Gut Microbiome: An Effector of Dietary Nitrate That Inhibits Cardiometabolic Disease?

Obesity is one of the most common cardiometabolic diseases. The prevalence of obesity is increasing due to increases in high-energy and low-fiber food consumption and sedentary lifestyle (1). One of the relevant obesity-related cardiac diseases is diabetic cardiomyopathy, which is diagnosed by the presence of insulin resistance and cardiac impairments while excluding other cardiomyopathies, including ischemic, inherited, and hypertensive cardiomyopathies (2). The clinical features of diabetic cardiomyopathy are diastolic dysfunction and an enlarged heart, a condition termed cardiac hypertrophy, leading to heart failure with preserved ejection fraction (HFpEF) (3). Given that obesity or diabetes complicates other cardiometabolic diseases, which often manifest as ischemic or hypertensive heart disease, diabetic cardiomyopathy may be underestimated in the clinical setting. Although the pathogenesis of diabetic cardiomyopathy is multifactorial, cardiac metabolic derangement is a primary underlying mechanism.

Based on the features of diastolic failure and ventricular stiffness in HFpEF, the therapeutic strategy to enhance nitric oxide (NO) bioavailability, which is essential for vascular smooth muscle relaxation and muscle contractile function, has been evaluated in clinical trials. Remarkably, dietary nitrate (NO₃⁻) consumption by beetroot juice increases exercise capacity in patients with HFpEF (4) and maximum rate of oxygen during exercise in patients with diabetes (5), suggesting that dietary nitrate could be a therapeutic option for diabetic cardiomyopathy as well. Nitrate is an inorganic anion, and its major sources for mammals are the NO synthase (NOS) pathway and diet. Nitrate is rich in leafy green vegetables, including spinach and arugula, and beetroot. The NO metabolism is regulated by the NOS pathways and the nitrate-nitrite (NO₂⁻)-NO reduction pathways. Dietary nitrate is quickly absorbed into circulation and tissues and can be reduced to nitrite and NO in tissues (6). Gut bacteria use host-delivered nitrate and oxygen to produce energy through redox reactions in the small intestine, facilitating the growth of facultatively anaerobic bacteria during gut homeostasis. In contrast, the host limits the availability of oxygen and nitrate in the colonic lumen to facilitate the growth of obligately anaerobic primary fermenters. Dietary behavior and medications pronouncedly affect the composition and function of gut microbiota, which in turn produces metabolites that influence host physiology and pathology, including obesity and cardiovascular disease (7,8) (Fig. 1). The high-energy, low-fiber dietary behavior increases the availability of host-derived oxygen and nitrate in the colonic lumen, causing microbial dysbiosis, where the gut microbial communities are imbalanced or lack diversity without regard to the presence or absence of harmful or beneficial microbes. Growing facultatively anaerobic bacteria in the large intestine adversely affects host health in part by dysbiosis-derived metabolites. Rodent studies reported the beneficial effect of dietary nitrate on high-fat-diet (HFD)–induced gut dysbiosis (9) and liver steatosis via microbiota (10). However, whether dietary nitrate protects the heart from HFD-induced metabolic stress and, if so, whether the gut microbiome is a hub of dietary nitrate action for diabetic cardiomyopathy have not yet been well understood.

In this issue of Diabetes, Petrick et al. (11) applied HFD (60% calories from fat) feeding to male C57BL/6N mice to induce cardiometabolic disease, which generally includes obesity, glucose intolerance, dyslipidemia, and diabetic cardiomyopathy (12). In the current study, blood pressure increased in mice fed an HFD, indicating that the study model does not faithfully represent diabetic cardiomyopathy but rather broadly defined obesity-related or metabolic cardiomyopathy. Petrick et al. (11) show that dietary nitrate via drinking water (4 mmol/L sodium nitrate) for 8 weeks can inhibit HFD-induced glucose intolerance, myocardial lipid accumulation, and cardiac hypertrophy and dysfunction without affecting body weight and blood pressure.

To better understand how dietary nitrate exerts cardioprotection against HFD, Petrick et al. (11) robustly examined bacterial taxonomy and metabolites of fecal microbiota. The abundance of Firmicutes and the ratio of Firmicutes to Bacteroidetes increased in mice fed an HFD, but this increase was prevented by dietary nitrate. These findings suggest that nitrate consumption contributes to gut homeostasis during HFD feeding. Notably, Petrick et al. (11) demonstrate that fecal microbiota transplant (FMT) from mice fed an HFD in the presence, but not absence, of dietary nitrate appears sufficient to prevent HFD-induced metabolic derangement and cardiac functional changes without increasing serum NO_x concentrations. These results indicate that dietary nitrate plays an important role in maintaining homeostasis of gut microbiota. In addition, this finding may uncover a critical role of gut dysbiosis in the pathology of diabetic cardiomyopathy.

Alongside the significance of this study, there remain some outstanding questions. First, it is uncertain whether the gut microbiome is required for the action of dietary nitrate on cardioprotection. Holloway and colleagues recently reported that dietary nitrate attenuates HFD-induced adipose inflammation (13) and improves skeletal muscle insulin signaling in a Sirt1-dependent manner (14). It is tempting to speculate that dietary nitrate affects metabolic signaling in multiple tissues as well as in gut microbiota. The effect of dietary nitrate on gut homeostasis may not be direct but rather secondary to the homeostasis of other organs being regulated in parallel by nitrate. Thus, it will be important to conduct experiments that disrupt the gut microbiota pathway, such as by using antibiotics or germ-free mice.

Second, how does the FMT from mice taking dietary nitrate mitigate HFD-induced diabetic cardiomyopathy? Neither fecal NO_x concentration of donor mice nor serum levels of NO_x in mice receiving the FMT was changed by dietary nitrate, and blood pressure was reported not to be altered by the FMT, suggesting very little involvement of NO bioavailability in FMT-mediated cardioprotection. A clinical study reported that administration of inhaled inorganic nitrite failed to improve exercise capacity among patients with HFpEF (15), supporting the current finding that dietary nitrate acts as a modulator of redox reactions for the gut microbiota.

The functions of multiple organs located far apart can be regulated in a coordinated manner, including the heart, muscle, adipose tissue, and liver, through circulating factors, such as cytokines (16). In recent years, the gut–heart axis through metabolites or other molecules produced by gut microbiota has been reported (8). Trimethylamine is a metabolite produced by microbiota with a high-energy, low-fiber dietary behavior and is converted into trimethylamine N-oxide (TMAO) in the liver (17). Elevated circulating TMAO causes atherosclerosis and is associated with mortality (18). Untargeted metabolomics identified phenylacetylglutamine as a gut microbiota-derived metabolite that enhances platelet activity and thrombosis, thereby promoting cardiovascular disease (19). In contrast, the current study failed to detect nitrate-mediated, significantly differentiated metabolites using targeted fecal metabolomics. Future studies will be needed to identify causative microbiota-derived metabolites or the compositional and functional changes of gut microbiota by dietary nitrate or FMT with a more in-depth and comprehensive analysis.

This study raises another important question. Patients with cardiometabolic disease are heavily medicated. Observational studies identified associative effects of drugs, drug combinations, and previous exposure to antibiotics on variations in the gut microbiome in cardiometabolic disease, which include statins and metformin (20,21). Accordingly, it will be interesting to test whether dietary nitrate has a synergistic or additive effect with other medications on gut homeostasis.

In summary, Petrick et al. (11) reveal that dietary nitrate contributes to gut homeostasis, which could prevent diet-induced diabetic cardiomyopathy. Further, the current work suggests a critical role of dysbiosis or metabolites produced by gut microbiota in the pathology of diabetic cardiomyopathy. Since no effective therapeutics to modulate the gut microbiome have been established yet, the current study may provide a microbial-targeted therapy using FMT or downstream metabolites using dietary nitrate for the treatment of diabetic cardiomyopathy.