Suppression of trimethylamine N-oxide with DMB mitigates vascular dysfunction, exercise intolerance, and frailty associated with a Western-style diet in mice.

Consumption of a Western-style diet (WD; high fat, high sugar, low fiber) is associated with impaired vascular function and increased risk of cardiovascular diseases (CVD), which could be mediated partly by increased circulating concentrations of the gut microbiome-derived metabolite trimethylamine N-oxide (TMAO). We investigated if suppression of TMAO with 3,3-dimethyl-1-butanol (DMB; inhibitor of microbial TMA lyase) in mice could prevent: 1) WD-induced vascular endothelial dysfunction and aortic stiffening and 2) WD-induced reductions in endurance exercise tolerance and increases in frailty, as both are linked to WD, vascular dysfunction, and increased CVD risk. C57BL/6N mice were fed standard chow or WD (41% fat, ∼25% sugar, 4% fiber) for 5 mo beginning at ∼2 mo of age. Within each diet, mice randomly received (n = 11-13/group) normal drinking water (control) or 1% DMB in drinking water for the last 8 wk (from 5 to 7 mo of age). Plasma TMAO was increased in WD-fed mice but suppressed by DMB. WD induced endothelial dysfunction, assessed as carotid artery endothelium-dependent dilation to acetylcholine, and progressive increases in aortic stiffness (measured serially in vivo as pulse wave velocity), both of which were fully prevented by supplementation with DMB. Endurance exercise tolerance, assessed as time to fatigue on a rotarod test, was impaired in WD-fed mice but partially recovered by DMB. Lastly, WD-induced increases in frailty (31-point index) were prevented by DMB. Our findings indicate DMB or other TMAO-lowering therapies may be promising for mitigating the adverse effects of WD on physiological function, and thereby reducing risk of chronic diseases.NEW & NOTEWORTHY 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.

The gut microbiome-derived metabolite trimethylamine N-oxide modulates neuroinflammation and cognitive function with aging.

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 r2 = 0.07, p < 0.05). Study 2: In mice, TMAO concentrations in plasma and the brain increased in parallel with aging (r2 = 0.60), suggesting TMAO crosses the blood-brain barrier. The greater TMAO concentrations in old mice (27 months) were associated with higher brain pro-inflammatory cytokines and markers of astrocyte activation vs. young adult mice (6 months). Study 3: To determine if TMAO independently induces an "aging-like" decline in cognitive function, young mice (6 months) were supplemented with TMAO in chow for 6 months. Compared with controls, TMAO-supplemented mice performed worse on the novel object recognition test, indicating impaired memory and learning, and had increased neuroinflammation and markers of astrocyte activation. Study 4: Human astrocytes cultured with TMAO vs. control media exhibited changes in cellular morphology and protein markers consistent with astrocyte activation, indicating TMAO directly acts on these cells. Our results provide translational insight into a novel pathway that modulates neuroinflammation and cognitive function with aging, and suggest that TMAO might be a promising target for prevention of neuroinflammation and cognitive decline with aging.

Gut Microbiome-Derived Metabolite Trimethylamine N-Oxide Induces Aortic Stiffening and Increases Systolic Blood Pressure With Aging in Mice and Humans.

[Figure: see text].

Short-term interleukin-37 treatment improves vascular endothelial function, endurance exercise capacity, and whole-body glucose metabolism in old mice.

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 < .05); this effect was accompanied by enhanced ACh-stimulated NO production and reduced levels of reactive oxygen species in endothelial cells cultured with plasma from IL-37-treated animals (p < .05 vs. vehicle plasma). RecIL-37 treatment increased endurance exercise capacity by 2.4-fold, which was accompanied by a 2.9-fold increase in the phosphorylated AMP-activated kinase (AMPK) to AMPK ratio (i.e., AMPK activation) in quadriceps muscle. RecIL-37 treatment also improved whole-body insulin sensitivity and glucose tolerance (p < .05 vs. vehicle). Improvements in physiological function occurred without significant changes in plasma, aortic, and skeletal muscle pro-inflammatory proteins (under resting conditions), whereas pro-/anti-inflammatory IL-6 was greater in recIL-37-treated animals. Plasma metabolomics analysis revealed that recIL-37 treatment altered metabolites related to pathways involved in NO synthesis (e.g., increased L-arginine and citrulline/arginine ratio) and fatty acid metabolism (e.g., increased pantothenol and free fatty acids). Our findings provide experimental support for IL-37 therapy as a novel strategy to improve diverse physiological functions in old age.

Trimethylamine-N-Oxide Promotes Age-Related Vascular Oxidative Stress and Endothelial Dysfunction in Mice and Healthy Humans.

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 (r2=0.29, P<0.00001). In young mice, 6 months of dietary supplementation with trimethylamine-N-oxide induced an aging-like impairment in carotid artery endothelium-dependent dilation to acetylcholine versus control feeding (peak dilation: 79±3% versus 95±3%, P<0.01). This impairment was accompanied by increased vascular nitrotyrosine, a marker of oxidative stress, and reversed by the superoxide dismutase mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl. Trimethylamine-N-oxide supplementation also reduced activation of endothelial nitric oxide synthase and impaired nitric oxide-mediated dilation, as assessed with the nitric oxide synthase inhibitor L-NAME (NG-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.