Search

Vascular dysfunction: develops progressively with ageing; increases the risk of cardiovascular diseases (CVD); and is characterized by endothelial dysfunction and arterial stiffening, which are primarily mediated by superoxide-driven oxidative stress and consequently reduced nitric oxide (NO) bioavailability and arterial structural changes. Interventions initiated before vascular dysfunction manifests may have more promise for reducing CVD risk than interventions targeting established dysfunction. Gut microbiome-derived trimethylamine N-oxide (TMAO) induces vascular dysfunction, is associated with higher CV risk, and can be suppressed by 3,3-dimethyl-1-butanol (DMB). We investigated whether DMB supplementation could prevent age-related vascular dysfunction in C57BL/6N mice when initiated prior to development of dysfunction. Mice received drinking water with 1% DMB or normal drinking water (control) from midlife (18 months) until being studied at 21, 24 or 27 months of age, and were compared to young adult (5 month) mice. Endothelial function [carotid artery endothelium-dependent dilatation (EDD) to acetylcholine; pressure myography] progressively declined with age in control mice, which was fully prevented by DMB via higher NO-mediated EDD and lower superoxide-related suppression of EDD (normalization of EDD with the superoxide dismutase mimetic TEMPOL). In vivo aortic stiffness (pulse wave velocity) increased progressively with age in controls, but DMB attenuated stiffening by ∼ 70%, probably due to preservation of endothelial function, as DMB did not affect aortic intrinsic mechanical (structural) stiffness (stress-strain testing) nor adventitial abundance of the arterial structural protein collagen. Our findings indicate that long-term DMB supplementation prevents/attenuates age-related vascular dysfunction, and therefore has potential for translation to humans for reducing CV risk with ageing. KEY POINTS: Vascular dysfunction, characterized by endothelial dysfunction and arterial stiffening, develops progressively with ageing and increases the risk of cardiovascular diseases (CVD). Interventions aimed at preventing the development of CV risk factors have more potential for preventing CVD relative to those aimed at reversing established dysfunction. The gut microbiome-derived metabolite trimethylamine N-oxide (TMAO) induces vascular dysfunction, is associated with higher CV risk and can be suppressed by supplementation with 3,3-dimethyl-1-butanol (DMB). In mice, DMB prevented the development of endothelial dysfunction and delayed and attenuated in vivo arterial stiffening with ageing when supplementation was initiated in midlife, prior to the development of dysfunction. DMB supplementation or other TMAO-suppressing interventions have potential for translation to humans for reducing CV risk with ageing.

We provide novel evidence that increased circulating concentrations of the gut microbiome-derived metabolite trimethylamine N-oxide (TMAO) contribute to vascular dysfunction associated with consumption of a Western-style diet and that this dysfunction can be prevented by suppressing TMAO with DMB, thereby supporting translation of this compound to humans. Furthermore, to our knowledge, we present the first evidence of the role of TMAO in mediating impairments in endurance exercise tolerance and increased frailty in any context.

Aging is associated with stiffening of the large elastic arteries and consequent increases in systolic blood pressure (SBP), which together increase cardiovascular disease risk; however, the upstream mechanisms are incompletely understood. Using complementary translational approaches in mice and humans, we investigated the role of the gut microbiome-derived metabolite trimethylamine N-oxide (TMAO) in age-related aortic stiffening and increased SBP. Aortic stiffness was measured using carotid-femoral or aortic pulse wave velocity (PWV) in humans and mice, respectively. Study 1: Plasma TMAO concentrations were elevated ( P r 2 =0.15, P r 2 =0.09, P

To determine the efficacy of inorganic nitrite supplementation on endothelial function in humans and mechanisms of action, we performed (1) a randomized, placebo-controlled, parallel-group clinical trial with sodium nitrite (80 mg/day, 12 weeks) in older adults (N=49, 68±1 year) and (2) reverse-translation experiments in young (6 months) and old (27 months) c57BL/6 mice. In the clinical trial, sodium nitrite increased plasma nitrite ( P P P P P P P Registration: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT02393742.

KEY POINTS The results of the present study establish the temporal pattern of age-related vascular dysfunction across the adult lifespan in sedentary mice consuming a non-Western diet, and the underlying mechanisms The results demonstrate that consuming a Western diet accelerates and exacerbates vascular ageing across the lifespan in sedentary mice They also show that lifelong voluntary aerobic exercise has remarkable protective effects on vascular function throughout the lifespan, in the setting of ageing alone, as well as ageing compounded by Western diet consumption Overall, the results indicate that amelioration of mitochondrial oxidative stress and inflammation are key mechanisms underlying the voluntary aerobic exercise-associated preservation of vascular function across the lifespan in both the presence and absence of a Western dietary pattern ABSTRACT: Advancing age is the major risk factor for cardiovascular diseases, driven largely by vascular endothelial dysfunction (impaired endothelium-dependent dilatation, EDD) and aortic stiffening (increased aortic pulse wave velocity, aPWV). In humans, vascular ageing occurs in the presence of differences in diet and physical activity, but the interactive effects of these factors are unknown. We assessed carotid artery EDD and aPWV across the lifespan in mice consuming standard (normal) low-fat chow (NC) or a high-fat/high-sucrose Western diet (WD) in the absence (sedentary, SED) or presence (voluntary wheel running, VWR) of aerobic exercise. Ageing impaired nitric oxide-mediated EDD (peak EDD 88 ± 12% 6 months P = 0.003 vs. 59 ± 9% 27 months NC-SED), which was accelerated by WD (60 ± 18% 6 months WD-SED). In NC mice, aPWV increased 32% with age (423 ± 13 cm/s at 24 months P

New findings What is the central question of this study? Obesity is associated with complex perturbations to iron homeostasis: is plasma ferritin concentration (a biomarker of whole-body iron stores) related to the abundance of ferritin (the key tissue iron storage protein) in skeletal muscle in adults with obesity? What is the main finding and its importance? Plasma ferritin concentration was tightly correlated with the abundance of ferritin in skeletal muscle, and this relationship persisted when accounting for sex, age, body mass index and plasma C-reactive protein concentration. Our findings suggest that skeletal muscle may be an important iron store. Abstract Obesity is associated with complex perturbations to whole-body and tissue iron homeostasis. Recent evidence suggests a potentially important influence of iron storage in skeletal muscle on whole-body iron homeostasis, but this association is not clearly resolved. The primary aim of this study was to assess the relationship between whole-body and skeletal muscle iron stores by measuring the abundance of the key iron storage (ferritin) and import (transferrin receptor) proteins in skeletal muscle, as well as markers of whole-body iron homeostasis in men (n = 19) and women (n = 43) with obesity. Plasma ferritin concentration (a marker of whole-body iron stores) was highly correlated with muscle ferritin abundance (r = 0.77, P = 2 × 10-13 ) and negatively associated with muscle transferrin receptor abundance (r = -0.76, P = 1 × 10-12 ). These relationships persisted when accounting for sex, age, BMI and plasma C-reactive protein concentration. In parallel with higher whole-body iron stores in our male versus female participants, men had 2.2-fold higher muscle ferritin abundance (P = 1 × 10-4 ) compared with women. In accordance with lower muscle iron storage, women had 2.7-fold higher transferrin receptor abundance (P = 7 × 10-10 ) compared with men. We conclude that muscle iron storage and import proteins are tightly and independently related to plasma ferritin concentration in adults with obesity, suggesting that skeletal muscle may be an underappreciated iron store.

Age-related vascular endothelial dysfunction is a major antecedent to cardiovascular diseases. We investigated whether increased circulating levels of the gut microbiome-generated metabolite trimethylamine-N-oxide induces endothelial dysfunction with aging. In healthy humans, plasma trimethylamine-N-oxide was higher in middle-aged/older (64±7 years) versus young (22±2 years) adults (6.5±0.7 versus 1.6±0.2 µmol/L) and inversely related to brachial artery flow-mediated dilation ( r 2 =0.29, P P G -nitro-L-arginine methyl ester). Acute incubation of carotid arteries with trimethylamine-N-oxide recapitulated these events. Next, treatment with 3,3-dimethyl-1-butanol for 8 to 10 weeks to suppress trimethylamine-N-oxide selectively improved endothelium-dependent dilation in old mice to young levels (peak: 90±2%) by normalizing vascular superoxide production, restoring nitric oxide-mediated dilation, and ameliorating superoxide-related suppression of endothelium-dependent dilation. Lastly, among healthy middle-aged/older adults, higher plasma trimethylamine-N-oxide was associated with greater nitrotyrosine abundance in biopsied endothelial cells, and infusion of the antioxidant ascorbic acid restored flow-mediated dilation to young levels, indicating tonic oxidative stress-related suppression of endothelial function with higher circulating trimethylamine-N-oxide. Using multiple experimental approaches in mice and humans, we demonstrate a clear role of trimethylamine-N-oxide in promoting age-related endothelial dysfunction via oxidative stress, which may have implications for prevention of cardiovascular diseases.

New findings What is the central question of this study? Does exercise training modify tissue iron storage in adults with obesity? What is the main finding and its importance? Twelve weeks of moderate-intensity exercise or high-intensity interval training lowered whole-body iron stores, decreased the abundance of the key iron storage protein in skeletal muscle (ferritin) and tended to lower hepatic iron content. These findings show that exercise training can reduce tissue iron storage in adults with obesity and might have important implications for obese individuals with dysregulated iron homeostasis. Abstract The regulation of iron storage is crucial to human health, because both excess and deficient iron storage have adverse consequences. Recent studies suggest altered iron storage in adults with obesity, with increased iron accumulation in their liver and skeletal muscle. Exercise training increases iron use for processes such as red blood cell production and can lower whole-body iron stores in humans. However, the effects of exercise training on liver and muscle iron stores in adults with obesity have not been assessed. The aim of this study was to determine the effects of 12 weeks of exercise training on whole-body iron stores, liver iron content and the abundance of ferritin (the key iron storage protein) in skeletal muscle in adults with obesity. Twenty-two inactive adults (11 women and 11 men; age, 31 ± 6 years; body mass index, 33 ± 3 kg/m2 ) completed 12 weeks (four sessions/week) of either moderate-intensity continuous training (MICT; 45 min at 70% of maximal heart rate; n = 11) or high-intensity interval training (HIIT; 10 × 1 min at 90% of maximal heart rate, interspersed with 1 min active recovery; n = 11). Whole-body iron stores were lower after training, as indicated by decreased plasma concentrations of ferritin (P = 3 × 10-5 ) and hepcidin (P = 0.02), without any change in C-reactive protein. Hepatic R2*, an index of liver iron content, was 6% lower after training (P = 0.06). Training reduced the skeletal muscle abundance of ferritin by 10% (P = 0.03), suggesting lower muscle iron storage. Interestingly, these adaptations were similar in MICT and HIIT groups. Our findings indicate that exercise training decreased iron storage in adults with obesity, which might have important implications for obese individuals with dysregulated iron homeostasis.

IntroductionObtaining informed consent from acute ischemic stroke patients poses many challenges, especially in the context of a research setting. Specifically, consenting for alternative acute ischemic stroke treatments to the standard of care, Tissue Plasminogen Activator (tPA), can cause delays leading to increased time to reperfusion and worse outcomes.ObjectivesWe sought to investigate the experiences of researchers in existing active-control trials in acute ischemic stroke comparing investigational therapy to tPA in order to identify the approaches and challenges in obtaining informed consent in this unique patient population.MethodsOut of 401 articles evaluated, 14 trials met inclusion criteria of patients receiving IV tPA vs alternative treatment within 4.5 hours of onset of symptoms for acute ischemic stroke. Trial representatives were emailed by the study team with a request for a copy of their patient consent form, other documents related to informed consent, and to complete a survey concerning aspects of the consent process.ResultsOf the 6 trials conducted across 6 continents that completed the survey in its entirety, 2 were ongoing, 4 were published between 2009 and 2016, and the median NIHSS for each published trial was at least an 8. All published trials in the sample stated that informed consent was obtained, but only half reported involvement of a research ethics committee. Although 3 trials performed in Europe or Asia reported directly consenting 75-100% of enrolled patients, the median NIHSS for these trials represented a moderate stroke. Trials with 75-100% of patients directly consented had shorter door to treatment (DTT) times than trials that directly consented less than 50% of enrolled patients. 5 trials allowed consent by proxy, but only 2 of those trials also required patient assent. 4 trials had translators available and translated consent documents, and these trials had longer DTT times. All trials relied on experienced providers or dedicated research coordinators to obtain informed consent; however, only 2 of 6 trials mentioned specific training with regards to informed consent skills.ConclusionsThe current informed consent process is not transparent and poses challenges to investigators in the USA directly comparing tPA to an alternative treatment. International differences in the standards of informed consent, such as deferred consent, may have allowed more patients with moderate strokes to provide direct consent after treatment administration without delaying DTT time. While targeted and innovative approaches for informed consent are needed to improve patient outcomes, we must balance protecting the autonomy of individuals whose willing involvement enables such pivotal discoveries. The stroke community must aim for efficiency, transparency, and inclusion of patients of diverse backgrounds in the informed consent process so that therapeutic advances are possible.

Aortic stiffening, assessed as pulse-wave velocity (PWV), increases with age and is an important antecedent to, and independent predictor of, cardiovascular diseases (CVD) and other clinical disorders of aging. Aerobic exercise promotes lower levels of aortic stiffness in older adults, but the underlying mechanisms are incompletely understood, largely due to inherent challenges of mechanistic studies of large elastic arteries in humans. Voluntary wheel running (VWR) is distinct among experimental animal exercise paradigms in that it allows investigation of the physiologic effects of aerobic training without potential confounding influences of aversive molecular signaling related to forced exercise. In this study, we investigated whether VWR in mice may be a suitable model for mechanistic studies (i.e., “reverse translation”) of the beneficial effects of exercise on arterial stiffness in humans. We found that 10 weeks of VWR in old mice (~ 28 months) reversed age-related elevations in aortic PWV assessed in vivo (Old VWR: 369 ± 19 vs. old sedentary: 439 ± 20 cm/s, P

Aging is associated with declines in cognitive performance, which are mediated in part by neuroinflammation, characterized by astrocyte activation and higher levels of pro-inflammatory cytokines; however, the upstream drivers are unknown. We investigated the potential role of the gut microbiome–derived metabolite trimethylamine N-oxide (TMAO) in modulating neuroinflammation and cognitive function with aging. Study 1: In middle-aged and older humans (65 ± 7 years), plasma TMAO levels were inversely related to performance on NIH Toolbox Cognition Battery tests of memory and fluid cognition (both r(2) = 0.07, p

Cardiovascular diseases (CVD) are the leading cause of death worldwide and aging is the primary risk factor for CVD. The development of vascular dysfunction, including endothelial dysfunction and stiffening of the large elastic arteries (i.e., the aorta and carotid arteries), contribute importantly to the age-related increase in CVD risk. Vascular aging is driven in large part by oxidative stress, which reduces bioavailability of nitric oxide and promotes alterations in the extracellular matrix. A key upstream driver of vascular oxidative stress is age-associated mitochondrial dysfunction. This review will focus on vascular mitochondria, mitochondrial dysregulation and mitochondrial reactive oxygen species (ROS) production and discuss current evidence for prevention and treatment of vascular aging via lifestyle and pharmacological strategies that improve mitochondrial health. We will also identify promising areas and important considerations (‘research gaps’) for future investigation.

Objective We compared the effects of high-intensity interval training (HIIT) and moderate-intensity continuous training (MICT) on insulin sensitivity and other important metabolic adaptations in adults with obesity. Methods Thirty-one inactive adults with obesity (age: 31 ± 6 years; body mass index: 33 ± 3 kg/m2) completed 12 weeks (4 sessions/week) of either HIIT (10 × 1-minute at 90%HRmax, 1-minute active recovery; n = 16) or MICT (45 minutes at 70%HRmax; n = 15). To assess the direct effects of exercise independent of weight/fat loss, participants were required to maintain body mass. Results Training increased peak oxygen uptake by ~10% in both HIIT and MICT (P Conclusion Despite large differences in training intensity and exercise time, 12 weeks of HIIT and MICT induce similar acute improvements in peripheral insulin sensitivity the day after exercise, and similar longer term metabolic adaptations in skeletal muscle in adults with obesity. These findings support the notion that the insulin-sensitizing effects of both HIIT and MICT are mediated by factors stemming from the most recent exercise session(s) rather than adaptations that accrue with training.

It is unknown if consumption of a Western diet (WD; high-fat/sucrose), versus a non-WD (healthy diet), accelerates declines in physical function over the adult lifespan, and whether regular voluntary activity attenuates age- and WD-associated declines in function. Accordingly, we studied 4 cohorts of mice that consumed either normal chow [NC] or WD with or without access (sedentary, Sed) to voluntary wheel running [VWR] beginning at 3 mo of age. We assessed coordination, grip strength and endurance every 6 mo throughout life, and measured skeletal muscle mass and inflammation at 3 pre-determined ages (6-7, 13-14 and 19-20 mo). Age-related declines (% change 3-18 mo) in physical function were accelerated in WD-Sed versus NC-Sed (coordination: +47 ± 5%; grip strength: +18 ± 2%; endurance: +32 ± 5%; all p < 0.05). VWR attenuated declines in physical function within diet group (coordination: -31 ± 3% with WD-VWR; -18 ± 2% with NC-VWR; grip strength: -26 ± 2% with WD-VWR; -24 ± 2% with NC-VWR; endurance: -48 ± 4% with WD-VWR; -23 ± 6% with NC-VWR; all p < 0.05). Skeletal muscle mass loss and pro-inflammatory cytokine abundance were exacerbated by WD throughout life (mass: NC-Sed [-]7-28%, WD-Sed [-]17-40%; inflammation: NC-Sed [+]40-65%, WD-Sed [+]40-84%, all p < 0.05 versus NC-Sed), and attenuated by VWR (mass: NC-VWR, [-]0-10%, WD-VWR [-]0-10%; inflammation: NC-VWR [+]0-30%, WD-VWR [+]0-42%, all p < 0.05 versus diet-matched Sed group). Our results depict the temporal impairment of physical function over the lifespan in mice, acceleration of dysfunction with WD, the protective effects of voluntary exercise, and the potential associations with skeletal muscle mass and inflammation.

A recent American Heart Association report shows that 34% of US adults ≥20 years of age have hypertension representing ≈86 million adults.1 A substantial increase in the prevalence of hypertension has occurred globally.2 The projected number of individuals with systolic blood pressure of ≥140 mm Hg has doubled from 442 million in 1990 to 874 million in 2015. Hypertension is one of the most prevalent risk factors for cardiovascular disease (CVD).1 Results of the recently concluded SPRINT (Systolic Blood Pressure Intervention Trial) funded by the National Heart, Lung, and Blood Institute showed that among older adults with hypertension but without diabetes mellitus, lowering systolic blood pressure to a target goal of 120 mm Hg, compared with the standard goal of 140 mm Hg, resulted in significantly lower rates of fatal and nonfatal cardiovascular events and death from any cause.3 Some groups such as blacks who display both disproportionately earlier onset and higher prevalence of hypertension have increased risk of blood pressure (BP)–related cardiovascular and renal disease complications compared with non-Hispanic whites. Despite intensive attempts to influence lifestyle changes, nutritional counseling, and intensive antihypertensive drug treatment strategies, ≈14% of all hypertension patients seem to be resistant to antihypertensive interventions.1 Resistant hypertension is defined as BP above goal (>140/90 mm Hg) on ≥3 BP-lowering medications or needing ≥4 medications prescribed at optimal dose to control BP to goal. Thus, both increased prevalence and inability to achieve BP goals in a large patient population with hypertension are creating a tremendous healthcare burden. In addition, no novel antihypertensive drugs have been added to our formulary since 1995, when the first angiotensin receptor antagonist was approved in the United States. The recent attempts to use percutaneous renal artery sympathetic denervation, as a novel means to control hypertension, have been largely unsuccessful to date.4 …

Aging is associated with vascular endothelial dysfunction, reduced exercise tolerance, and impaired whole‐body glucose metabolism. Interleukin‐37 (IL‐37), an anti‐inflammatory cytokine of the interleukin‐1 family, exerts salutary physiological effects in young mice independent of its inflammation‐suppressing properties. Here, we assess the efficacy of IL‐37 treatment for improving physiological function in older age. Old mice (26–28 months) received daily intraperitoneal injections of recombinant human IL‐37 (recIL‐37; 1 µg/200 ml PBS) or vehicle (200 ml PBS) for 10–14 days. Vascular endothelial function (ex vivo carotid artery dilation to increasing doses of acetylcholine, ACh) was enhanced in recIL‐37 vs. vehicle‐treated mice via increased nitric oxide (NO) bioavailability (all p, Physiological function declines with aging, increasing risk of chronic diseases and disability. We show for the first time that treatment with a novel anti‐inflammatory compound interleukin‐37 (IL‐37) improves multiple physiological functions in old mice, and identify potential roles of reduced superoxide production, improved oxidative metabolism and circulating factors in mediating improvements. Our findings support IL‐37 therapy as a novel strategy for improving diverse physiological functions in old age.

KEY POINTS: Age‐related arterial dysfunction, characterized by oxidative stress‐ and inflammation‐mediated endothelial dysfunction and arterial stiffening, is the primary risk factor for cardiovascular diseases. To investigate whether age‐related changes in the gut microbiome may mediate arterial dysfunction, we suppressed gut microbiota in young and old mice with a cocktail of broad‐spectrum, poorly‐absorbed antibiotics in drinking water for 3–4 weeks. In old mice, antibiotic treatment reversed endothelial dysfunction and arterial stiffening and attenuated vascular oxidative stress and inflammation. To provide insight into age‐related changes in gut microbiota that may underlie these observations, we show that ageing altered the abundance of microbial taxa associated with gut dysbiosis and increased plasma levels of the adverse gut‐derived metabolite trimethylamine N‐oxide. The results of the present study provide the first proof‐of‐concept evidence that the gut microbiome is an important mediator of age‐related arterial dysfunction and therefore may be a promising therapeutic target for preserving arterial function with ageing, thereby reducing the risk of cardiovascular diseases. . ABSTRACT: Oxidative stress‐mediated arterial dysfunction (e.g. endothelial dysfunction and large elastic artery stiffening) is the primary mechanism driving age‐related cardiovascular diseases. Accumulating evidence suggests the gut microbiome modulates host physiology because dysregulation (‘gut dysbiosis’) has systemic consequences, including promotion of oxidative stress. The present study aimed to determine whether the gut microbiome modulates arterial function with ageing. We measured arterial function in young and older mice after 3–4 weeks of treatment with broad‐spectrum, poorly‐absorbed antibiotics to suppress the gut microbiome. To identify potential mechanistic links between the gut microbiome and age‐related arterial dysfunction, we sequenced microbiota from young and older mice and measured plasma levels of the adverse gut‐derived metabolite trimethylamine N‐oxide (TMAO). In old mice, antibiotics reversed endothelial dysfunction [area‐under‐the‐curve carotid artery dilatation to acetylcholine in young: 345 ± 16 AU vs. old control (OC): 220 ± 34 AU, P

Excess reactive oxygen species production by mitochondria is a key mechanism of age-related vascular dysfunction. Our laboratory has shown that supplementation with the mitochondrial-targeted antioxidant MitoQ improves vascular endothelial function by reducing mitochondrial reactive oxygen species and ameliorates arterial stiffening in old mice, but the effects in humans are unknown. Here, we sought to translate our preclinical findings to humans and determine the safety and efficacy of MitoQ. Twenty healthy older adults (60–79 years) with impaired endothelial function (brachial artery flow–mediated dilation P P P P 7.60 m/s; n=11). Plasma oxidized LDL (low-density lipoprotein), a marker of oxidative stress, also was lower after MitoQ versus placebo ( P P >0.1). These findings in humans extend earlier preclinical observations and suggest that MitoQ and other therapeutic strategies targeting mitochondrial reactive oxygen species may hold promise for treating age-related vascular dysfunction. Clinical Trial Registration— URL: http://www.clinicaltrials.gov . Unique identifier: NCT02597023.

Accumulating evidence suggests obese women maintain higher insulin sensitivity than obese men, but factors underlying this sex difference in insulin resistance remain unclear. We recently developed a semi-automated method for quantifying visceral and hepatic fat using advanced chemical shift-encoded magnetic resonance imaging (MRI). Using this method, we explored whether sex differences in ectopic fat accumulation may contribute to differences in insulin resistance. Twenty-one obese women (32 ± 6 year, 34.5 ± 3.1 kg/m2, 43.1 ± 5.5 kg fat mass (FM)) and a well-matched cohort of fifteen obese men (29 ± 6 year, 34.5 ± 3.3 kg/m2, 42.0 ± 7.8 kg FM) underwent an MRI scan during which 5mm axial slices were acquired from the abdominal region. Using 3D Slicer software, visceral fat area was quantified on three axial slices from the L2-L3 vertebrae region and averaged. Hepatic fat was quantified as the average percentage fat from three axial slices of the liver. On a separate day, subjects underwent a hyperinsulinemic-euglycemic clamp to quantify insulin sensitivity. Compared with men, women had lower hepatic (4.2 ± 5.5 vs. 13.1 ± 9.7%, p Disclosure J.B. Gillen: None. B.J. Ryan: None. R.A. Gioscia-Ryan: None. A. Ludzki: None. T.L. Chenevert: None. J.F. Horowitz: None.

High body iron stores are positively related to insulin resistance in humans, but the underlying mechanisms remain unresolved. Iron stimulates lipolysis in murine adipocytes in vitro, but the influence of iron stores on lipolytic rate in vivo is unknown. Our aim was to determine if body iron stores are associated with lipolytic rate and whole-body insulin sensitivity in obese, pre-menopausal women. We studied 20 subjects with clinically-normal (20-200 ng/mL) plasma [ferritin], an index of body iron stores. Fatty acid rate of appearance into systemic circulation (FA Ra) was assessed by 13C-palmitate isotope dilution, insulin resistance was assessed by hyperinsulinemic-euglycemic clamp, and abdominal subcutaneous adipose tissue protein expression was assessed via immunoblot. [Ferritin] was significantly (p < 0.01), positively correlated with FA Ra (r2 = 0.42), adipose hormone sensitive lipaseser660 phosphorylation (p-HSLser660, a marker of lipolytic activation in adipose tissue; r2 = 0.41), and whole-body insulin resistance (r2 = 0.33). Importantly, [ferritin] remained a significant, independent predictor for these parameters in multiple regression models including body mass index, plasma [adiponectin], and plasma [CRP] as covariates. We stratified subjects into tertiles based on [ferritin] to compare high-normal and low-normal [ferritin] groups (138 ± 30 vs. 40 ± 15 ng/mL). Compared with the low-normal [ferritin] group, the high-normal [ferritin] group had significantly (p < 0.02) higher FA Ra (15.6 ± 5.5 vs. 8.8 ± 2.4 µmol/kg fat mass/min), adipose p-HSLser660 (1.3 ± 0.4 vs. 0.6 ± 0.2 AU) and whole-body insulin resistance (glucose infusion rate 9.2 ± 2.3 vs. 14.3 ± 3.5 mg/kg fat free mass/min). There were no between-group differences in anthropometric characteristics, plasma [adiponectin], or plasma [CRP]. Our findings suggest that iron-mediated activation of adipose lipolysis may be a mechanism linking body iron stores and insulin resistance in obese humans. Disclosure B.J. Ryan: None. D.W. Van Pelt: None. L.M. Guth: None. A. Ludzki: None. R.A. Gioscia-Ryan: None. C. Ahn: None. J.F. Horowitz: None.

Aging is associated with progressive declines in physical function. However, it is unknown if consumption of a western-style diet (WD; high-fat and sucrose, low fiber), compared with a non-WD (healthy diet), accelerates declines in physical function over the adult lifespan, and whether regular voluntary exercise attenuates age- and WD-associated declines in function. To determine this, we studied 4 cohorts of male C57BL/6 mice that consumed either normal chow [NC] or WD with or without access to voluntary running [VR] wheels beginning at 3 mo of age and assessed strength (grip strength normalized to body mass) and endurance (rota-rod distance) every 3 mo throughout life. WD decreased average lifespan by 30% (WD: 18.6±0.5 vs. NC: 26.7±0.8 mo); therefore, function was compared from 3-18 mo of age in all groups. Age-related declines (% change over 3-18 mo) in physical function were accelerated by WD (strength: WD -61.2±10.1%, NC -43.2±10.2%; endurance: WD -97.4±5.1%, NC -65.1±6.3%; all p

Endothelial dysfunction develops with age and increases the risk of age-associated vascular disorders. Nitric oxide insufficiency, oxidative stress, and chronic low-grade inflammation, induced by upregulation of adverse cellular signaling processes and imbalances in stress resistance pathways, mediate endothelial dysfunction with aging. Healthy lifestyle behaviors preserve endothelial function with aging by inhibiting these mechanisms, and novel nutraceutical compounds that favorably modulate these pathways hold promise as a complementary approach for preserving endothelial health.

Age-related arterial endothelial dysfunction, a key antecedent of the development of cardiovascular disease (CVD), is largely caused by a reduction in nitric oxide (NO) bioavailability as a consequence of oxidative stress. Mitochondria are a major source and target of vascular oxidative stress when dysregulated. Mitochondrial dysregulation is associated with primary ageing, but its role in age-related endothelial dysfunction is unknown. Our aim was to determine the efficacy of a mitochondria-targeted antioxidant, MitoQ, in ameliorating vascular endothelial dysfunction in old mice. Ex vivo carotid artery endothelium-dependent dilation (EDD) to increasing doses of acetylcholine was impaired by ∼30% in old (∼27 months) compared with young (∼8 months) mice as a result of reduced NO bioavailability (P < 0.05). Acute (ex vivo) and chronic (4 weeks in drinking water) administration of MitoQ completely restored EDD in older mice by improving NO bioavailability. There were no effects of age or MitoQ on endothelium-independent dilation to sodium nitroprusside. The improvements in endothelial function with MitoQ supplementation were associated with the normalization of age-related increases in total and mitochondria-derived arterial superoxide production and oxidative stress (nitrotyrosine abundance), as well as with increases in markers of vascular mitochondrial health, including antioxidant status. MitoQ also reversed the age-related increase in endothelial susceptibility to acute mitochondrial damage (rotenone-induced impairment in EDD). Our results suggest that mitochondria-derived oxidative stress is an important mechanism underlying the development of endothelial dysfunction in primary ageing. Mitochondria-targeted antioxidants such as MitoQ represent a promising novel strategy for the preservation of vascular endothelial function with advancing age and the prevention of age-related CVD.

Motor function in humans can be characterized with tests of locomotion, strength, balance, and endurance. The aim of our project was to establish an analogous test battery to assess motor function in mice. Male C57BL/6 mice were studied at 3 (n = 87), 20 (n = 48) and 26 (n = 43) months of age. Tests assessed locomotion, strength, balance/coordination, and endurance capacity in mice. Motor function was reduced in the older groups of mice for the locomotion, strength, and endurance subdomains (p

Arterial aging, characterized by stiffening of large elastic arteries and the development of arterial endothelial dysfunction, increases cardiovascular disease (CVD) risk. We tested the hypothesis that spermidine, a nutrient associated with the anti-aging process autophagy, would improve arterial aging. Aortic pulse wave velocity (aPWV), a measure of arterial stiffness, was ~20% greater in old (O, 28 months) compared with young C57BL6 mice (Y, 4 months, P < 0.05). Arterial endothelium-dependent dilation (EDD), a measure of endothelial function, was ~25% lower in O (P < 0.05 vs. Y) due to reduced nitric oxide (NO) bioavailability. These impairments were associated with greater arterial oxidative stress (nitrotyrosine), superoxide production, and protein cross-linking (advanced glycation end-products, AGEs) in O (all P < 0.05). Spermidine supplementation normalized aPWV, restored NO-mediated EDD and reduced nitrotyrosine, superoxide, AGEs and collagen in O. These effects of spermidine were associated with enhanced arterial expression of autophagy markers, and in vitro experiments demonstrated that vascular protection by spermidine was autophagy-dependent. Our results indicate that spermidine exerts a potent anti-aging influence on arteries by increasing NO bioavailability, reducing oxidative stress, modifying structural factors and enhancing autophagy. Spermidine may be a promising nutraceutical treatment for arterial aging and prevention of age-associated CVD.

Mitochondrial dysregulation and associated excessive reactive oxygen species (mtROS) production is a key source of oxidative stress in aging arteries that reduces baseline function and may influence resilience (ability to withstand stress). We hypothesized that voluntary aerobic exercise would increase arterial resilience in old mice. An acute mitochondrial stressor (rotenone) caused greater (further) impairment in peak carotid EDD in old (~27 mo., OC, n=12; -32.5±-10.5%) versus young (~7 mo., YC n=11; -5.4±- 3.7%) control male mice, whereas arteries from young and old exercising (YVR n=10 and OVR n=11, 10-wk voluntary running; -0.8±-2.1% and -8.0±4.9%, respectively) mice were protected. Ex-vivo simulated Western diet (WD, high glucose and palmitate) caused greater impairment in EDD in OC (-28.5±8.6%) versus YC (-16.9±5.2%) and YVR (-15.3±2.3%), whereas OVR (-8.9±3.9%) were more resilient (not different versus YC). Simultaneous ex-vivo treatment with mitochondria-specific antioxidant MitoQ attenuated WD-induced impairments in YC and OC, but not YVR or OVR, suggesting that exercise improved resilience to mtROS-mediated stress. Exercise normalized age-related alterations in aortic mitochondrial protein markers PGC-1α, SIRT-3 and Fis1 and augmented cellular antioxidant and stress response proteins. Our results indicate that arterial aging is accompanied by reduced resilience and mitochondrial health, which are restored by voluntary aerobic exercise.

Aging is associated with motor declines that lead to functional limitations and disability, necessitating the development of therapies to slow or reverse these events. We tested the hypothesis that sodium nitrite supplementation attenuates declines in motor function in older C57BL/6 mice. Motor function was assessed using a battery of tests (grip strength, open-field distance, rota-rod endurance) in old animals (age 20–24 mo) at baseline and after 8 wk of sodium nitrite (old nitrite, n = 22, 50 mg/liter) or no treatment (old control, n = 40), and in young reference animals (3 mo, n = 87). Eight weeks of sodium nitrite supplementation improved grip strength (old nitrite, +12.0 ± 14.9% vs. old control, +1.5 ± 15.2%, P < 0.05) and open field distance (old nitrite, +9.5 ± 7.7%, P < 0.01 vs. old control, −28.1 ± 2.0%) and completely restored rota-rod endurance-run time (old nitrite, +3.2 ± 7.1%, P < 0.01 vs. old control, −21.5 ± 7.2%; old nitrite after treatment P > 0.05 vs. young reference). Inflammatory cytokines were markedly increased in quadriceps of old compared with young reference animals (by ELISA, interleukin-1β [IL-1β] 3.86 ± 2.34 vs. 1.11 ± 0.74, P < 0.05; interferon-gamma [INF-γ] 8.31 ± 1.59 vs. 3.99 ± 2.59, P < 0.01; tumor necrosis factor-alpha [TNF-α] 1.69 ± 0.44 vs. 0.76 ± 0.30 pg/ml, P < 0.01), but were reduced to young reference levels after treatment (old nitrite, IL-1β 0.67 ± 0.95; INF-γ 5.22 ± 2.01, TNF-α 1.21 ± 0.39 pg/ml, P < 0.05 vs. old control, P > 0.05 vs. young reference). Cytokine expression and treatment (old nitrite vs. old control) predicted strength ( R2 = 0.822, P < 0.001, IL-1β, INF-γ, group), open field distance ( R2 = 0.574, P < 0.01, IL-1β, group) and endurance run time ( R2 = 0.477, P < 0.05, INF-γ). Our results suggest that sodium nitrite improves motor function in old mice, in part by reducing low-grade inflammation in muscle.

Age-related arterial endothelial dysfunction, a key antecedent of the development of cardiovascular disease (CVD), is largely caused by a reduction in nitric oxide (NO) bioavailability as a consequence of oxidative stress. Mitochondria are a major source and target of vascular oxidative stress when dysregulated. Mitochondrial dysregulation is associated with primary ageing, but its role in age-related endothelial dysfunction is unknown. Our aim was to determine the efficacy of a mitochondria-targeted antioxidant, MitoQ, in ameliorating vascular endothelial dysfunction in old mice. Ex vivo carotid artery endothelium-dependent dilation (EDD) to increasing doses of acetylcholine was impaired by ∼30% in old (∼27 months) compared with young (∼8 months) mice as a result of reduced NO bioavailability (P < 0.05). Acute (ex vivo) and chronic (4 weeks in drinking water) administration of MitoQ completely restored EDD in older mice by improving NO bioavailability. There were no effects of age or MitoQ on endothelium-independent dilation to sodium nitroprusside. The improvements in endothelial function with MitoQ supplementation were associated with the normalization of age-related increases in total and mitochondria-derived arterial superoxide production and oxidative stress (nitrotyrosine abundance), as well as with increases in markers of vascular mitochondrial health, including antioxidant status. MitoQ also reversed the age-related increase in endothelial susceptibility to acute mitochondrial damage (rotenone-induced impairment in EDD). Our results suggest that mitochondria-derived oxidative stress is an important mechanism underlying the development of endothelial dysfunction in primary ageing. Mitochondria-targeted antioxidants such as MitoQ represent a promising novel strategy for the preservation of vascular endothelial function with advancing age and the prevention of age-related CVD.

The gut microbiome, an emerging mediator of host physiological function, is adversely altered by aging and many diseases, termed “gut dysbiosis.” One consequence of gut dysbiosis is elevated circulating levels of the gut-derived metabolite trimethylamine N-oxide (TMAO), which has been directly linked to cardiovascular (CV) risk, including the development of atherosclerosis. However, it is unknown whether TMAO mediates arterial dysfunction that precedes the onset of clinical disease, and if so, the underlying mechanisms. Purpose: To determine whether TMAO independently induces large elastic artery stiffening and endothelial dysfunction via increased superoxide-related oxidative stress. Method: Twenty young (6 mo) male C57BL/6 mice were fed a chemically-defined choline (0.08–0.09%) diet supplemented without (Control; N = 9) or with (N = 11) 0.12% TMAO for 6 months. Arterial stiffness was assessed as aortic pulse wave velocity (aPWV). Endothelial function was evaluated ex vivo as carotid artery endothelium-dependent dilation (EDD) to increasing doses of acetylcholine (10−9 to 10−4M) in the absence or presence of the superoxide dismutase mimetic TEMPOL. Results: TMAO increased aPWV (Control: 392 ± 20 vs. TMAO: 483 ± 32 cm/sec, p = 0.04) and impaired EDD (peak dilation, Control: 93.7 ± 3.2 vs. TMAO: 79.9 ± 3.4%, p = 0.01). Suppression of oxidative stress with TEMPOL restored EDD in TMAO-treated animals (peak dilation: 92.1 ± 4.7%, p = 0.46 vs. Control). Conclusions: TMAO independently induces large elastic artery stiffening and endothelial dysfunction in mice. Dysfunction appears to occur through increases in oxidative stress. These data may explain, at least in part, why TMAO increases CV risk and provide a potential target for prevention/treatment of arterial dysfunction. Supported by R01 HL134887 & T32 HL007822.

Aging leads to large elastic artery stiffening mediated by increases in superoxide-dependent oxidative stress and related changes/damage to structural proteins. Mitochondria are a major source of c...