Neuronal CRTC-1 governs systemic mitochondrial metabolism and lifespan via a catecholamine signal.

Low energy states delay aging in multiple species, yet mechanisms coordinating energetics and longevity across tissues remain poorly defined. The conserved energy sensor AMP-activated protein kinase (AMPK) and its corresponding phosphatase calcineurin modulate longevity via the CREB regulated transcriptional coactivator (CRTC)-1 in C. elegans. We show that CRTC-1 specifically uncouples AMPK/calcineurin-mediated effects on lifespan from pleiotropic side effects by reprogramming mitochondrial and metabolic function. This pro-longevity metabolic state is regulated cell nonautonomously by CRTC-1 in the nervous system. Neuronal CRTC-1/CREB regulates peripheral metabolism antagonistically with the functional PPARα ortholog, NHR-49, drives mitochondrial fragmentation in distal tissues, and suppresses the effects of AMPK on systemic mitochondrial metabolism and longevity via a cell-nonautonomous catecholamine signal. These results demonstrate that while both local and distal mechanisms combine to modulate aging, distal regulation overrides local contribution. Targeting central perception of energetic state is therefore a potential strategy to promote healthy aging.

SnapShot: Mammalian Sirtuins.

The mammalian sirtuins have emerged as critical regulators of cellular stress resistance, energy metabolism, and tumorigenesis. In some contexts, they delay the onset of age-related diseases and promote a healthy lifespan. The seven mammalian sirtuins, SIRT1-7, share a highly conserved NAD+-binding catalytic core domain although they exhibit distinct expression patterns, catalytic activities, and biological functions. This SnapShot provides an overview of these properties, with an emphasis on their relevance to aging.

Discovering the landscape of protein modifications.

Protein modifications modulate nearly every aspect of cell biology in organisms, ranging from Archaea to Eukaryotes. The earliest evidence of covalent protein modifications was found in the early 20th century by studying the amino acid composition of proteins by chemical hydrolysis. These discoveries challenged what defined a canonical amino acid. The advent and rapid adoption of mass-spectrometry-based proteomics in the latter part of the 20th century enabled a veritable explosion in the number of known protein modifications, with more than 500 discrete modifications counted today. Now, new computational tools in data science, machine learning, and artificial intelligence are poised to allow researchers to make significant progress in discovering new protein modifications and determining their function. In this review, we take an opportunity to revisit the historical discovery of key post-translational modifications, quantify the current landscape of covalent protein adducts, and assess the role that new computational tools will play in the future of this field.

NAD metabolism modulates inflammation and mitochondria function in diabetic kidney disease.

Diabetes mellitus is the leading cause of cardiovascular and renal disease in the United -States. Despite the beneficial interventions available for patients with diabetes, there remains a need for additional therapeutic targets and therapies in diabetic kidney disease (DKD). Inflammation and oxidative stress are increasingly recognized as important causes of renal diseases. Inflammation is closely associated with mitochondrial damage. The molecular connection between inflammation and mitochondrial metabolism remains to be elucidated. Recently, nicotinamide adenine nucleotide (NAD+) metabolism has been found to regulate immune function and inflammation. In the present studies, we tested the hypothesis that enhancing NAD metabolism could prevent inflammation in and progression of DKD. We found that treatment of db/db mice with type 2 diabetes with nicotinamide riboside (NR) prevented several manifestations of kidney dysfunction (i.e., albuminuria, increased urinary kidney injury marker-1 (KIM1) excretion, and pathologic changes). These effects were associated with decreased inflammation, at least in part via inhibiting the activation of the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) signaling pathway. An antagonist of the serum stimulator of interferon genes (STING) and whole-body STING deletion in diabetic mice showed similar renoprotection. Further analysis found that NR increased SIRT3 activity and improved mitochondrial function, which led to decreased mitochondrial DNA damage, a trigger for mitochondrial DNA leakage which activates the cGAS-STING pathway. Overall, these data show that NR supplementation boosted NAD metabolism to augment mitochondrial function, reducing inflammation and thereby preventing the progression of diabetic kidney disease.

Deglutarylation of glutaryl-CoA dehydrogenase by deacylating enzyme SIRT5 promotes lysine oxidation in mice.

A wide range of protein acyl modifications has been identified on enzymes across various metabolic processes; however, the impact of these modifications remains poorly understood. Protein glutarylation is a recently identified modification that can be nonenzymatically driven by glutaryl-CoA. In mammalian systems, this unique metabolite is only produced in the lysine and tryptophan oxidative pathways. To better understand the biology of protein glutarylation, we studied the relationship between enzymes within the lysine/tryptophan catabolic pathways, protein glutarylation, and regulation by the deglutarylating enzyme sirtuin 5 (SIRT5). Here, we identify glutarylation on the lysine oxidation pathway enzyme glutaryl-CoA dehydrogenase (GCDH) and show increased GCDH glutarylation when glutaryl-CoA production is stimulated by lysine catabolism. Our data reveal that glutarylation of GCDH impacts its function, ultimately decreasing lysine oxidation. We also demonstrate the ability of SIRT5 to deglutarylate GCDH, restoring its enzymatic activity. Finally, metabolomic and bioinformatic analyses indicate an expanded role for SIRT5 in regulating amino acid metabolism. Together, these data support a feedback loop model within the lysine/tryptophan oxidation pathway in which glutaryl-CoA is produced, in turn inhibiting GCDH function via glutaryl modification of GCDH lysine residues and can be relieved by SIRT5 deacylation activity.

Reactive Acyl-CoA Species Modify Proteins and Induce Carbon Stress.

In recent years, our understanding of the scope and diversity of protein post-translational modifications (PTMs) has rapidly expanded. In particular, mitochondrial proteins are decorated with an array of acyl groups that can occur non-enzymatically. Interestingly, these modifying chemical moieties are often associated with intermediary metabolites from core metabolic pathways. In this Review, we describe biochemical reactions and biological mechanisms that activate carbon metabolites for protein PTM. We explore the emerging links between the intrinsic reactivity of metabolites, non-enzymatic protein acylation, and possible signaling roles for this system. Finally, we propose a model of 'carbon stress', similar to oxidative stress, as an effective way to conceptualize the relationship between widespread protein acylation, nutrient sensing, and metabolic homeostasis.

A cell-nonautonomous mechanism of yeast chronological aging regulated by caloric restriction and one-carbon metabolism.

Caloric restriction (CR) improves health span and life span of organisms ranging from yeast to mammals. Understanding the mechanisms involved will uncover future interventions for aging-associated diseases. In budding yeast, Saccharomyces cerevisiae, CR is commonly defined by reduced glucose in the growth medium, which extends both replicative and chronological life span (CLS). We found that conditioned media collected from stationary-phase CR cultures extended CLS when supplemented into nonrestricted (NR) cultures, suggesting a potential cell-nonautonomous mechanism of CR-induced life span regulation. Chromatography and untargeted metabolomics of the conditioned media, as well as transcriptional responses associated with the longevity effect, pointed to specific amino acids enriched in the CR conditioned media (CRCM) as functional molecules, with L-serine being a particularly strong candidate. Indeed, supplementing L-serine into NR cultures extended CLS through a mechanism dependent on the one-carbon metabolism pathway, thus implicating this conserved and central metabolic hub in life span regulation.

Nonenzymatic protein acylation as a carbon stress regulated by sirtuin deacylases.

Cellular proteins are decorated with a wide range of acetyl and other acyl modifications. Many studies have demonstrated regulation of site-specific acetylation by acetyltransferases and deacetylases. Acylation is emerging as a new type of lysine modification, but less is known about its overall regulatory role. Furthermore, the mechanisms of lysine acylation, its overlap with protein acetylation, and how it influences cellular function are major unanswered questions in the field. In this review, we discuss the known roles of acetyltransferases and deacetylases and the sirtuins as a conserved family of a nicotinamide adenine dinucleotide (NAD⁺)-dependent protein deacylases that are important for response to cellular stress and homeostasis. We also consider the evidence for an emerging idea of nonenzymatic protein acylation. Finally, we put forward the hypothesis that protein acylation is a form of protein "carbon stress" that the deacylases evolved to remove as a part of a global protein quality-control network.

ß-Cell-specific ablation of sirtuin 4 does not affect nutrient-stimulated insulin secretion in mice.

Sirtuins are a family of proteins that regulate biological processes such as cellular stress and aging by removing posttranslational modifications (PTMs). We recently identified several novel PTMs that can be removed by sirtuin 4 (SIRT4), which is found in mitochondria. We showed that mice with a global loss of SIRT4 [SIRT4-knockout (KO) mice] developed an increase in glucose- and leucine-stimulated insulin secretion, and this was followed by accelerated age-induced glucose intolerance and insulin resistance. Because whole body SIRT4-KO mice had alterations to nutrient-stimulated insulin secretion, we hypothesized that SIRT4 plays a direct role in regulating pancreatic ß-cell function. Thus, we tested whether ß-cell-specific ablation of SIRT4 would recapitulate the elevated insulin secretion seen in mice with a global loss of SIRT4. Tamoxifen-inducible ß-cell-specific SIRT4-KO mice were generated, and their glucose tolerance and glucose- and leucine-stimulated insulin secretion were measured over time. These mice exhibited normal glucose- and leucine-stimulated insulin secretion and maintained normal glucose tolerance even as they aged. Furthermore, 832/13 ß-cells with a CRISPR/Cas9n-mediated loss of SIRT4 did not show any alterations in nutrient-stimulated insulin secretion. Despite the fact that whole body SIRT4-KO mice demonstrated an age-induced increase in glucose- and leucine-stimulated insulin secretion, our current data indicate that the loss of SIRT4 specifically in pancreatic ß-cells, both in vivo and in vitro, does not have a significant impact on nutrient-stimulated insulin secretion. These data suggest that SIRT4 controls nutrient-stimulated insulin secretion during aging by acting on tissues external to the ß-cell, which warrants further study.

Investigating RNA expression profiles altered by nicotinamide mononucleotide therapy in a chronic model of alcoholic liver disease.

BACKGROUND: Chronic alcohol consumption is a significant cause of liver disease worldwide. Several biochemical mechanisms have been linked to the initiation and progression of alcoholic liver disease (ALD) such as oxidative stress, inflammation, and metabolic dysregulation, including the disruption of NAD+/NADH. Indeed, an ethanol-mediated reduction in hepatic NAD+ levels is thought to be one factor underlying ethanol-induced steatosis, oxidative stress, steatohepatitis, insulin resistance, and inhibition of gluconeogenesis. Therefore, we applied a NAD+ boosting supplement to investigate alterations in the pathogenesis of early-stage ALD. METHODS: To examine the impact of NAD+ therapy on the early stages of ALD, we utilized nicotinamide mononucleotide (NMN) at 500 mg/kg intraperitoneal injection every other day, for the duration of a Lieber-DeCarli 6-week chronic ethanol model in mice. Numerous strategies were employed to characterize the effect of NMN therapy, including the integration of RNA-seq, immunoblotting, and metabolomics analysis. RESULTS: Our findings reveal that NMN therapy increased hepatic NAD+ levels, prevented an ethanol-induced increase in plasma ALT and AST, and changed the expression of 25% of the genes that were modulated by ethanol metabolism. These genes were associated with a number of pathways including the MAPK pathway. Interestingly, our analysis revealed that NMN treatment normalized Erk1/2 signaling and prevented an induction of Atf3 overexpression. CONCLUSIONS: These findings reveal previously unreported mechanisms by which NMN supplementation alters hepatic gene expression and protein pathways to impact ethanol hepatotoxicity in an early-stage murine model of ALD. Overall, our data suggest further research is needed to fully characterize treatment paradigms and biochemical implications of NAD+-based interventions.

Quantifying Competition among Mitochondrial Protein Acylation Events Induced by Ethanol Metabolism.

Mitochondrial dysfunction is one of many key factors in the etiology of alcoholic liver disease (ALD). Lysine acetylation is known to regulate numerous mitochondrial metabolic pathways, and recent reports demonstrate that alcohol-induced protein acylation negatively impacts these processes. To identify regulatory mechanisms attributed to alcohol-induced protein post-translational modifications, we employed a model of alcohol consumption within the context of wild type (WT), sirtuin 3 knockout (SIRT3 KO), and sirtuin 5 knockout (SIRT5 KO) mice to manipulate hepatic mitochondrial protein acylation. Mitochondrial fractions were examined by label-free quantitative HPLC-MS/MS to reveal competition between lysine acetylation and succinylation. A class of proteins defined as "differential acyl switching proteins" demonstrate select sensitivity to alcohol-induced protein acylation. A number of these proteins reveal saturated lysine-site occupancy, suggesting a significant level of differential stoichiometry in the setting of ethanol consumption. We hypothesize that ethanol downregulates numerous mitochondrial metabolic pathways through differential acyl switching proteins. Data are available via ProteomeXchange with identifier PXD012089.

Ablation of Sirtuin5 in the postnatal mouse heart results in protein succinylation and normal survival in response to chronic pressure overload.

Mitochondrial Sirtuin 5 (SIRT5) is an NAD+-dependent demalonylase, desuccinylase, and deglutarylase that controls several metabolic pathways. A number of recent studies point to SIRT5 desuccinylase activity being important in maintaining cardiac function and metabolism under stress. Previously, we described a phenotype of increased mortality in whole-body SIRT5KO mice exposed to chronic pressure overload compared with their littermate WT controls. To determine whether the survival phenotype we reported was due to a cardiac-intrinsic or cardiac-extrinsic effect of SIRT5, we developed a tamoxifen-inducible, heart-specific SIRT5 knockout (SIRT5KO) mouse model. Using our new animal model, we discovered that postnatal cardiac ablation of Sirt5 resulted in persistent accumulation of protein succinylation up to 30 weeks after SIRT5 depletion. Succinyl proteomics revealed that succinylation increased on proteins of oxidative metabolism between 15 and 31 weeks after ablation. Heart-specific SIRT5KO mice were exposed to chronic pressure overload to induce cardiac hypertrophy. We found that, in contrast to whole-body SIRT5KO mice, there was no difference in survival between heart-specific SIRT5KO mice and their littermate controls. Overall, the data presented here suggest that survival of SIRT5KO mice may be dictated by a multitissue or prenatal effect of SIRT5.

Fructose and glucose can regulate mammalian target of rapamycin complex 1 and lipogenic gene expression via distinct pathways.

Although calorically equivalent to glucose, fructose appears to be more lipogenic, promoting dyslipidemia, fatty liver disease, cardiovascular disease, and diabetes. To better understand how fructose induces lipogenesis, we compared the effects of fructose and glucose on mammalian target of rapamycin complex 1 (mTORC1), which appeared to have both positive and negative effects on lipogenic gene expression. We found that fructose acutely and transiently suppressed mTORC1 signaling in vitro and in vivo The constitutive activation of mTORC1 reduced hepatic lipogenic gene expression and produced hypotriglyceridemia after 1 week of fructose feeding. In contrast, glucose did not suppress mTORC1, and the constitutive activation of mTORC1 failed to suppress plasma triglycerides after 1 week of glucose feeding. Thus, these data reveal fundamental differences in the signaling pathways used by fructose and glucose to regulate lipid metabolism.

SIRT3 deficiency and mitochondrial protein hyperacetylation accelerate the development of the metabolic syndrome.

Acetylation is increasingly recognized as an important metabolic regulatory posttranslational protein modification, yet the metabolic consequence of mitochondrial protein hyperacetylation is unknown. We find that high-fat diet (HFD) feeding induces hepatic mitochondrial protein hyperacetylation in mice and downregulation of the major mitochondrial protein deacetylase SIRT3. Mice lacking SIRT3 (SIRT3KO) placed on a HFD show accelerated obesity, insulin resistance, hyperlipidemia, and steatohepatitis compared to wild-type (WT) mice. The lipogenic enzyme stearoyl-CoA desaturase 1 is highly induced in SIRT3KO mice, and its deletion rescues both WT and SIRT3KO mice from HFD-induced hepatic steatosis and insulin resistance. We further identify a single nucleotide polymorphism in the human SIRT3 gene that is suggestive of a genetic association with the metabolic syndrome. This polymorphism encodes a point mutation in the SIRT3 protein, which reduces its overall enzymatic efficiency. Our findings show that loss of SIRT3 and dysregulation of mitochondrial protein acetylation contribute to the metabolic syndrome.

Sirtuin 5 is required for mouse survival in response to cardiac pressure overload.

In mitochondria, the sirtuin SIRT5 is an NAD+-dependent protein deacylase that controls several metabolic pathways. Although a wide range of SIRT5 targets have been identified, the overall function of SIRT5 in organismal metabolic homeostasis remains unclear. Given that SIRT5 expression is highest in the heart and that sirtuins are commonly stress-response proteins, we used an established model of pressure overload-induced heart muscle hypertrophy caused by transverse aortic constriction (TAC) to determine SIRT5's role in cardiac stress responses. Remarkably, SIRT5KO mice had reduced survival upon TAC compared with wild-type mice but exhibited no mortality when undergoing a sham control operation. The increased mortality with TAC was associated with increased pathological hypertrophy and with key abnormalities in both cardiac performance and ventricular compliance. By combining high-resolution MS-based metabolomic and proteomic analyses of cardiac tissues from wild-type and SIRT5KO mice, we found several biochemical abnormalities exacerbated in the SIRT5KO mice, including apparent decreases in fatty acid oxidation and glucose oxidation as well as an overall decrease in mitochondrial NAD+/NADH. Together, these abnormalities suggest that SIRT5 deacylates protein substrates involved in cellular oxidative metabolism to maintain mitochondrial energy production. Overall, the functional and metabolic results presented here suggest an accelerated development of cardiac dysfunction in SIRT5KO mice in response to TAC, explaining increased mortality upon cardiac stress. Our findings reveal a key role for SIRT5 in maintaining cardiac oxidative metabolism under pressure overload to ensure survival.

Loss of sirtuin 4 leads to elevated glucose- and leucine-stimulated insulin levels and accelerated age-induced insulin resistance in multiple murine genetic backgrounds.

Several inherited metabolic disorders are associated with an accumulation of reactive acyl-CoA metabolites that can non-enzymatically react with lysine residues to modify proteins. While the role of acetylation is well-studied, the pathophysiological relevance of more recently discovered acyl modifications, including those found in inherited metabolic disorders, warrants further investigation. We recently showed that sirtuin 4 (SIRT4) removes glutaryl, 3-hydroxy-3-methylglutaryl, 3-methylglutaryl, and 3-methylglutaconyl modifications from lysine residues. Thus, we used SIRT4 knockout mice, which can accumulate these novel post-translational modifications, as a model to investigate their physiological relevance. Since SIRT4 is localized to mitochondria and previous reports have shown SIRT4 influences metabolism, we thoroughly characterized glucose and lipid metabolism in male and female SIRT4KO mice across different genetic backgrounds. While only minor perturbations in overall lipid metabolism were observed, we found SIRT4KO mice consistently had elevated glucose- and leucine-stimulated insulin levels in vivo and developed accelerated age-induced insulin resistance. Importantly, elevated leucine-stimulated insulin levels in SIRT4KO mice were dependent upon genetic background since SIRT4KO mice on a C57BL/6NJ genetic background had elevated leucine-stimulated insulin levels but not SIRT4KO mice on the C57BL/6J background. Taken together, the data suggest that accumulation of acyl modifications on proteins in inherited metabolic disorders may contribute to the overall metabolic dysfunction seen in these patients.

Metabolic control by sirtuins and other enzymes that sense NAD+, NADH, or their ratio.

NAD+ is a dinucleotide cofactor with the potential to accept electrons in a variety of cellular reduction-oxidation (redox) reactions. In its reduced form, NADH is a ubiquitous cellular electron donor. NAD+, NADH, and the NAD+/NADH ratio have long been known to control the activity of several oxidoreductase enzymes. More recently, enzymes outside those participating directly in redox control have been identified that sense these dinucleotides, including the sirtuin family of NAD+-dependent protein deacylases. In this review, we highlight examples of non-redox enzymes that are controlled by NAD+, NADH, or NAD+/NADH. In particular, we focus on the sirtuin family and assess the current evidence that the sirtuin enzymes sense these dinucleotides and discuss the biological conditions under which this might occur; we conclude that sirtuins sense NAD+, but neither NADH nor the ratio. Finally, we identify future studies that might be informative to further interrogate physiological and pathophysiological changes in NAD+ and NADH, as well as enzymes like sirtuins that sense and respond to redox changes in the cell.

Investigating the Sensitivity of NAD+-dependent Sirtuin Deacylation Activities to NADH.

Protein lysine posttranslational modification by an increasing number of different acyl groups is becoming appreciated as a regulatory mechanism in cellular biology. Sirtuins are class III histone deacylases that use NAD(+)as a co-substrate during amide bond hydrolysis. Several studies have described the sirtuins as sensors of the NAD(+)/NADH ratio, but it has not been formally tested for all the mammalian sirtuinsin vitro To address this problem, we first synthesized a wide variety of peptide-based probes, which were used to identify the range of hydrolytic activities of human sirtuins. These probes included aliphatic ϵ-N-acyllysine modifications with hydrocarbon lengths ranging from formyl (C1) to palmitoyl (C16) as well as negatively charged dicarboxyl-derived modifications. In addition to the well established activities of the sirtuins, "long chain" acyllysine modifications were also shown to be prone to hydrolytic cleavage by SIRT1-3 and SIRT6, supporting recent findings. We then tested the ability of NADH, ADP-ribose, and nicotinamide to inhibit these NAD(+)-dependent deacylase activities of the sirtuins. In the commonly used 7-amino-4-methylcoumarin-coupled fluorescence-based assay, the fluorophore has significant spectral overlap with NADH and therefore cannot be used to measure inhibition by NADH. Therefore, we turned to an HPLC-MS-based assay to directly monitor the conversion of acylated peptides to their deacylated forms. All tested sirtuin deacylase activities showed sensitivity to NADH in this assay. However, the inhibitory concentrations of NADH in these assays are far greater than the predicted concentrations of NADH in cells; therefore, our data indicate that NADH is unlikely to inhibit sirtuinsin vivo These data suggest a re-evaluation of the sirtuins as direct sensors of the NAD(+)/NADH ratio.

Chronic Ethanol Metabolism Inhibits Hepatic Mitochondrial Superoxide Dismutase via Lysine Acetylation.

BACKGROUND: Chronic ethanol (EtOH) consumption is a major cause of liver disease worldwide. Oxidative stress is a known consequence of EtOH metabolism and is thought to contribute significantly to alcoholic liver disease (ALD). Therefore, elucidating pathways leading to sustained oxidative stress and downstream redox imbalances may reveal how EtOH consumption leads to ALD. Recent studies suggest that EtOH metabolism impacts mitochondrial antioxidant processes through a number of proteomic alterations, including hyperacetylation of key antioxidant proteins. METHODS: To elucidate mechanisms of EtOH-induced hepatic oxidative stress, we investigate a role for protein hyperacetylation in modulating mitochondrial superoxide dismutase (SOD2) structure and function in a 6-week Lieber-DeCarli murine model of EtOH consumption. Our experimental approach includes immunoblotting immunohistochemistry (IHC), activity assays, mass spectrometry, and in silico modeling. RESULTS: We found that EtOH metabolism significantly increased the acetylation of SOD2 at 2 functionally relevant lysine sites, K68 and K122, resulting in a 40% decrease in enzyme activity while overall SOD2 abundance was unchanged. In vitro studies also reveal which lysine residues are more susceptible to acetylation. IHC analysis demonstrates that SOD2 hyperacetylation occurs near zone 3 within the liver, which is the main EtOH-metabolizing region of the liver. CONCLUSIONS: Overall, the findings presented in this study support a role for EtOH-induced lysine acetylation as an adverse posttranslational modification within the mitochondria that directly impacts SOD2 charge state and activity. Last, the data presented here indicate that protein hyperacetylation may be a major factor contributing to an imbalance in hepatic redox homeostasis due to chronic EtOH metabolism.

Metabolic Regulation by Lysine Malonylation, Succinylation, and Glutarylation.

Protein acetylation is a well-studied regulatory mechanism for several cellular processes, ranging from gene expression to metabolism. Recent discoveries of new post-translational modifications, including malonylation, succinylation, and glutarylation, have expanded our understanding of the types of modifications found on proteins. These three acidic lysine modifications are structurally similar but have the potential to regulate different proteins in different pathways. The deacylase sirtuin 5 (SIRT5) catalyzes the removal of these modifications from a wide range of proteins in different subcellular compartments. Here, we review these new modifications, their regulation by SIRT5, and their emerging role in cellular regulation and diseases.