Visualizing subcellular changes in the NAD(H) pool size versus redox state using fluorescence lifetime imaging microscopy of NADH.

NADH autofluorescence imaging is a promising approach for visualizing energy metabolism at the single-cell level. However, it is sensitive to the redox ratio and the total NAD(H) amount, which can change independently from each other, for example with aging. Here, we evaluate the potential of fluorescence lifetime imaging microscopy (FLIM) of NADH to differentiate between these modalities.We perform targeted modifications of the NAD(H) pool size and ratio in cells and mice and assess the impact on NADH FLIM. We show that NADH FLIM is sensitive to NAD(H) pool size, mimicking the effect of redox alterations. However, individual components of the fluorescence lifetime are differently impacted by redox versus pool size changes, allowing us to distinguish both modalities using only FLIM. Our results emphasize NADH FLIM's potential for evaluating cellular metabolism and relative NAD(H) levels with high spatial resolution, providing a crucial tool for our understanding of aging and metabolism.

Single-cell NAD(H) levels predict clonal lymphocyte expansion dynamics.

Adaptive immunity requires the expansion of high-affinity lymphocytes from a heterogeneous pool. Whereas current models explain this through signal transduction, we hypothesized that antigen affinity tunes discrete metabolic pathways to license clonal lymphocyte dynamics. Here, we identify nicotinamide adenine dinucleotide (NAD) biosynthesis as a biochemical hub for the T cell receptor affinity-dependent metabolome. Through this central anabolic role, we found that NAD biosynthesis governs a quiescence exit checkpoint, thereby pacing proliferation. Normalizing cellular NAD(H) likewise normalizes proliferation across affinities, and enhancing NAD biosynthesis permits the expansion of lower affinity clones. Furthermore, single-cell differences in NAD(H) could predict division potential for both T and B cells, before the first division, unmixing proliferative heterogeneity. We believe that this supports a broader paradigm in which complex signaling networks converge on metabolic pathways to control single-cell behavior.

Brain energy metabolism: A roadmap for future research.

Although we have learned much about how the brain fuels its functions over the last decades, there remains much still to discover in an organ that is so complex. This article lays out major gaps in our knowledge of interrelationships between brain metabolism and brain function, including biochemical, cellular, and subcellular aspects of functional metabolism and its imaging in adult brain, as well as during development, aging, and disease. The focus is on unknowns in metabolism of major brain substrates and associated transporters, the roles of insulin and of lipid droplets, the emerging role of metabolism in microglia, mysteries about the major brain cofactor and signaling molecule NAD+ , as well as unsolved problems underlying brain metabolism in pathologies such as traumatic brain injury, epilepsy, and metabolic downregulation during hibernation. It describes our current level of understanding of these facets of brain energy metabolism as well as a roadmap for future research.

Antibody blockade of activin type II receptors preserves skeletal muscle mass and enhances fat loss during GLP-1 receptor agonism.

OBJECTIVE: Glucagon-like peptide 1 (GLP-1) receptor agonists reduce food intake, producing remarkable weight loss in overweight and obese individuals. While much of this weight loss is fat mass, there is also a loss of lean mass, similar to other approaches that induce calorie deficit. Targeting signaling pathways that regulate skeletal muscle hypertrophy is a promising avenue to preserve lean mass and modulate body composition. Myostatin and Activin A are TGFß-like ligands that signal via the activin type II receptors (ActRII) to antagonize muscle growth. Pre-clinical and clinical studies demonstrate that ActRII blockade induces skeletal muscle hypertrophy and reduces fat mass. In this manuscript, we test the hypothesis that combined ActRII blockade and GLP-1 receptor agonism will preserve muscle mass, leading to improvements in skeletomuscular and metabolic function and enhanced fat loss. METHODS: In this study, we explore the therapeutic potential of bimagrumab, a monoclonal antibody against ActRII, to modify body composition alone and during weight loss induced by GLP-1 receptor agonist semaglutide in diet-induced obese mice. Mechanistically, we define the specific role of the anabolic kinase Akt in mediating the hypertrophic muscle effects of ActRII inhibition in vivo. RESULTS: Treatment of obese mice with bimagrumab induced a ∼10 % increase in lean mass while simultaneously decreasing fat mass. Daily treatment of obese mice with semaglutide potently decreased body weight; this included a significant decrease in both muscle and fat mass. Combination treatment with bimagrumab and semaglutide led to superior fat mass loss while simultaneously preserving lean mass despite reduced food intake. Treatment with both drugs was associated with improved metabolic outcomes, and increased lean mass was associated with improved exercise performance. Deletion of both Akt isoforms in skeletal muscle modestly reduced, but did not prevent, muscle hypertrophy driven by ActRII inhibition. CONCLUSIONS: Collectively, these data demonstrate that blockade of ActRII signaling improves body composition and metabolic parameters during calorie deficit driven by GLP-1 receptor agonism and demonstrate the existence of Akt-independent pathways supporting muscle hypertrophy in the absence of ActRII signaling.

ACSS2 gene variants determine kidney disease risk by controlling de novo lipogenesis in kidney tubules.

Worldwide, over 800 million people are affected by kidney disease, yet its pathogenesis remains elusive, hindering the development of novel therapeutics. In this study, we used kidney-specific expression of quantitative traits and single-nucleus open chromatin analysis to show that genetic variants linked to kidney dysfunction on chromosome 20 target the acyl-CoA synthetase short-chain family 2 (ACSS2). By generating ACSS2-KO mice, we demonstrated their protection from kidney fibrosis in multiple disease models. Our analysis of primary tubular cells revealed that ACSS2 regulated de novo lipogenesis (DNL), causing NADPH depletion and increasing ROS levels, ultimately leading to NLRP3-dependent pyroptosis. Additionally, we discovered that pharmacological inhibition or genetic ablation of fatty acid synthase safeguarded kidney cells against profibrotic gene expression and prevented kidney disease in mice. Lipid accumulation and the expression of genes related to DNL were elevated in the kidneys of patients with fibrosis. Our findings pinpoint ACSS2 as a critical kidney disease gene and reveal the role of DNL in kidney disease.

Sirt3 Deletion Increases Inflammation and Mortality in Polymicrobial Sepsis.

Background: Sirtuin 3 (SIRT3) is a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase that confers resilience to cellular stress by promoting mitochondrial activity. Mitochondrial dysfunction is a major driver of inflammation during sepsis. We hypothesize that Sirt3 expression improves survival in polymicrobial sepsis by mitigating the inflammatory response. Materials and Methods: Sirt3 knockout (S3KO) and wild-type (WT) mice underwent cecal ligation and puncture (CLP) or sham surgery. mRNA expression was quantified using quantitative polymerase chain reaction (qPCR) and protein expression was quantified using enzyme-linked immunosorbent assay (ELISA). Spectrophotometric assays were used to quantify serum markers of organ dysfunction. For in vitro studies, bone marrow-derived macrophages (BMDMs) were harvested from S3KO and WT mice and treated with lipopolysaccharide (LPS). Results: After CLP, hepatic Sirt3 levels decreased from baseline by nine hours and remained depressed at 24 hours. Peak serum interleukin-6 (IL-6) protein levels were higher in S3KO mice. In LPS-treated BMDMs, IL-6 mRNA levels peaked earlier in S3KO cells, although peak levels were comparable to WT. Although S3KO mice had decreased median survival after CLP compared with WT, there was no difference in five-day survival or organ dysfunction. Conclusions: Although S3KO mice initially had increased inflammation and mortality, this difference abated with time, and overall survival was comparable between the groups. This pattern is consistent with the timeline of sepsis-induced Sirt3 downregulation in WT mice, and suggests that Sirt3 downregulation occurring in sepsis is at least partially responsible for the initial hyperinflammatory response and subsequent mortality. Our data support upregulation of Sirt3 as a promising therapeutic strategy for further research in sepsis.

A subpopulation of lipogenic brown adipocytes drives thermogenic memory.

Sustained responses to transient environmental stimuli are important for survival. The mechanisms underlying long-term adaptations to temporary shifts in abiotic factors remain incompletely understood. Here, we find that transient cold exposure leads to sustained transcriptional and metabolic adaptations in brown adipose tissue, which improve thermogenic responses to secondary cold encounter. Primary thermogenic challenge triggers the delayed induction of a lipid biosynthesis programme even after cessation of the original stimulus, which protects from subsequent exposures. Single-nucleus RNA sequencing and spatial transcriptomics reveal that this response is driven by a lipogenic subpopulation of brown adipocytes localized along the perimeter of Ucp1hi adipocytes. This lipogenic programme is associated with the production of acylcarnitines, and supplementation of acylcarnitines is sufficient to recapitulate improved secondary cold responses. Overall, our data highlight the importance of heterogenous brown adipocyte populations for 'thermogenic memory', which may have therapeutic implications for leveraging short-term thermogenesis to counteract obesity.

Sleep deprivation and aging are metabolically linked across tissues.

STUDY OBJECTIVES: Insufficient sleep is a concerning hallmark of modern society because sleep deprivation (SD) is a risk factor for neurodegenerative and cardiometabolic disorders. SD imparts an aging-like effect on learning and memory, although little is known about possible common molecular underpinnings of SD and aging. Here, we examine this question by profiling metabolic features across different tissues after acute SD in young adult and aged mice. METHODS: Young adult and aged mice were subjected to acute SD for 5 hours. Blood plasma, hippocampus, and liver samples were subjected to UPLC-MS/MS-based metabolic profiling. RESULTS: SD preferentially impacts peripheral plasma and liver profiles (e.g. ketone body metabolism) whereas the hippocampus is more impacted by aging. We further demonstrate that aged animals exhibit SD-like metabolic features at baseline. Hepatic alterations include parallel changes in nicotinamide metabolism between aging and SD in young animals. Overall, metabolism in young adult animals is more impacted by SD, which in turn induces aging-like features. A set of nine metabolites was classified (79% correct) based on age and sleep status across all four groups. CONCLUSIONS: Our metabolic observations demonstrate striking parallels to previous observations in studies of learning and memory and define a molecular metabolic signature of sleep loss and aging.

CAR T therapy beyond cancer: the evolution of a living drug.

Engineering a patient's own T cells to selectively target and eliminate tumour cells has cured patients with untreatable haematologic cancers. These results have energized the field to apply chimaeric antigen receptor (CAR) T therapy throughout oncology. However, evidence from clinical and preclinical studies underscores the potential of CAR T therapy beyond oncology in treating autoimmunity, chronic infections, cardiac fibrosis, senescence-associated disease and other conditions. Concurrently, the deployment of new technologies and platforms provides further opportunity for the application of CAR T therapy to noncancerous pathologies. Here we review the rationale behind CAR T therapy, current challenges faced in oncology, a synopsis of preliminary reports in noncancerous diseases, and a discussion of relevant emerging technologies. We examine potential applications for this therapy in a wide range of contexts. Last, we highlight concerns regarding specificity and safety and outline the path forward for CAR T therapy beyond cancer.

Drugs Targeting Mechanisms of Aging to Delay Age-Related Disease and Promote Healthspan: Proceedings of a National Institute on Aging Workshop.

The geroscience hypothesis posits that by targeting key hallmarks of aging we may simultaneously prevent or delay several age-related diseases and thereby increase healthspan, or life span spent free of significant disease and disability. Studies are underway to examine several possible pharmacological interventions for this purpose. As part of a National Institute on Aging workshop on the development of function-promoting therapies, scientific content experts provided literature reviews and state-of-the-field assessments for the studies of senolytics, nicotinamide adenine dinucleotide (NAD+) boosters, and metformin. Cellular senescence increases with age, and preclinical studies demonstrate that the use of senolytic drugs improves healthspan in rodents. Human studies using senolytics are in progress. NAD+ and its phosphorylated form, NADP+, play vital roles in metabolism and cellular signaling. Increasing NAD+ by supplementation with precursors including nicotinamide riboside and nicotinamide mononucleotide appears to extend healthspan in model organisms, but human studies are limited and results are mixed. Metformin is a biguanide widely used for glucose lowering, which is believed to have pleiotropic effects targeting several hallmarks of aging. Preclinical studies suggest it improves life span and healthspan, and observational studies suggest benefits for the prevention of several age-related diseases. Clinical trials are underway to examine metformin for healthspan and frailty prevention. Preclinical and emerging clinical studies suggest there is potential to improve healthspan through the use of pharmacologic agents reviewed. However, much further research is needed to demonstrate benefits and general safety for wider use, the appropriate target populations, and longer-term outcomes.

Nicotinamide Adenine Dinucleotide in Aging Biology: Potential Applications and Many Unknowns.

Recent research has unveiled an expansive role of NAD+ in cellular energy generation, redox reactions, and as a substrate or cosubstrate in signaling pathways that regulate health span and aging. This review provides a critical appraisal of the clinical pharmacology and the preclinical and clinical evidence for therapeutic effects of NAD+ precursors for age-related conditions, with a particular focus on cardiometabolic disorders, and discusses gaps in current knowledge. NAD+ levels decrease throughout life; age-related decline in NAD+ bioavailability has been postulated to be a contributor to many age-related diseases. Raising NAD+ levels in model organisms by administration of NAD+ precursors improves glucose and lipid metabolism; attenuates diet-induced weight gain, diabetes, diabetic kidney disease, and hepatic steatosis; reduces endothelial dysfunction; protects heart from ischemic injury; improves left ventricular function in models of heart failure; attenuates cerebrovascular and neurodegenerative disorders; and increases health span. Early human studies show that NAD+ levels can be raised safely in blood and some tissues by oral NAD+ precursors and suggest benefit in preventing nonmelanotic skin cancer, modestly reducing blood pressure and improving lipid profile in older adults with obesity or overweight; preventing kidney injury in at-risk patients; and suppressing inflammation in Parkinson disease and SARS-CoV-2 infection. Clinical pharmacology, metabolism, and therapeutic mechanisms of NAD+ precursors remain incompletely understood. We suggest that these early findings provide the rationale for adequately powered randomized trials to evaluate the efficacy of NAD+ augmentation as a therapeutic strategy to prevent and treat metabolic disorders and age-related conditions.

Taurine linked with healthy aging.

Reversing age-associated taurine loss improves mouse longevity and monkey health.

NAD+ precursor supplementation prevents mtRNA/RIG-I-dependent inflammation during kidney injury.

Our understanding of how global changes in cellular metabolism contribute to human kidney disease remains incompletely understood. Here we show that nicotinamide adenine dinucleotide (NAD+) deficiency drives mitochondrial dysfunction causing inflammation and kidney disease development. Using unbiased global metabolomics in healthy and diseased human kidneys, we identify NAD+ deficiency as a disease signature. Furthermore using models of cisplatin- or ischaemia-reperfusion induced kidney injury in male mice we observed NAD+ depletion Supplemental nicotinamide riboside or nicotinamide mononucleotide restores NAD+ levels and improved kidney function. We find that cisplatin exposure causes cytosolic leakage of mitochondrial RNA (mtRNA) and activation of the cytosolic pattern recognition receptor retinoic acid-inducible gene I (RIG-I), both of which can be ameliorated by restoring NAD+. Male mice with RIG-I knock-out (KO) are protected from cisplatin-induced kidney disease. In summary, we demonstrate that the cytosolic release of mtRNA and RIG-I activation is an NAD+-sensitive mechanism contributing to kidney disease.

Limiting Mrs2-dependent mitochondrial Mg2+ uptake induces metabolic programming in prolonged dietary stress.

The most abundant cellular divalent cations, Mg2+ (mM) and Ca2+ (nM-µM), antagonistically regulate divergent metabolic pathways with several orders of magnitude affinity preference, but the physiological significance of this competition remains elusive. In mice consuming a Western diet, genetic ablation of the mitochondrial Mg2+ channel Mrs2 prevents weight gain, enhances mitochondrial activity, decreases fat accumulation in the liver, and causes prominent browning of white adipose. Mrs2 deficiency restrains citrate efflux from the mitochondria, making it unavailable to support de novo lipogenesis. As citrate is an endogenous Mg2+ chelator, this may represent an adaptive response to a perceived deficit of the cation. Transcriptional profiling of liver and white adipose reveals higher expression of genes involved in glycolysis, ß-oxidation, thermogenesis, and HIF-1α-targets, in Mrs2-/- mice that are further enhanced under Western-diet-associated metabolic stress. Thus, lowering mMg2+ promotes metabolism and dampens diet-induced obesity and metabolic syndrome.

Regulation of metabolic health by dietary histidine in mice.

Low-protein (LP) diets are associated with a decreased risk of diabetes in humans, and promote leanness and glycaemic control in both rodents and humans. While the effects of an LP diet on glycaemic control are mediated by reduced levels of the branched-chain amino acids, we have observed that reducing dietary levels of the other six essential amino acids leads to changes in body composition. Here, we find that dietary histidine plays a key role in the response to an LP diet in male C57BL/6J mice. Specifically reducing dietary levels of histidine by 67% reduces the weight gain of young, lean male mice, reducing both adipose and lean mass without altering glucose metabolism, and rapidly reverses diet-induced obesity and hepatic steatosis in diet-induced obese male mice, increasing insulin sensitivity. This normalization of metabolic health was associated not with caloric restriction or increased activity, but with increased energy expenditure. Surprisingly, the effects of histidine restriction do not require the energy balance hormone Fgf21. Histidine restriction that was started in midlife promoted leanness and glucose tolerance in aged males but not females, but did not affect frailty or lifespan in either sex. Finally, we demonstrate that variation in dietary histidine levels helps to explain body mass index differences in humans. Overall, our findings demonstrate that dietary histidine is a key regulator of weight and body composition in male mice and in humans, and suggest that reducing dietary histidine may be a translatable option for the treatment of obesity. KEY POINTS: Protein restriction (PR) promotes metabolic health in rodents and humans and extends rodent lifespan. Restriction of specific individual essential amino acids can recapitulate the benefits of PR. Reduced histidine promotes leanness and increased energy expenditure in male mice. Reduced histidine does not extend the lifespan of mice when begun in midlife. Dietary levels of histidine are positively associated with body mass index in humans.

A microbiome-dependent gut-brain pathway regulates motivation for exercise.

Exercise exerts a wide range of beneficial effects for healthy physiology1. However, the mechanisms regulating an individual's motivation to engage in physical activity remain incompletely understood. An important factor stimulating the engagement in both competitive and recreational exercise is the motivating pleasure derived from prolonged physical activity, which is triggered by exercise-induced neurochemical changes in the brain. Here, we report on the discovery of a gut-brain connection in mice that enhances exercise performance by augmenting dopamine signalling during physical activity. We find that microbiome-dependent production of endocannabinoid metabolites in the gut stimulates the activity of TRPV1-expressing sensory neurons and thereby elevates dopamine levels in the ventral striatum during exercise. Stimulation of this pathway improves running performance, whereas microbiome depletion, peripheral endocannabinoid receptor inhibition, ablation of spinal afferent neurons or dopamine blockade abrogate exercise capacity. These findings indicate that the rewarding properties of exercise are influenced by gut-derived interoceptive circuits and provide a microbiome-dependent explanation for interindividual variability in exercise performance. Our study also suggests that interoceptomimetic molecules that stimulate the transmission of gut-derived signals to the brain may enhance the motivation for exercise.

NAD precursors cycle between host tissues and the gut microbiome.

Nicotinamide adenine dinucleotide (NAD) is an essential redox cofactor in mammals and microbes. Here we use isotope tracing to investigate the precursors supporting NAD synthesis in the gut microbiome of mice. We find that dietary NAD precursors are absorbed in the proximal part of the gastrointestinal tract and not available to microbes in the distal gut. Instead, circulating host nicotinamide enters the gut lumen and supports microbial NAD synthesis. The microbiome converts host-derived nicotinamide into nicotinic acid, which is used for NAD synthesis in host tissues and maintains circulating nicotinic acid levels even in the absence of dietary consumption. Moreover, the main route from oral nicotinamide riboside, a widely used nutraceutical, to host NAD is via conversion into nicotinic acid by the gut microbiome. Thus, we establish the capacity for circulating host micronutrients to feed the gut microbiome, and in turn be transformed in a manner that enhances host metabolic flexibility.

Nicotinamide riboside alleviates exercise intolerance in ANT1-deficient mice.

OBJECTIVE: Mitochondrial disorders are often characterized by muscle weakness and fatigue. Null mutations in the heart-muscle adenine nucleotide translocator isoform 1 (ANT1) of both humans and mice cause cardiomyopathy and myopathy associated with exercise intolerance and muscle weakness. Here we decipher the molecular underpinnings of ANT1-deficiency-mediated exercise intolerance. METHODS: This was achieved by correlating exercise physiology, mitochondrial function and metabolomics of mice deficient in ANT1 and comparing this to control mice. RESULTS: We demonstrate a peripheral limitation of skeletal muscle mitochondrial respiration and a reduced complex I respiration in ANT1-deficient mice. Upon exercise, this results in a lack of NAD+ leading to a substrate limitation and stalling of the TCA cycle and mitochondrial respiration, further limiting skeletal muscle mitochondrial respiration. Treatment of ANT1-deficient mice with nicotinamide riboside increased NAD+ levels in skeletal muscle and liver, which increased the exercise capacity and the mitochondrial respiration. CONCLUSION: Increasing NAD+ levels with nicotinamide riboside can alleviate the exercise intolerance associated to ANT1-deficiency, indicating the therapeutic potential of NAD+-stimulating compounds in mitochondrial myopathies.

Sirtuin 1 deletion increases inflammation and mortality in sepsis.

BACKGROUND: Sepsis is a hyperinflammatory response to infection that can lead to multiorgan failure and eventually death. Often, the onset of multiorgan failure is heralded by renal dysfunction. Sirtuin 1 (SIRT1) promotes cellular stress resilience by inhibiting inflammation and promoting mitochondrial function. We hypothesize that SIRT1 plays an important role in limiting the inflammatory responses that drive organ failure in sepsis, predominantly via expression in myeloid cells. METHODS: We performed cecal ligation and puncture (CLP) on whole body SIRT1 knockout (S1KO) and myeloid cell-specific S1KO (S1KO-LysMCre) mice on a C57BL/6J background. Serum interleukin (IL)-6 was quantified by enzyme-linked immunosorbent assay. Renal mitochondrial complex activity was measured using Oxygraph-2k (Oroboros Instruments, Innsbruck, Austria). Blood urea nitrogen (BUN) was measured from serum. Survival was monitored for up to 5 days. RESULTS: Following CLP, S1KO mice had decreased renal mitochondrial complex I-dependent respiratory capacity (241.7 vs. 418.3 mmolO2/mg/min, p = 0.018) and renal mitochondrial complex II-dependent respiratory capacity (932.3 vs. 1,178.4, p = 0.027), as well as reduced rates of fatty acid oxidation (187.3 vs. 250.3, p = 0.022). Sirtuin 1 knockout mice also had increased BUN (48.0 mg/dL vs. 16.0 mg/dL, p = 0.049). Interleukin-6 levels were elevated in S1KO mice (96.5 ng/mL vs. 45.6 ng/mL, p = 0.028) and S1KO-LysMCre mice (35.8 ng/mL vs. 24.5 ng/mL, p = 0.033) compared with controls 12 hours after surgery. Five-day survival in S1KO (33.3% vs. 83.3%, p = 0.025) and S1KO-LysMCre (60% vs. 100%, p = 0.049) mice was decreased compared with controls. CONCLUSION: Sirtuin 1 deletion increases systemic inflammation in sepsis. Renal mitochondrial dysfunction, kidney injury, and mortality following CLP were all exacerbated by SIRT1 deletion. Similar effects on inflammation and survival were seen following myeloid cell-specific SIRT1 deletion, indicating that SIRT1 activity in myeloid cells may be a significant contributor for the protective effects of SIRT1 in sepsis.