ATP biosensor reveals microbial energetic dynamics and facilitates bioproduction.

Adenosine-5'-triphosphate (ATP), the primary energy currency in cellular processes, drives metabolic activities and biosynthesis. Despite its importance, understanding intracellular ATP dynamics' impact on bioproduction and exploiting it for enhanced bioproduction remains largely unexplored. Here, we harness an ATP biosensor to dissect ATP dynamics across different growth phases and carbon sources in multiple microbial strains. We find transient ATP accumulations during the transition from exponential to stationary growth phases in various conditions, coinciding with fatty acid (FA) and polyhydroxyalkanoate (PHA) production in Escherichia coli and Pseudomonas putida, respectively. We identify carbon sources (acetate for E. coli, oleate for P. putida) that elevate steady-state ATP levels and boost FA and PHA production. Moreover, we employ ATP dynamics as a diagnostic tool to assess metabolic burden, revealing bottlenecks that limit limonene bioproduction. Our results not only elucidate the relationship between ATP dynamics and bioproduction but also showcase its value in enhancing bioproduction in various microbial species.

Loss of Macrophage mTORC2 Drives Atherosclerosis via FoxO1 and IL-1ß Signaling.

BACKGROUND: The mTOR (mechanistic target of rapamycin) pathway is a complex signaling cascade that regulates cellular growth, proliferation, metabolism, and survival. Although activation of mTOR signaling has been linked to atherosclerosis, its direct role in lesion progression and in plaque macrophages remains poorly understood. We previously demonstrated that mTORC1 (mTOR complex 1) activation promotes atherogenesis through inhibition of autophagy and increased apoptosis in macrophages. METHODS: Using macrophage-specific Rictor- and mTOR-deficient mice, we now dissect the distinct functions of mTORC2 pathways in atherogenesis. RESULTS: In contrast to the atheroprotective effect seen with blockade of macrophage mTORC1, macrophage-specific mTORC2-deficient mice exhibit an atherogenic phenotype, with larger, more complex lesions and increased cell death. In cultured macrophages, we show that mTORC2 signaling inhibits the FoxO1 (forkhead box protein O1) transcription factor, leading to suppression of proinflammatory pathways, especially the inflammasome/IL (interleukin)-1ß response, a key mediator of vascular inflammation and atherosclerosis. In addition, administration of FoxO1 inhibitors efficiently rescued the proinflammatory response caused by mTORC2 deficiency both in vitro and in vivo. Interestingly, collective deletion of macrophage mTOR, which ablates mTORC1- and mTORC2-dependent pathways, leads to minimal change in plaque size or complexity, reflecting the balanced yet opposing roles of these signaling arms. CONCLUSIONS: Our data provide the first mechanistic details of macrophage mTOR signaling in atherosclerosis and suggest that therapeutic measures aimed at modulating mTOR need to account for its dichotomous functions.

Trehalose causes low-grade lysosomal stress to activate TFEB and the autophagy-lysosome biogenesis response.

The autophagy-lysosome system is an important cellular degradation pathway that recycles dysfunctional organelles and cytotoxic protein aggregates. A decline in this system is pathogenic in many human diseases including neurodegenerative disorders, fatty liver disease, and atherosclerosis. Thus there is intense interest in discovering therapeutics aimed at stimulating the autophagy-lysosome system. Trehalose is a natural disaccharide composed of two glucose molecules linked by a ɑ-1,1-glycosidic bond with the unique ability to induce cellular macroautophagy/autophagy and with reported efficacy on mitigating several diseases where autophagy is dysfunctional. Interestingly, the mechanism by which trehalose induces autophagy is unknown. One suggested mechanism is its ability to activate TFEB (transcription factor EB), the master transcriptional regulator of autophagy-lysosomal biogenesis. Here we describe a potential mechanism involving direct trehalose action on the lysosome. We find trehalose is endocytically taken up by cells and accumulates within the endolysosomal system. This leads to a low-grade lysosomal stress with mild elevation of lysosomal pH, which acts as a potent stimulus for TFEB activation and nuclear translocation. This process appears to involve inactivation of MTORC1, a known negative regulator of TFEB which is sensitive to perturbations in lysosomal pH. Taken together, our data show the trehalose can act as a weak inhibitor of the lysosome which serves as a trigger for TFEB activation. Our work not only sheds light on trehalose action but suggests that mild alternation of lysosomal pH can be a novel method of inducing the autophagy-lysosome system.Abbreviations: ASO: antisense oligonucleotide; AU: arbitrary units; BMDM: bone marrow-derived macrophages; CLFs: crude lysosomal fractions; CTSD: cathepsin D; LAMP: lysosomal associated membrane protein; LIPA/LAL: lipase A, lysosomal acid type; MAP1LC3: microtubule-associated protein 1 light chain 3; MFI: mean fluorescence intensity; MTORC1: mechanistic target of rapamycin kinase complex 1; pMAC: peritoneal macrophages; SLC2A8/GLUT8: solute carrier family 2, (facilitated glucose transporter), member 8; TFEB: transcription factor EB; TMR: tetramethylrhodamine; TREH: trehalase.

Late-life voluntary wheel running reverses age-related aortic stiffness in mice: a translational model for studying mechanisms of exercise-mediated arterial de-stiffening.

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 < 0.05). The de-stiffening effects of VWR were accompanied by normalization of age-related increases in ex vivo mechanical stiffness of aortic segments and aortic accumulation of collagen-I and advanced glycation end products, as well as lower levels of aortic superoxide and nitrotyrosine. Our results suggest that late-life VWR in mice recapitulates the aortic de-stiffening effects of exercise in humans and indicates important mechanistic roles for decreased oxidative stress and extracellular matrix remodeling. Therefore, VWR is a suitable model for further study of the mechanisms underlying beneficial effects of exercise on arterial stiffness.

Author Correction: High-protein diets increase cardiovascular risk by activating macrophage mTOR to suppress mitophagy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

TFEB is a master regulator of tumor-associated macrophages in breast cancer.

BACKGROUND: Tumor-associated macrophages (TAMs) play key roles in the development of many malignant solid tumors including breast cancer. They are educated in the tumor microenvironment (TME) to promote tumor growth, metastasis, and therapy resistance. However, the phenotype of TAMs is elusive and how to regulate them for therapeutic purpose remains unclear; therefore, TAM-targeting therapies have not yet achieved clinical success. The purposes of this study were to examine the role of transcription factor EB (TFEB) in regulating TAM gene expression and function and to determine if TFEB activation can halt breast tumor development. METHODS: Microarrays were used to analyze the gene expression profile of macrophages (MΦs) in the context of breast cancer and to examine the impact of TFEB overexpression. Cell culture studies were performed to define the mechanisms by which TFEB affects MΦ gene expression and function. Mouse studies were carried out to investigate the impact of MΦ TFEB deficiency or activation on breast tumor growth. Human cancer genome data were analyzed to reveal the prognostic value of TFEB and its regulated genes. RESULTS: TAM-mimic MΦs display a unique gene expression profile, including significant reduction in TFEB expression. TFEB overexpression favorably modulates TAM gene expression through multiple signaling pathways. Specifically, TFEB upregulates suppressor of cytokine signaling 3 (SOCS3) and peroxisome proliferator-activated receptor γ (PPARγ) expression and autophagy/lysosome activities, inhibits NLRP3 (NLR Family Pyrin Domain Containing 3) inflammasome and hypoxia-inducible factor (HIF)-1α mediated hypoxia response, and thereby suppresses an array of effector molecules in TAMs including arginase 1, interleukin (IL)-10, IL-1ß, IL-6 and prostaglandin E2. MΦ-specific TFEB deficiency promotes, while activation of TFEB using the natural disaccharide trehalose halts, breast tumor development by modulating TAMs. Analysis of human patient genome database reveals that expression levels of TFEB, SOCS3 and PPARγ are positive prognostic markers, while HIF-1α is a negative prognostic marker of breast cancer. CONCLUSIONS: Our study identifies TFEB as a master regulator of TAMs in breast cancer. TFEB controls TAM gene expression and function through multiple autophagy/lysosome-dependent and independent pathways. Therefore, pharmacological activation of TFEB would be a promising therapeutic approach to improve the efficacy of existing treatment including immune therapies for breast cancer by favorably modulating TAM function and the TME.

Bacterial metabolic heterogeneity: origins and applications in engineering and infectious disease.

Bacteria within an isoclonal population display significant heterogeneity in metabolism, even under tightly controlled environmental conditions. Metabolic heterogeneity enables influential functions not possible or measurable at the ensemble scale. Several molecular and cellular mechanisms are likely to give rise to metabolic heterogeneity including molecular noise in metabolic enzyme expression, positive feedback loops, and asymmetric partitioning of cellular components during cell division. Dissection of the mechanistic origins of metabolic heterogeneity has been enabled by recent developments in single-cell analytical tools. Finally, we provide a discussion of recent studies examining the importance of metabolic heterogeneity in applied settings such as infectious disease and metabolic engineering.

High-protein diets increase cardiovascular risk by activating macrophage mTOR to suppress mitophagy.

High protein diets are commonly utilized for weight loss, yet have been reported to raise cardiovascular risk. The mechanisms underlying this risk are unknown. Here, we show that dietary protein drives atherosclerosis and lesion complexity. Protein ingestion acutely elevates amino acid levels in blood and atherosclerotic plaques, stimulating macrophage mTOR signaling. This is causal in plaque progression as the effects of dietary protein are abrogated in macrophage-specific Raptor-null mice. Mechanistically, we find amino acids exacerbate macrophage apoptosis induced by atherogenic lipids, a process that involves mTORC1-dependent inhibition of mitophagy, accumulation of dysfunctional mitochondria, and mitochondrial apoptosis. Using macrophage-specific mTORC1- and autophagy-deficient mice we confirm this amino acid-mTORC1-autophagy signaling axis in vivo. Our data provide the first insights into the deleterious impact of excessive protein ingestion on macrophages and atherosclerotic progression. Incorporation of these concepts in clinical studies will be important to define the vascular effects of protein-based weight loss regimens.

TFEB drives PGC-1α expression in adipocytes to protect against diet-induced metabolic dysfunction.

TFEB is a basic helix-loop-helix transcription factor that confers protection against metabolic diseases such as atherosclerosis by targeting a network of genes involved in autophagy-lysosomal biogenesis and lipid catabolism. In this study, we sought to characterize the role of TFEB in adipocyte and adipose tissue physiology and evaluate the therapeutic potential of adipocyte-specific TFEB overexpression in obesity. We demonstrated that mice with adipocyte-specific TFEB overexpression (Adipo-TFEB) were protected from diet-induced obesity, insulin resistance, and metabolic sequelae. Adipo-TFEB mice were lean primarily through increased metabolic rate, suggesting a role for adipose tissue browning and enhanced nonshivering thermogenesis in fat. Transcriptional characterization revealed that TFEB targeted genes involved in adipose tissue browning rather than those involved in autophagy. One such gene encoded PGC-1α, an established target of TFEB that promotes adipocyte browning. To dissect the role of PGC-1α in mediating the downstream effects of TFEB overexpression, we generated mice with adipocyte-specific PGC-1α deficiency and TFEB overexpression. Without PGC-1α, the ability of TFEB overexpression to brown adipose tissue and to elicit beneficial metabolic effects was blunted. Overall, these data implicate TFEB as a PGC-1α-dependent regulator of adipocyte browning and suggest its therapeutic potential in treating metabolic disease.

Functional Characterization of LIPA (Lysosomal Acid Lipase) Variants Associated With Coronary Artery Disease.

OBJECTIVE: LIPA (lysosomal acid lipase) mediates cholesteryl ester hydrolysis, and patients with rare loss-of-function mutations develop hypercholesterolemia and severe disease. Genome-wide association studies of coronary artery disease have identified several tightly linked, common intronic risk variants in LIPA which unexpectedly associate with increased mRNA expression. However, an exonic variant (rs1051338 resulting in T16P) in linkage with intronic variants lies in the signal peptide region and putatively disrupts trafficking. We sought to functionally investigate the net impact of this locus on LIPA and whether rs1051338 could disrupt LIPA processing and function to explain coronary artery disease risk. Approach and Results: In monocytes isolated from a large cohort of healthy individuals, we demonstrate both exonic and intronic risk variants are associated with increased LIPA enzyme activity coincident with the increased transcript levels. To functionally isolate the impact of rs1051338, we studied several in vitro overexpression systems and consistently observed no differences in LIPA expression, processing, activity, or secretion. Further, we characterized a second common exonic coding variant (rs1051339), which is predicted to alter LIPA signal peptide cleavage similarly to rs1051338, yet is not linked to intronic variants. rs1051339 also does not impact LIPA function in vitro and confers no coronary artery disease risk. CONCLUSIONS: Our findings show that common LIPA exonic variants in the signal peptide are of minimal functional significance and suggest coronary artery disease risk is instead associated with increased LIPA function linked to intronic variants. Understanding the mechanisms and cell-specific contexts of LIPA function in the plaque is necessary to understand its association with cardiovascular risk.

p62/SQSTM1 and Selective Autophagy in Cardiometabolic Diseases.

Significance: p62/SQSTM1 is a multifunctional scaffolding protein involved in the regulation of various signaling pathways as well as autophagy. In particular, p62/SQSTM1 serves as an essential adaptor to identify and deliver specific organelles and protein aggregates to autophagosomes for degradation, a process known as selective autophagy. Critical Issues: With the emergence of autophagy as a critical process in cellular metabolism and the development of cardiometabolic diseases, it is increasingly important to understand p62's role in the integration of signaling and autophagic pathways. Recent Advances: This review first discusses the features that make p62/SQSTM1 an ideal chaperone in integrating signaling pathways with autophagy and details the current understanding of its diverse roles in selective autophagy processes. Distinct and overlapping roles of other chaperones with similar functions are then discussed in the context of p62/SQSTM1. Finally, the recent literature focusing on p62 and selective autophagy in metabolism and the spectrum of cardiometabolic diseases including atherosclerosis, fatty liver disease, and obesity is evaluated. Future Directions: A comprehensive understanding of the nuanced roles p62/SQSTM1 plays in mediating distinct autophagy pathways would provide new insights into the mechanisms of this critical degradative pathway. This will, in turn, facilitate our understanding of cardiovascular and cardiometabolic disease pathology and the development of novel autophagy-modulating therapeutic strategies.

Classical and alternative roles for autophagy in lipid metabolism.

PURPOSE OF REVIEW: Intracellular lipid metabolism is a complex interplay of exogenous lipid handling, trafficking, storage, lipolysis, and export. Recent work has implicated the cellular degradative process called autophagy in several aspects of lipid metabolism. We will discuss both the classical and novel roles of autophagy and the autophagic machinery in this setting. RECENT FINDINGS: The delivery of lipid droplets to lysosomes for hydrolysis, named lipophagy, was the first described functional role for autophagy in lipid metabolism. The molecular machinery and regulation of this selective form of macroautophagy is beginning to be discovered and has the potential to shed enormous light on intracellular lipolysis. Yet, the autophagic machinery appears to also be coopted for alternative roles that include interaction with cytosolic lipolysis pathways, supply and expansion of lipid droplets, and lipoprotein trafficking. Additionally, lesser studied forms of autophagy called microautophagy and chaperone-mediated autophagy have distinct roles in lipid handling that also intersect with classical macroautophagy. The integration of current knowledge in these areas into a holistic understanding of intracellular lipid metabolism will be a goal of this review. SUMMARY: As the field of autophagy has evolved and expanded to include functional roles in various aspects of cellular degradation, so has its role in intracellular lipid metabolism. Understanding the mechanisms underlying these classical and alternative roles of autophagy will not only enhance our knowledge in lipid biology but also provide new avenues of translation to human lipid disorders.

TFEB and trehalose drive the macrophage autophagy-lysosome system to protect against atherosclerosis.

In the atherosclerotic plaque, macrophages are the key catabolic workhorse responsible for clearing lipid and dead cell debris. To survive the highly proinflammatory and lipotoxic plaque environment, macrophages must adopt strategies for maintaining tight homeostasis and self-renewal. Macroautophagy/autophagy is a pro-survival cellular pathway wherein damaged or excess cellular cargoes are encapsulated by a double-membrane compartment and delivered to the lysosome for hydrolysis. Previously, macrophage-specific autophagy deficiency has been shown to be atherogenic through several complementary mechanisms including hyperactivation of the inflammasome, defective efferocytosis, accumulation of cytotoxic protein aggregates, and impaired lipid degradation. Conversely, in a recent study we hypothesized that enhancing the macrophage autophagy-lysosomal system through genetic or pharmacological means could protect against atherosclerosis. We demonstrated that TFEB, a transcription factor master regulator of autophagy and lysosome biogenesis, coordinately enhances the function of this system to reduce atherosclerotic plaque burden. Further, we characterized the disaccharide trehalose as a novel inducer of TFEB with similar atheroprotective effects. Overall, these findings mechanistically interrogate the importance and therapeutic promise of a functional autophagy-lysosome degradation system in plaque macrophage biology.

Transcriptional factor EB regulates macrophage polarization in the tumor microenvironment.

Tumor microenvironment (TME) contains a variety of infiltrating immune cells. Among them, tumor-associated macrophages (TAMs) and their alternative activation contribute greatly to the progression of tumors. The mechanisms governing macrophage polarization in the TME are unclear. Here, we show that in TAMs or macrophages under tumor-conditioned medium treatment, the expression of transcription factor EB (TFEB) is reduced and more of the TFEB protein is in an inactive cytosolic form. Transforming growth factor (TGF)-ß is identified as a main driving force for the reduced TFEB expression and activity in TAMs via activating ERK signaling. TFEB interference in macrophages significantly enhanced their alternative activation, with reduced expression of MHC-II and co-stimulatory molecule CD80, decreased ability to activate T cells, and increased ability to attract tumor cells. When co-inoculated with tumor cells, macrophages with TFEB knockdown significantly enhanced tumor growth with increased infiltration of M2-like macrophages, reduced infiltration of CD8+ T cells, and enhanced angiogenesis in the tumors. Mechanistic studies revealed that TFEB downregulation resulted in macrophage M2 polarization through reducing SOCS3 production and enhancing STAT3 activation. We further demonstrate that the activation of TFEB by hydroxypropyl-ß-cyclodextrin in macrophages suppressed their M2 polarization and tumor-promoting capacity, and that macrophage-specific TFEB overexpression inhibited breast tumor growth in mice. Therefore, our data suggest that TFEB plays critical roles in macrophage polarization, and the downregulation of TFEB expression and activation is an integral part of tumor-induced immune editing in the TME. This study provides a rationale for a new cancer treatment strategy by modulating macrophage polarization through activating TFEB.

Exploiting macrophage autophagy-lysosomal biogenesis as a therapy for atherosclerosis.

Macrophages specialize in removing lipids and debris present in the atherosclerotic plaque. However, plaque progression renders macrophages unable to degrade exogenous atherogenic material and endogenous cargo including dysfunctional proteins and organelles. Here we show that a decline in the autophagy-lysosome system contributes to this as evidenced by a derangement in key autophagy markers in both mouse and human atherosclerotic plaques. By augmenting macrophage TFEB, the master transcriptional regulator of autophagy-lysosomal biogenesis, we can reverse the autophagy dysfunction of plaques, enhance aggrephagy of p62-enriched protein aggregates and blunt macrophage apoptosis and pro-inflammatory IL-1ß levels, leading to reduced atherosclerosis. In order to harness this degradative response therapeutically, we also describe a natural sugar called trehalose as an inducer of macrophage autophagy-lysosomal biogenesis and show trehalose's ability to recapitulate the atheroprotective properties of macrophage TFEB overexpression. Our data support this practical method of enhancing the degradative capacity of macrophages as a therapy for atherosclerotic vascular disease.

Target acquired: Selective autophagy in cardiometabolic disease.

The accumulation of damaged or excess proteins and organelles is a defining feature of metabolic disease in nearly every tissue. Thus, a central challenge in maintaining metabolic homeostasis is the identification, sequestration, and degradation of these cellular components, including protein aggregates, mitochondria, peroxisomes, inflammasomes, and lipid droplets. A primary route through which this challenge is met is selective autophagy, the targeting of specific cellular cargo for autophagic compartmentalization and lysosomal degradation. In addition to its roles in degradation, selective autophagy is emerging as an integral component of inflammatory and metabolic signaling cascades. In this Review, we focus on emerging evidence and key questions about the role of selective autophagy in the cell biology and pathophysiology of metabolic diseases such as obesity, diabetes, atherosclerosis, and steatohepatitis. Essential players in these processes are the selective autophagy receptors, defined broadly as adapter proteins that both recognize cargo and target it to the autophagosome. Additional domains within these receptors may allow integration of information about autophagic flux with critical regulators of cellular metabolism and inflammation. Details regarding the precise receptors involved, such as p62 and NBR1, and their predominant interacting partners are just beginning to be defined. Overall, we anticipate that the continued study of selective autophagy will prove to be informative in understanding the pathogenesis of metabolic diseases and to provide previously unrecognized therapeutic targets.

Inclusion bodies enriched for p62 and polyubiquitinated proteins in macrophages protect against atherosclerosis.

Autophagy is a catabolic cellular mechanism that degrades dysfunctional proteins and organelles. Atherosclerotic plaque formation is enhanced in mice with macrophages deficient for the critical autophagy protein ATG5. We showed that exposure of macrophages to lipids that promote atherosclerosis increased the abundance of the autophagy chaperone p62 and that p62 colocalized with polyubiquitinated proteins in cytoplasmic inclusions, which are characterized by insoluble protein aggregates. ATG5-null macrophages developed further p62 accumulation at the sites of large cytoplasmic ubiquitin-positive inclusion bodies. Aortas from atherosclerotic mice and plaques from human endarterectomy samples showed increased abundance of p62 and polyubiquitinated proteins that colocalized with plaque macrophages, suggesting that p62-enriched protein aggregates were characteristic of atherosclerosis. The formation of the cytoplasmic inclusions depended on p62 because lipid-loaded p62-null macrophages accumulated polyubiquitinated proteins in a diffuse cytoplasmic pattern. Lipid-loaded p62-null macrophages also exhibited increased secretion of interleukin-1ß (IL-1ß) and had an increased tendency to undergo apoptosis, which depended on the p62 ubiquitin-binding domain and at least partly involved p62-mediated clearance of NLRP3 inflammasomes. Consistent with our in vitro observations, p62-deficient mice formed greater numbers of more complex atherosclerotic plaques, and p62 deficiency further increased atherosclerotic plaque burden in mice with a macrophage-specific ablation of ATG5. Together, these data suggested that sequestration of cytotoxic ubiquitinated proteins by p62 protects against atherogenesis, a condition in which the clearance of protein aggregates is disrupted.

Effects of sodium nitrite supplementation on vascular function and related small metabolite signatures in middle-aged and older adults.

Insufficient nitric oxide (NO) bioavailability plays an important role in endothelial dysfunction and arterial stiffening with aging. Supplementation with sodium nitrite, a precursor of NO, ameliorates age-related vascular endothelial dysfunction and arterial stiffness in mice, but effects on humans, including the metabolic pathways altered, are unknown. The purpose of this study was to determine the safety, feasibility, and efficacy of oral sodium nitrite supplementation for improving vascular function in middle-aged and older adults and to identify related circulating metabolites. Ten weeks of sodium nitrite (80 or 160 mg/day, capsules, TheraVasc; randomized, placebo control, double blind) increased plasma nitrite acutely (5- to 15-fold, P < 0.001 vs. placebo) and chronically (P < 0.10) and was well tolerated without symptomatic hypotension or clinically relevant elevations in blood methemoglobin. Endothelial function, measured by brachial artery flow-mediated dilation, increased 45-60% vs. baseline (P < 0.10) without changes in body mass or blood lipids. Measures of carotid artery elasticity (ultrasound and applanation tonometry) improved (decreased ß-stiffness index, increased cross-sectional compliance, P < 0.05) without changes in brachial or carotid artery blood pressure. Aortic pulse wave velocity was unchanged. Nitrite-induced changes in vascular measures were significantly related to 11 plasma metabolites identified by untargeted analysis. Baseline abundance of multiple metabolites, including glycerophospholipids and fatty acyls, predicted vascular changes with nitrite. This study provides evidence that sodium nitrite supplementation is well tolerated, increases plasma nitrite concentrations, improves endothelial function, and lessens carotid artery stiffening in middle-aged and older adults, perhaps by altering multiple metabolic pathways, thereby warranting a larger clinical trial.

Improved motor and cognitive performance with sodium nitrite supplementation is related to small metabolite signatures: a pilot trial in middle-aged and older adults.

Advancing age is associated with reductions in nitric oxide bioavailability and changes in metabolic activity, which are implicated in declines in motor and cognitive function. In preclinical models, sodium nitrite supplementation (SN) increases plasma nitrite and improves motor function, whereas other nitric oxide-boosting agents improve cognitive function. This pilot study was designed to translate these findings to middle-aged and older (MA/O) humans to provide proof-of-concept support for larger trials. SN (10 weeks, 80 to 160 mg/day capsules, TheraVasc, Inc.) acutely and chronically increased plasma nitrite and improved performance on measures of motor and cognitive outcomes (all p<0.05 or better) in healthy MA/O adults (62 ± 7 years). Untargeted metabolomics analysis revealed that SN significantly altered 33 (160 mg/day) to 45 (80 mg/day) different metabolites, 13 of which were related to changes in functional outcomes; baseline concentrations of 99 different metabolites predicted functional improvements with SN. This pilot study provides the first evidence that SN improves aspects of motor and cognitive function in healthy MA/O adults, and that these improvements are associated with, and predicted by, the plasma metabolome. Our findings provide the necessary support for larger clinical trials on this promising pharmacological strategy for preserving physiological function with aging.