (CD) for 16 wk. WD feeding resulted in DD characterized by increased left ventricle (LV) filling pressure ( E/ e′) and diastolic stiffness [ E/ e′/LV inner diameter at end diastole (LVIDd)]. Compared with CD, WD in LM resulted in increased myocardial macrophage infiltration, oxidative stress, and increased myocardial phosphorylation of Akt, in concert with decreased phospholamban phosphorylation. WD also resulted in focal cardiomyocyte remodeling, characterized by areas of sarcomeric disorganization, loss of mitochondrial electron density, and mitochondrial fragmentation. Conversely, WD-induced DD and associated biochemical and structural abnormalities were prevented by ECMR deletion. In contrast with our previously reported observations in females, WD-fed male mice exhibited enhanced Akt signaling and a lower magnitude of cardiac injury. Collectively, our data support a critical role for ECMR in obesity-induced DD and suggest critical mechanistic differences in the genesis of DD between males and females.

Background: Transglutaminase 2 (TG2) is an enzyme involved in the pathogenesis of vascular remodeling in conditions of chronic over-nutrition and activation of the renin angiotensin aldosterone system (RAAS). Furthermore, it has been shown that pharmacologic inhibition of TG2 significantly reduces blood pressure in male rodents. However, the cell-specific role of TG2 activation in vascular smooth muscle cells (VSMC) in mediating the hypertensive response to RAAS overactivation has not been explored. Methods: We generated a novel inducible model (tamoxifen dose: 50 mg/kg/d oral for 6 weeks) of TG2 deletion in VSMC (VSMCTG2KO) by sequentially crossing Tgm2t/t floxed mice (loxP sites flanking exons 6-8 of the TG2, C polypeptide [Tgm2] gene) with male Myh11-CreERT2 positive mice. Effectiveness of the TG2 deletion was evaluated by immunohistochemistry and TG2 activity was determined by incorporation of Alexa Fluor488 Cadaverine.A cohort of fifteen-month-old wild-type and VSMC TG2KO male mice were implanted with deoxycorticosterone (DOCA; 50 mg, 21-day releasing pellets) and 1 % salt added to the drinking water. Systolic blood pressure was assessed by tail-cuff method prior to sacrifice. Ex vivo aortic stiffness was determined by atomic force microscopy. Student t-test or two-way ANOVA were used for statistical analysis as appropriate. The results were considered significant when p Results: administration of DOCA/salt induced increases in systolic blood pressure, augmented expression of TG2 in aorta, as well as increased aortic stiffness in wild-type mice (10.47±1.27kPA DOCA/salt vs. 3.91 + 1.01 kPa placebo). The VSMCTG2KO model exhibited 77% downregulation of TG2 expression in VSMC. VSMC TG2 KO suppressed DOCA/salt induced TG2 activity (29.13 + 2.22 arbitrary units (AU) in DOCA/salt vs 14.10 + 2.90 AU in KO cohort). This was accompanied by a significant improvement in DOCA/ salt-induced hypertension (SBP 142 ± 5.4 mmHg in DOCA/salt treated mice vs. 111.8 ± 6.2 mmHg in VSMCTG2KO). Conclusions: Our data demonstrate for the first-time the key role of TG2 activation in VSMC, in the regulation of hypertensive responses in in a male rodent model of RAAS activation.

Excess energy intake by spectators at a sporting event (i.e., a tailgate) might cause acute negative health effects. However, limited data exist regarding the effects of overeating and alcohol consumption on lipid metabolism and the potential to gain intrahepatic triacylglycerols (IHTG). We tested the hypothesis that overconsumption of food and alcohol would significantly increase both hepatic de novo lipogenesis (DNL) and IHTG.Eighteen males (mean ± SD, age: 31.4 ± 7.3 years, BMI: 32.1 ± 5.9 kg/mSubjects consumed 5087 ± 149 kcal (191 ± 25% excess of total daily energy needs including 171 ± 24 g alcohol), which increased plasma insulin, glucose, TG, and decreased NEFA (ANOVA p ≤ 0.003 for all). Both DNL and TRL-TG increased (p 0.001), while IHTG did not change in the group as a whole (p = 0.229). Individual subject data revealed remarkably differing responses for IHTG (nine increased, five decreased, one did not change). Despite maintaining equal breath alcohol levels, subjects with IHTG elevations exhibited higher DNL, consumed 90% less alcohol (p = 0.048), tended to consume more carbohydrates, and exhibited lower whole-body fat oxidation (not significant) compared to those whose IHTG was reduced.This study demonstrates that acute excess energy intake may have differing effects on an individual's DNL and IHTG, and dietary carbohydrate may influence DNL more than alcohol.

Resumen Introducción: La alteración de la vitamina D es un factor de riesgo para enfermar de tuberculosis (TBC). Objetivo: Evaluar la concentración pulmonar y sérica del compuesto 25-hidroxi-vitamina D (25OHD) en pacientes con y sin TBC pulmonar. Metodología: Estudio de dos etapas: la primera parte fue de corte transversal, retrospectiva, y la segunda prospectiva. Se incluyeron > 18 años a los que se les realizó fibrobroncoscopia por sospecha de TBC pulmonar y en quienes se confirmó la infección. Se tomaron como controles a pacientes con otro tipo de infección no TBC, y enfermedades no infecciosas para la primera etapa y controles infecciosos sin TBC en la fase prospectiva. La medición de 25OHD se realizó mediante ELFA (ensayo de fluorescencia ligado a enzima). Se empleó la prueba de Kruskal-Wallis para evaluar asociación considerando significativo un valor de p < 0,05. Los datos se procesaron con el programa SPSS versión 23. Resultados: La muestra total fue de 77 pacientes (35 en la primera etapa y 42 en la segunda). Las características entre los grupos fueron homogéneas. Las concentraciones en suero (segunda fase) como en el lavado bronco-alveolar (primera y segunda fase) de 25OHD fueron más bajas en pacientes con TBC comparado con los controles e independientes de la concentración de calcio sérico (suero: 22,4 ng/mL vs 33 ng/mL, p = 0,006 y lavado bronco-alveolar: 9,7 ng/mL vs 12,2 ng/mL; p = 0,012). Conclusiones: Hubo una diferencia significativa entre las concentraciones de 25OHD, tanto en suero como en lavado bronco-alveolar, en pacientes con TBC pulmonar con relación a sus controles.

Consumption of a Western diet (WD) induces central aortic stiffening that contributes to the transmittance of pulsatile blood flow to end organs, including the kidney. Our recent work supports that endothelial epithelial Na+ channel (EnNaC) expression and activation enhances aortic endothelial cell stiffening through reductions in endothelial nitric oxide (NO) synthase (eNOS) and bioavailable NO that result in inflammatory and oxidant responses and perivascular fibrosis. However, the role that EnNaC activation has on endothelial responses in the renal circulation remains unknown. We hypothesized that cell-specific deletion of the α-subunit of EnNaC would prevent WD-induced central aortic stiffness and protect the kidney from endothelial dysfunction and vascular stiffening. Twenty-eight-week-old female αEnNaC knockout and wild-type mice were fed either mouse chow or WD containing excess fat (46%), sucrose, and fructose (17.5% each). WD feeding increased fat mass, indexes of vascular stiffening in the aorta and renal artery (in vivo pulse wave velocity and ultrasound), and renal endothelial cell stiffening (ex vivo atomic force microscopy). WD further impaired aortic endothelium-dependent relaxation and renal artery compliance (pressure myography) without changes in blood pressure. WD-induced renal arterial stiffening occurred in parallel to attenuated eNOS activation, increased oxidative stress, and aortic and renal perivascular fibrosis. αEnNaC deletion prevented these abnormalities and support a novel mechanism by which WD contributes to renal arterial stiffening that is endothelium and Na+ channel dependent. These results demonstrate that cell-specific EnNaC is important in propagating pulsatility into the renal circulation, generating oxidant stress, reduced bioavailable NO, and renal vessel wall fibrosis and stiffening.

Objective Cardiac diastolic dysfunction (DD) and arterial stiffness are early manifestations of obesity-associated prediabetes, and both serve as risk factors for the development of heart failure with preserved ejection fraction (HFpEF). Since the incidence of DD and arterial stiffness are increasing worldwide due to exponential growth in obesity, an effective treatment is urgently needed to blunt their development and progression. Here we investigated whether the combination of an inhibitor of neprilysin (sacubitril), a natriuretic peptide-degrading enzyme, and an angiotensin II type 1 receptor blocker (valsartan), suppresses DD and arterial stiffness in an animal model of prediabetes more effectively than valsartan monotherapy. Methods Sixteen-week-old male Zucker Obese rats (ZO; n = 64) were assigned randomly to 4 different groups: Group 1: saline control (ZOC); Group 2: sacubitril/valsartan (sac/val; 68 mg•kg−1•day−1; ZOSV); Group 3: valsartan (31 mg•kg−1•day−1; ZOV) and Group 4: hydralazine, an anti-hypertensive drug (30 mg•kg−1•day−1; ZOH). Six Zucker Lean (ZL) rats that received saline only (Group 5) served as lean controls (ZLC). Drugs were administered daily for 10 weeks by oral gavage. Results Sac/val improved echocardiographic parameters of impaired left ventricular (LV) stiffness in untreated ZO rats, without altering the amount of food consumed or body weight gained. In addition to improving DD, sac/val decreased aortic stiffness and reversed impairment in nitric oxide-induced vascular relaxation in ZO rats. However, sac/val had no impact on LV hypertrophy. Notably, sac/val was more effective than val in ameliorating DD. Although, hydralazine was as effective as sac/val in improving these parameters, it adversely affected LV mass index. Further, cytokine array revealed distinct effects of sac/val, including marked suppression of Notch-1 by both valsartan and sac/val, suggesting that cardiovascular protection afforded by both share some common mechanisms; however, sac/val, but not val, increased IL-4, which is increasingly recognized for its cardiovascular protection, possibly contributing, in part, to more favorable effects of sac/val over val alone in improving obesity-associated DD. Conclusions These studies suggest that sac/val is superior to val in reversing obesity-associated DD. It is an effective drug combination to blunt progression of asymptomatic DD and vascular stiffness to HFpEF development in a preclinical model of obesity-associated prediabetes.

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During exercise, there is coordination between various hormonal systems to ensure glucoregulation. This study examined if hypoglycemia occurs during moderate-intensity exercise in non-obese and obese individuals with and without type 2 diabetes (T2D). Eighteen non-obese, 18 obese, and 10 obese with T2D completed 2 study days that included a meal at 1,800 h followed by rest (NOEX) or exercise (PMEX; 45 min/55% of VO2 max 2 h post meal). Glucose, insulin, and glucagon concentrations were measured throughout this 5.5 h period. Subjects with T2D had elevated glucose responses to the meal on both study days, compared to non-obese and obese subjects (P < 0.05). During evening exercise (PMEX), subjects with T2D had a greater drop in glucose concentration (−98.4 ± 13.3 mg/dL) compared to obese (−44.8 ± 7.1 mg/dL) and non-obese (−39.3 ± 6.1 mg/dL; P < 0.01) subjects. Glucose levels decreased more so in females than males in both conditions (P < 0.01). Nadir glucose levels

Widespread consumption of diets high in fat, sugars and salt (Western diet, WD) is associated arterial stiffening, which is a major independent risk factor for cardiovascular disease (CVD). Notably, while WD feeding increases the risk of CVD in both males and females, the latter are more prone to develop arterial stiffening. However, the mechanisms underlying WD-induced arterial stiffening are poorly understood, particularly in females, and there are currently no specific treatments targeted at vascular stiffening.Tissue transglutaminase 2 (TG2) is an enzyme that mediates the cross-linking and stabilization of extracellular matrix proteins such as collagen, and promotes the polymerization of actin stress fibers of the cytoskeleton. It is ubiquitously expressed and abundantly present in the vasculature. Mounting evidence implicates TG2 activation in the pathogenesis of arterial stiffening and vascular fibrosis. Herein we propose that TG2 activation is central to WD-induced arterial stiffening and sought to determine the efficacy of cystamine (a non-specific competitive inhibitor of TG2) for reducing arterial stiffening in the setting of WD consumption. Accordingly, we fed 20 female mice (4 weeks old) a WD (4.65 kcal/g of food, fat 46% kcals, high-fructose corn syrup 17.5%, sucrose 17.5%, protein 17.6%, salt 1.6%) for 43 weeks. Ten of these mice received cystamine (40 mg/Kg/d in the drinking water) during their last 8 weeks on the WD. Another group of female mice (n=10) fed regular chow was used as reference controls. Aortic stiffness was measured in vivo via ultrasound-based pulse wave velocity and ex vivo by aortic explant atomic force microscopy. Vasomotor responses were assessed in isolated aortic rings via wire myography.Cystamine did not influence glucose homeostasis (intraperitoneal glucose tolerance test) or blood pressure (tail-cuff) (control 77.208±2.229 mm Hg versus WD 77.208±6.077 versus WD+Cystamine 76.297±7.894), but it was associated with increased body weight (control 26.860±2.215 grams versus WD 25.320±2.889 versus WD+Cystamine 33.220±4.848, p

Alteration of vitamin D is a risk factor for tuberculosis (TB).To evaluate the pulmonary and serum levels of 25-hydroxy vitamin D (25OHD) in patients with and without pulmonary TB.Two-stage study: the first part was retrospective cross-sectional and the second prospective. Those18 years of age who underwent fiberoptic bronchoscopy for suspected pulmonary TB and in whom the infection was confirmed were included. Patients with another type of infection without TB and non-infectious diseases were taken as controls for the first stage and infectious controls without TB in the prospective phase. The measurement of 25OHD was performed by ELFA (enzyme-linked fluorescence assay). The Kruskal-Wallis test was used to evaluate association, considering a value of p0.05 to be significant. The data were processed with the SPSS version 23 program.The total sample was 77 patients (35 in the first stage and 42 in the second). The characteristics between the groups were homogeneous. Serum (second phase) and broncho-alveolar lavage (first and second phase) levels of 25OHD were lower in TB patients compared to controls and were independent of serum calcium level (serum: 22.4 ng/mL vs 33 ng/mL, p = 0.006 and broncho-alveolar lavage: 9.7 ng/mL vs 12.2 ng/mL; p = 0.012).There was a significant difference between the levels of 25OHD in both serum and broncho-alveolar lavage in patients with pulmonary TB in relation to their controls.

Consumption of a high-fat, high-fructose diet [Western diet (WD)] promotes vascular stiffness, a critical factor in the development of cardiovascular disease (CVD). Obese and diabetic women exhibit greater arterial stiffness than men, which contributes to the increased incidence of CVD in these women. Furthermore, high-fructose diets result in elevated plasma concentrations of uric acid via xanthine oxidase (XO) activation, and uric acid elevation is also associated with increased vascular stiffness. However, the mechanisms by which increased xanthine oxidase activity and uric acid contribute to vascular stiffness in obese females remain to be fully uncovered. Accordingly, we examined the impact of XO inhibition on endothelial function and vascular stiffness in female C57BL/6J mice fed a WD or regular chow for 16 wk. WD feeding resulted in increased arterial stiffness, measured by atomic force microscopy in aortic explants (16.19 ± 1.72 vs. 5.21 ± 0.54 kPa, P < 0.05), as well as abnormal aortic endothelium-dependent and -independent vasorelaxation. XO inhibition with allopurinol (widely utilized in the clinical setting) substantially improved vascular relaxation and attenuated stiffness (16.9 ± 0.50 vs. 3.44 ± 0.50 kPa, P < 0.05) while simultaneously lowering serum uric acid levels (0.55 ± 0.98 vs. 0.21 ± 0.04 mg/dL, P < 0.05). In addition, allopurinol improved WD-induced markers of fibrosis and oxidative stress in aortic tissue, as analyzed by immunohistochemistry and transmission electronic microscopy. Collectively, these results demonstrate that XO inhibition protects against WD-induced vascular oxidative stress, fibrosis, impaired vasorelaxation, and aortic stiffness in females. Furthermore, excessive oxidative stress resulting from XO activation appears to play a key role in mediating vascular dysfunction induced by chronic exposure to WD consumption in females.

Obesity and insulin resistance stiffen the vasculature, with females appearing to be more adversely affected. As augmented arterial stiffness is an independent predictor of cardiovascular disease (CVD), the increased predisposition of women with obesity and insulin resistance to arterial stiffening may explain their heightened risk for CVD. However, the cellular mechanisms by which females are more vulnerable to arterial stiffening associated with obesity and insulin resistance remain largely unknown. In this study, we provide evidence that female mice are more susceptible to Western diet–induced endothelial cell stiffening compared with age-matched males. Mechanistically, we show that the increased stiffening of the vascular intima in Western diet–fed female mice is accompanied by enhanced epithelial sodium channel (ENaC) activity in endothelial cells (EnNaC). Our data further indicate that: (i) estrogen signaling through estrogen receptor α (ERα) increases EnNaC activity to a larger extent in females compared with males, (ii) estrogen-induced activation of EnNaC is mediated by the serum/glucocorticoid inducible kinase 1 (SGK-1), and (iii) estrogen signaling stiffens endothelial cells when nitric oxide is lacking and this stiffening effect can be reduced with amiloride, an ENaC inhibitor. In aggregate, we demonstrate a sexual dimorphism in obesity-associated endothelial stiffening, whereby females are more vulnerable than males. In females, endothelial stiffening with obesity may be attributed to estrogen signaling through the ERα–SGK-1–EnNaC axis, thus establishing a putative therapeutic target for female obesity-related vascular stiffening.

Consumption of a Western Diet (WD) high in saturated fat and refined carbohydrates causes obesity and leads to insulin resistance and associated vascular stiffening in females earlier than males. This diet-induced obesity is characterized by enhanced activation of vascular mineralocorticoid receptors (MRs). Our recent work suggests that enhanced MR signaling in female mice prompts increased expression and translocation of the α-subunit of the epithelial sodium channel to the endothelial cell (EC) surface (EnNaC). However, the influence of EnNaC on endothelial function in the kidney remains unknown. Therefore, we hypothesized that WD feeding would induce kidney microvessel dysfunction and kidney injury through inflammatory pathways. Using a model of cell specific deletion of the α-subunit of EnNaC, female KO mice and littermate controls were fed a diet high in fat (46%) and fructose (17.5%) for 12 weeks. There were increases in indices of weight gain and fat mass in WD fed mice along with increased measures of central aortic stiffness, renal arteriolar remodeling, perivascular fibrosis and aortic and kidney endothelial stiffening in the absence of increases in blood pressure or proteinuria. The central aortic stiffness, renal arteriolar remodeling, perivascular fibrosis and aortic and kidney endothelial stiffening occurred in temporal relation to reductions in bioavailable nitric oxide. All of these abnormalities were attenuated in the EnNaC α-subunit knockout (endothelial stiffness, WD, 9.2 ± 0.65 kPA; WD-KO, 5.38 ± 0.75kPA). Our observations suggest, that central aortic and kidney vessel stiffening along with arterial wall remodeling occur in conjunction to renal injury.

Obesity is associated with a metabolic cardiomyopathy characterized by diastolic dysfunction with preserved systolic function. Although premenopausal non-obese women show relative protection against development of metabolic cardiomyopathy and cardiovascular disease compared to men, this relationship is lost in obese women. Diastolic dysfunction in such cardiomyopathy is caused not only by extrinsic extracellular remodeling and interstitial fibrosis but also through alterations in intrinsic stiffness of cardiomyocytes. One mechanism by which intrinsic cardiomyocyte stiffness is increased in obesity is decreased coronary endothelial cell production of nitric oxide. We have described a clinically relevant diet induced obesity murine model that exhibits increased vascular stiffness associated with cardiac dysfunction. In this model, female mice have high plasma aldosterone levels and increased mineralocorticoid receptor (MR) expression in both the vasculature and heart. One of the mechanisms by which MR activation promotes endothelial stiffness is through increased expression and activation of epithelial sodium channel (ENaC) in ECs (EnNaC). In this study, we tested the hypothesis that specific deletion of EnNaC ameliorates increases in endothelial stiffness and decreases diastolic dysfunction occurring during diet induced obesity (DIO) in female mice. To produce cell specific deletion of the EnNaC gene,”floxed” EnNaC mice were serially crossed with Tie 2-Cre transgene mice. This resulted in marked suppression of EnNaC expression in ECs. Female KO mice and littermate controls were fed a diet high in fat (46%) and fructose (17.5%) for 12 to 16 weeks. Compared to mice fed a control diet (CD), whole cell sodium currents in pulmonary ECs and aortic EC stiffness were significantly increased in DIO mice and this was prevented in EnNaC KO mice with DIO. Moreover, deletion of EnNaC also prevented DIO increases in stiffness of isolated cardiomyocytes (WD, 7.43 ± 1.21 kPA, WD-KO, 3.47 ± 0.14 kPA) as well as normalizing impaired diastolic relaxation. Collectively, these findings support the notion that DIO promotes coronary ECMR mediated activation of EnNaC and associated increases in intrinsic cardiomyocyte stiffness and diastolic dysfunction.

Objective Mineralocorticoid receptor activation of the epithelial sodium channel in endothelial cells (ECs) (EnNaC) is accompanied by aldosterone induced endothelial stiffening and impaired nitric oxide (NO)-mediated arterial relaxation. Recent data support enhanced activity of the alpha subunit of EnNaC (αEnNaC) mediates this aldosterone induced endothelial stiffening and associated endothelial NO synthase (eNOS) activation. There is mounting evidence that diet induced obesity diminishes expression and activation of AMP-activated protein kinase α (AMPKα), sirtuin 1 (Sirt1), which would be expected to lead to impaired downstream eNOS activation. Thereby, we posited that enhanced EnNaC activation contributes to diet induced obesity related increases in stiffness of the endothelium and diminished NO mediated vascular relaxation by increasing oxidative stress and related inhibition of AMPKα, Sirt1, and associated eNOS inactivation. Materials/Methods Sixteen to twenty week-old αEnNaC knockout (αEnNaC−/−) and wild type littermate (EnNaC+/+) female mice were fed a mouse chow or an obesogenic western diet (WD) containing excess fat (46%) and fructose (17.5%) for 16 weeks. Sodium currents of ECs, endothelial stiffness and NO mediated aortic relaxation were examined along with indices of aortic oxidative stress, vascular remodeling and fibrosis. Results Enhanced EnNaC activation-mediated WD-induced increases in sodium currents in isolated lung ECs, increased endothelial stiffness and impaired aortic endothelium-dependent relaxation to acetylcholine (10−9–10−4 mol/L). These abnormalities occurred in conjunction with WD-mediated aortic tissue oxidative stress, inflammation, and decreased activation of AMPKα, Sirt1, and downstream eNOS were substantially mitigated in αEnNaC−/− mice. Importantly, αEnNaC−/− prevented WD induced increases in endothelial stiffness and related impairment of endothelium-dependent relaxation as well as aortic fibrosis and remodeling. However, EnNaC signaling was not involved in diet-induced abnormal expression of adipokines and CYP11b2 in abdominal aortic perivascular adipose tissue. Conclusion These data suggest that endothelial specific EnNaC activation mediates WD-induced endothelial stiffness, impaired eNOS activation, aortic fibrosis and remodeling through increased aortic oxidative stress and increased inflammation related to a reduction of AMPKα and Sirt 1 mediated eNOS phosphorylation/activation and NO production.

Excessive epithelial sodium channel activation in endothelium (EnNaC) increase oxidative stress and inflammation with associated cardiovascular abnormalities. Our recent data has shown that activation of EnNaC mediates aortic endoplasmic reticulum stress, redox oxidative stress, expression of pro-inflammatory cytokines, and aortic remodeling. These abnormalities are potentially related to abnormal expression and activation of sirtuin 1 (Sirt1) and AMP-activated protein kinase (AMPK) in metabolic disorders such as obesity. We hypothesized that EnNaC mediates Western diet (WD)-induced endothelium and aortic stiffness by increasing aortic oxidative stress and associated oxidative stress related inhibition of Sirt1 and AMPK expression and activation. Accordingly, sixteen to twenty week-old EnNaC-/- and wild type littermate female mice were fed a mouse chow or an obesogenic WD containing excess fat (46%) and fructose (17.5%) for 16 weeks. EnNaC activation mediated WD-induced increase of Na+ currents in isolated lung endothelial cells, reduction of phosphorylation of endothelial nitric oxide synthase (eNOS), as well as impairment of aortic endothelium-dependent relaxation to acetylcholine (10-9-10-4 mol/L) in a wire myograph. The expression and activation of Sirt1 and AMPK were inhibited in WD EnNaC+/+ mice and these abnormalities were prevented in EnNaC-/- mice. Importantly, EnNaC specific KO prevented WD induced aortic oxidative stress and related endothelium stiffness and impairment of endothelium (NO)-dependent relaxation. These data suggest that endothelial specific EnNaC activation mediates WD-induced aortic oxidative stress, decreased expression and activation of Sirt1, AMPK, eNOS, as well as endothelial and aortic vessel stiffness. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. Abstracts presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Over the last decades, our understanding about the role of estrogens has increased dramatically. Estrogens impact several tissues, organs, and systems well beyond fertility. In particular, estrogens impact the cardiovascular system and are responsible for the well-accepted protective role against vascular dysfunction and cardiovascular dysfunction in females relative to males. Conversely, lack of estrogens results in increased incidence of atherosclerosis and CVD which result in worse outcomes in postmenopausal women compared to men. However, estrogen replacement therapy in this scenario not only fails to restore protection against CVD, but can worsen its clinical manifestation, an unexpected observation that has prompted further research and investigation about the mechanisms of estrogen action in the vasculature. In this chapter, we try to uncover the complex mechanisms underlying the actions of estrogens in the vasculature as well as in the cardiovascular system that explain the role of estrogens in cardiovascular disease.

Consumption of a diet high in fat and refined carbohydrates (Western diet [WD]) is associated with obesity and insulin resistance, both major risk factors for cardiovascular disease (CVD). In women, obesity and insulin resistance abrogate the protection against CVD likely afforded by estrogen signaling through estrogen receptor (ER)α. Indeed, WD in females results in increased vascular stiffness, which is independently associated with CVD. We tested the hypothesis that loss of ERα signaling in the endothelium exacerbates WD-induced vascular stiffening in female mice. We used a novel model of endothelial cell (EC)-specific ERα knockout (EC-ERαKO), obtained after sequential crossing of the ERα double floxed mice and VE-Cadherin Cre-recombinase mice. Ten-week-old females, EC-ERαKO and aged-matched genopairs were fed either a regular chow diet (control diet) or WD for 8 weeks. Vascular stiffness was measured in vivo by pulse wave velocity and ex vivo in aortic explants by atomic force microscopy. In addition, vascular reactivity was assessed in isolated aortic rings. Initial characterization of the model fed a control diet did not reveal changes in whole-body insulin sensitivity, aortic vasoreactivity, or vascular stiffness in the EC-ERαKO mice. Interestingly, ablation of ERα in ECs reduced WD-induced vascular stiffness and improved endothelial-dependent dilation. In the setting of a WD, endothelial ERα signaling contributes to vascular stiffening in females. The precise mechanisms underlying the detrimental effects of endothelial ERα in the setting of a WD remain to be elucidated.

Objective Obesity is associated with myocardial fibrosis and impaired diastolic relaxation, abnormalities that are especially prevalent in women. Normal coronary vascular endothelial function is integral in mediating diastolic relaxation, and recent work suggests increased activation of the endothelial cell (EC) mineralocorticoid receptor (ECMR) is associated with impaired diastolic relaxation. As the endothelial Na+ channel (EnNaC) is a downstream target of the ECMR, we sought to determine whether EC-specific deletion of the critical alpha subunit, αEnNaC, would prevent diet induced-impairment of diastolic relaxation in female mice. Methods and materials Female αEnNaC KO mice and littermate controls were fed a Western diet (WD) high in fat (46%), fructose corn syrup (17.5%) and sucrose (17.5%) for 12–16 weeks. Measurements were conducted for in vivo cardiac function, in vitro cardiomyocyte stiffness and EnNaC activity in primary cultured ECs. Additional biochemical studies examined indicators of oxidative stress, including aspects of antioxidant Nrf2 signaling, in cardiac tissue. Results Deletion of αEnNaC in female mice fed a WD significantly attenuated WD mediated impairment in diastolic relaxation. Improved cardiac relaxation was accompanied by decreased EnNaC-mediated Na+ currents in ECs and reduced myocardial oxidative stress. Further, deletion of αEnNaC prevented WD-mediated increases in isolated cardiomyocyte stiffness. Conclusion Collectively, these findings support the notion that WD feeding in female mice promotes activation of EnNaC in the vasculature leading to increased cardiomyocyte stiffness and diastolic dysfunction.

PURPOSE OF THE REVIEW: In the present review, we will discuss the evidence and the mechanisms underlying the complex interplay between obesity, mineralocorticoid receptor activation, and cardiovascular dysfunction with special emphasis on the pathogenesis of cardiovascular disease (CVD) in obese and insulin-resistant females. RECENT FINDINGS: Since the initial isolation of aldosterone in 1953 and the cloning of the mineralocorticoid receptor (MR) decades later, our understanding has expanded tremendously regarding their involvement in the pathogenesis of CVD. Recent results from both pre-clinical and clinical studies support a close correlation between increase adiposity and enhanced aldosterone production (MR activation). SUMMARY: Importantly, insulin resistance and obese females are more prone to the deleterious cardiovascular effects of MR activation, and enhanced MR activation in females has emerged as an important causative event in the genesis of a more severe CVD in diabetic women. Different clinical trials have been completed examining the effect of MR blockade in subjects with CVD. Despite its important beneficial mortality impact, side effects are frequent and a newer MR antagonist, finerenone, with less risk of hyperkalemia is currently being tested in large clinical trials.

Novel therapies are needed for treating the increasing prevalence of hepatic steatosis in Western populations. In this regard, dipeptidyl peptidase-4 (DPP-4) inhibitors have recently been reported to attenuate the development of hepatic steatosis, but the potential mechanisms remain poorly defined. In the current study, 4-week-old C57Bl/6 mice were fed a high-fat/high-fructose Western diet (WD) or a WD containing the DPP-4 inhibitor, MK0626, for 16 weeks. The DPP-4 inhibitor prevented WD-induced hepatic steatosis and reduced hepatic insulin resistance by enhancing insulin suppression of hepatic glucose output. WD-induced accumulation of hepatic triacylglycerol (TAG) and diacylglycerol (DAG) content was significantly attenuated with DPP-4 inhibitor treatment. In addition, MK0626 significantly reduced mitochondrial incomplete palmitate oxidation and increased indices of pyruvate dehydrogenase activity, TCA cycle flux, and hepatic TAG secretion. Furthermore, DPP-4 inhibition rescued WD-induced decreases in hepatic PGC-1α and CPT-1 mRNA expression and hepatic Sirt1 protein content. Moreover, plasma uric acid levels in mice fed the WD were decreased after MK0626 treatment. These studies suggest that DPP-4 inhibition ameliorates hepatic steatosis and insulin resistance by suppressing hepatic TAG and DAG accumulation through enhanced mitochondrial carbohydrate utilization and hepatic TAG secretion/export with a concomitant reduction of uric acid production.

A western diet (WD), high in fructose and fat, is often accompanied by insulin resistance and cardiovascular disease characterized by endothelial cell (EC) dysfunction, increased arterial and cardiac stiffness, and diastolic dysfunction. Although premenopausal non-obese women are protected against cardiovascular disease, arterial stiffness and diastolic dysfunction, in obese women these abnormalities are more pronounced than in men. We have recently developed a clinically relevant, WD fed, murine model that exhibits increased aortic stiffness associated with vascular and cardiac dysfunction. In this model, female mice have high plasma aldosterone levels and increased mineralocorticoid receptor expression (MR) in both the vasculature and heart. One of the mechanisms by which MR activation promotes endothelial stiffness is through increased expression and activation of epithelial sodium channel (ENaC) in ECs (EnNaC). We reported increased aortic EC stiffness associated with increased expression of EnNaC in WD fed mice and suppression of aortic stiffness and improved diastolic function by treatment with a low dose of an MR antagonist or the ENaC inhibitor, amiloride. In this study, we tested the hypothesis that specific deletion of EnNaC, decreases aortic EC stiffness and improves vascular relaxation and diastolic function in WD fed female mice. To produce cell specific deletion of the EnNaC gene,”floxed” EnNaC mice were serially crossed with Tie 2-Cre transgene mice. This resulted in marked suppression of EnNaC expression in ECs. Female KO mice and littermate controls were fed a WD with high in fat (46%) and fructose (17.5%) for 12 weeks. Compared to mice fed a control diet (CD), aortic EC stiffness, measured ex vivo by atomic force microscopy (AFM) was significantly increased in WD fed mice and this was prevented in EnNaC KO mice fed WD. Decreased EC stiffness was associated with improved endothelial-dependent aortic relaxation in response to acetylcholine. Moreover, deletion of EnNaC also prevented WD induced impairment of diastolic function. Taken together, these findings support the notion that a WD promotes ECMR mediated activation of EnNaC and associated aortic stiffness, cardiac stiffness and diastolic dysfunction.

Excessive activation of endothelial cell (EC) mineralocorticoid receptor (ECMR) signaling induces EC epithelial sodium channel (EnNaC) activity to promote cardiovascular stiffness. Our previous study has demonstrated that activated ECMR signaling prompts expression and translocation of EnNaC to the EC surface inducing fibrosis, inflammation, and macrophage infiltration in the vasculature of female mice fed a western diet (WD). As ECMR KO also prevented these abnormalities, we posit that ECMR/EnNaC activation was critical. Accordingly, we hypothesized that EC-specific EnNaC activation would mediate endothelium dysfunction, vascular stiffness, and impair flow-mediated vasodilation through reduction of bioavailable NO. Four week old C57BL6/J mice were fed a WD containing high fat (46%), sucrose (17.5%), and high fructose corn syrup (17.5%) with or without a low dose of amiloride (1 mg/kg/day) for 16 weeks. Female EnNaC KO and wild-type littermate females were treated with aldosterone (250 μg/kg/day) via osmotic minipumps for 3 weeks. Amiloride, an antagonist for EnNaC, significantly inhibited inward Na+ currents and EnNaC activity in the cultured endothelial cells. Amiloride treatment significantly attenuated WD-induced increases in aortic stiffness in vivo as measured by pulse wave velocity and in vitro endothelial stiffness measured by atomic force microscopy. In addition, amiloride improved flow mediated dilation in mesenteric arteries and endothelium-dependent relaxation in response to acetylcholine (10 -9 -10 -4 mol/L). Furthermore, amiloride prevented WD-induced increases in coronary endothelium permeability that were associated with decreased expression of claudin-5 and occluding. This also resulted in reduction of total macrophage recruitment (CD11b) and M1 polarization (CD11c). Importantly, genetic knock-out EnNaC KO also prevented aldosterone-induced endothelium stiffening and impairment of endothelium-dependent relaxation. These data indicate that EC specific EnNaC activation decreases bioavailable NO, increases vascular endothelium dysfunction, and prompts vascular stiffening in obese female mice.

Therapies to prevent renal injury in obese hypertensive individuals are being actively sought due to the obesity epidemic arising from the Western diet (WD), which is high in fructose and fat. Recently, activation of the immune system and hyperuricemia, observed with high fructose intake, have been linked to the pathophysiology of hypertension and renal injury. Because dipeptidyl peptidase 4 (DPP4) is a driver of maladaptive T-cell/macrophage responses, renal-protective benefits of DPP4 inhibition in the WD-fed mice were examined. Mice fed a WD for 16 weeks were given the DPP4 inhibitor MK0626 in their diet beginning at 4 weeks of age. WD-fed mice were obese, hypertensive, and insulin-resistant and manifested proteinuria and increased plasma DPP4 activity and uric acid levels. WD-fed mice also had elevated kidney DPP4 activity and monocyte chemoattractant protein-1 and IL-12 levels and suppressed IL-10 levels in the kidney, suggesting macrophage-driven inflammation, glomerular and tubulointerstitial injury. WD-induced increases in DPP4 activation in the plasma and kidney and proteinuria in WD mice were abrogated by MK0626, although blood pressure and systemic insulin sensitivity were not improved. Contemporaneously, MK0626 reduced serum uric acid levels, renal oxidative stress, and IL-12 levels and increased IL-10 levels, suggesting that suppression of DPP4 activity leads to suppression of renal immune/inflammatory injury responses to a WD. Taken together, these results demonstrate that DPP4 inhibition prevents high-fructose/high-fat diet-induced glomerular and tubular injury independent of blood pressure/insulin sensitivity and offers a potentially novel therapy for diabetic and obesity-related kidney disease.

Patients with hypertension and type 2 diabetes are at increased risk of cardiovascular and chronic renal disease. Factors involved in the pathogenesis of both hypertension and type 2 diabetes include inappropriate activation of the renin-angiotensin-aldosterone system, oxidative stress, inflammation, impaired insulin-mediated vasodilatation, augmented sympathetic nervous system activation, altered innate and adaptive immunity, and abnormal sodium processing by the kidney. The renin-angiotensin-aldosterone system blockade is a key therapeutic strategy in the treatment of hypertension in type 2 diabetes. Emerging therapies for resistant hypertension as often exists in patients with diabetes, include renal denervation and carotid body denervation.

Obesity continues to increase dramatically in both industrialized and nonindustrialized countries. The scope of the problem in Hispanic populations follows a similar epidemic pattern to industrialized countries, but the unfavorable social and economic features of these populations have placed them at increased risk of developing cardiometabolic syndrome and cardiovascular disease. Some pathophysiologic elements of obesity appear to have a differential biologic behavior in these populations, suggesting that not only environmental factors, but also genetically determined conditions, can influence obesity-related cardiometabolic risk. However, the high epidemiologic diversity of these populations and the lack of reliable statistics make it difficult to ascertain the characteristics of obesity in Hispanics, especially as most of the available data come from Hispanics living in industrialized countries. The management of obesity in Hispanic populations is similar to that in non-Hispanics, and is based on lifestyle changes, weight-reducing medication and bariatric surgery. Novel research related to gut-derived hormones and the endocannabinoid network will potentially contribute to treat obesity better. Newer studies taking into account ethnic differences will result in improved therapeutic alternatives for the prevention of obesity and cardiometabolic syndrome-related cardiovascular disease if they can be coupled with improved living conditions.

Nonalcoholic fatty liver disease (NAFLD), a common clinical disorder, is increasing largely due to increasing consumption of diets high in fructose and fat (1–3). Indeed, NAFLD has been reported to be present in up to 30% of the U.S. population (3), closely mirroring the prevalence of obesity. Its presence carries an increased risk for type 2 diabetes, end-stage liver disease, and cardiovascular disease (2). Obesity and type 2 diabetes are characterized by chronic low-grade inflammation in which maladaptive immune responses contribute to the development of resistance to the metabolic actions of insulin, thus generating a proinflammatory milieu that leads to steatosis and fibrosis of the liver (4). A critical event in the pathophysiology of tissue inflammation is the shift in the polarization status of macrophages from an anti-inflammatory (M2) to a proinflammatory (M1) phenotype of macrophages (5,6). M1 polarization of hepatic Kupffer cells (KCs) plays an important role in the genesis of hepatic steatosis and insulin resistance in response to chronic overnutrition (6). M1 polarization results in increased expression of proinflammatory mediators, such as CD11c, tumor necrosis factor α, interleukin (IL)-6, and inducible nitric oxide synthase (7). Conversely, the anti-inflammatory M2 phenotype secretes preferentially CD206, arginase1, and IL-10 (8). The role of macrophage M1/M2 polarization status in conditions of insulin resistance has been explored using myeloid-specific knockout …