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Brown adipose tissue (BAT) engages futile fatty acid synthesis-oxidation cycling, the purpose of which has remained elusive. Here, we show that ATP-citrate lyase (ACLY), which generates acetyl-CoA for fatty acid synthesis, promotes thermogenesis by mitigating metabolic stress. Without ACLY, BAT overloads the tricarboxylic acid cycle, activates the integrated stress response (ISR) and suppresses thermogenesis. ACLY's role in preventing BAT stress becomes critical when mice are weaned onto a carbohydrate-plentiful diet, while removing dietary carbohydrates prevents stress induction in ACLY-deficient BAT. ACLY loss also upregulates fatty acid synthase (Fasn); yet while ISR activation is not caused by impaired fatty acid synthesis per se, deleting Fasn and Acly unlocks an alternative metabolic programme that overcomes tricarboxylic acid cycle overload, prevents ISR activation and rescues thermogenesis. Overall, we uncover a previously unappreciated role for ACLY in mitigating mitochondrial stress that links dietary carbohydrates to uncoupling protein 1-dependent thermogenesis and provides fundamental insight into the fatty acid synthesis-oxidation paradox in BAT.

While the placenta regulates nutritional exchange between mother and fetus, Yu et al. reveal that human placental development is itself nutrient-sensitive. They elucidate entwined metabolic and epigenetic transitions driving syncytialization and pinpoint a requirement for the metabolite acetyl-CoA, which is sensitive to glucose metabolism.

Propiogenic substrates and gut bacteria produce propionate, a post-translational protein modifier. In this study, we used a mouse model of propionic acidaemia (PA) to study how disturbances to propionate metabolism result in histone modifications and changes to gene expression that affect cardiac function. Plasma propionate surrogates were raised in PA mice, but female hearts manifested more profound changes in acyl-CoAs, histone propionylation and acetylation, and transcription. These resulted in moderate diastolic dysfunction with raised diastolic Ca, expanded end-systolic ventricular volume and reduced stroke volume. Propionate was traced to histone H3 propionylation and caused increased acetylation genome-wide, including at promoters of and , genes related to contractile dysfunction through downscaled cGMP signaling. The less severe phenotype in male hearts correlated with β-alanine buildup. Raising β-alanine in cultured myocytes treated with propionate reduced propionyl-CoA levels, indicating a mechanistic relationship. Thus, we linked perturbed propionate metabolism to epigenetic changes that impact cardiac function.

Endothelial-to-mesenchymal transition (EndMT), a process initiated by activation of endothelial TGF-β signaling, underlies numerous chronic vascular diseases and fibrotic states. Once induced, EndMT leads to a further increase in TGF-β signaling, thus establishing a positive-feedback loop with EndMT leading to more EndMT. Although EndMT is understood at the cellular level, the molecular basis of TGF-β-driven EndMT induction and persistence remains largely unknown. Here, we show that metabolic modulation of the endothelium, triggered by atypical production of acetate from glucose, underlies TGF-β-driven EndMT. Induction of EndMT suppresses the expression of the enzyme PDK4, which leads to an increase in ACSS2-dependent Ac-CoA synthesis from pyruvate-derived acetate. This increased Ac-CoA production results in acetylation of the TGF-β receptor ALK5 and SMADs 2 and 4 leading to activation and long-term stabilization of TGF-β signaling. Our results establish the metabolic basis of EndMT persistence and unveil novel targets, such as ACSS2, for the potential treatment of chronic vascular diseases.

The ability of cells to store and rapidly mobilize energy reserves in response to nutrient availability is essential for survival. Breakdown of carbon stores produces acetyl-coenzyme-A (AcCoA), which fuels essential metabolic pathways and is also the acyl donor for protein lysine acetylation. Histones are abundant and highly acetylated proteins, accounting for 40% - 75% of cellular protein acetylation. Notably, histone acetylation is sensitive to AcCoA availability and nutrient replete conditions induce a substantial accumulation of acetylation on histones. Deacetylation releases acetate, which can be recycled to AcCoA, suggesting that deacetylation could be mobilized as an AcCoA source to feed downstream metabolic processes under nutrient depletion. While the notion of histones as a metabolic reservoir has been frequently proposed, experimental evidence has been lacking. Therefore, to test this concept directly, we used acetate-dependent ATP citrate lyase-deficient fibroblasts (Acly MEFs) and designed a pulse-chase experimental system to trace deacetylation-derived acetate and its incorporation into AcCoA. We found that dynamic protein deacetylation in Acly MEFs contributed carbons to AcCoA and proximal downstream metabolites. However, deacetylation had no significant effect on acyl-CoA pool sizes, and even at maximal acetylation, deacetylation transiently supplied less than 10% of cellular AcCoA. Together, our data reveal that although histone acetylation is dynamic and nutrient-sensitive, its potential for maintaining cellular AcCoA-dependent metabolic pathways is limited compared to cellular demand.

The metabolite acetyl-CoA is necessary for both lipid synthesis in the cytosol and histone acetylation in the nucleus. The two canonical precursors to acetyl-CoA in the nuclear-cytoplasmic compartment are citrate and acetate, which are processed to acetyl-CoA by ATP-citrate lyase (ACLY) and acyl-CoA synthetase short-chain 2 (ACSS2), respectively. It is unclear whether other substantial routes to nuclear-cytosolic acetyl-CoA exist. To investigate this, we generated cancer cell lines lacking both ACLY and ACSS2 [double knockout (DKO) cells]. Using stable isotope tracing, we show that both glucose and fatty acids contribute to acetyl-CoA pools and histone acetylation in DKO cells and that acetylcarnitine shuttling can transfer two-carbon units from mitochondria to cytosol. Further, in the absence of ACLY, glucose can feed fatty acid synthesis in a carnitine responsive and carnitine acetyltransferase (CrAT)-dependent manner. The data define acetylcarnitine as an ACLY- and ACSS2-independent precursor to nuclear-cytosolic acetyl-CoA that can support acetylation, fatty acid synthesis, and cell growth.

Anabolic metabolism of carbon in mammals is mediated via the one- and two-carbon carriers S-adenosyl methionine and acetyl-coenzyme A. In contrast, anabolic metabolism of three-carbon units via propionate has not been shown to extensively occur. Mammals are primarily thought to oxidize the three-carbon short chain fatty acid propionate by shunting propionyl-CoA to succinyl-CoA for entry into the TCA cycle. Here, we found that this may not be absolute as, in mammals, one nonoxidative fate of propionyl-CoA is to condense to two three-carbon units into a six-carbon trans-2-methyl-2-pentenoyl-CoA (2M2PE-CoA). We confirmed this reaction pathway using purified protein extracts provided limited substrates and verified the product via LC-MS using a synthetic standard. In whole-body in vivo stable isotope tracing following infusion of C-labeled valine at steady state, 2M2PE-CoA was found to form via propionyl-CoA in multiple murine tissues, including heart, kidney, and to a lesser degree, in brown adipose tissue, liver, and tibialis anterior muscle. Using ex vivo isotope tracing, we found that 2M2PE-CoA also formed in human myocardial tissue incubated with propionate to a limited extent. While the complete enzymology of this pathway remains to be elucidated, these results confirm the in vivo existence of at least one anabolic three- to six-carbon reaction conserved in humans and mice that utilizes propionate.

Glioblastomas (GBMs) preferentially generate acetyl-CoA from acetate as a fuel source to promote tumor growth. O-GlcNAcylation has been shown to be elevated by increasing O-GlcNAc transferase (OGT) in many cancers and reduced O-GlcNAcylation can block cancer growth. Here, we identify a novel mechanism whereby OGT regulates acetate-dependent acetyl-CoA and lipid production by regulating phosphorylation of acetyl-CoA synthetase 2 (ACSS2) by cyclin-dependent kinase 5 (CDK5). OGT is required and sufficient for GBM cell growth and regulates acetate conversion to acetyl-CoA and lipids. Elevating O-GlcNAcylation in GBM cells increases phosphorylation of ACSS2 on Ser-267 in a CDK5-dependent manner. Importantly, we show that ACSS2 Ser-267 phosphorylation regulates its stability by reducing polyubiquitination and degradation. ACSS2 Ser-267 is critical for OGT-mediated GBM growth as overexpression of ACSS2 Ser-267 phospho-mimetic rescues growth in vitro and in vivo. Importantly, we show that pharmacologically targeting OGT and CDK5 reduces GBM growth ex vivo. Thus, the OGT/CDK5/ACSS2 pathway may be a way to target altered metabolic dependencies in brain tumors.

Quantitative subcellular metabolomic measurements can explain the roles of metabolites in cellular processes but are subject to multiple confounding factors. We developed stable isotope labeling of essential nutrients in cell culture-subcellular fractionation (SILEC-SF), which uses isotope-labeled internal standard controls that are present throughout fractionation and processing to quantify acyl-coenzyme A (acyl-CoA) thioesters in subcellular compartments by liquid chromatography-mass spectrometry. We tested SILEC-SF in a range of sample types and examined the compartmentalized responses to oxygen tension, cellular differentiation, and nutrient availability. Application of SILEC-SF to the challenging analysis of the nuclear compartment revealed a nuclear acyl-CoA profile distinct from that of the cytosol, with notable nuclear enrichment of propionyl-CoA. Using isotope tracing, we identified the branched chain amino acid isoleucine as a major metabolic source of nuclear propionyl-CoA and histone propionylation, thus revealing a new mechanism of crosstalk between metabolism and the epigenome.

Tumors frequently exhibit aberrant glycosylation, which can impact cancer progression and therapeutic responses. The hexosamine biosynthesis pathway (HBP) produces uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a major substrate for glycosylation in the cell. Prior studies have identified the HBP as a promising therapeutic target in pancreatic ductal adenocarcinoma (PDA). The HBP requires both glucose and glutamine for its initiation. The PDA tumor microenvironment is nutrient poor, however, prompting us to investigate how nutrient limitation impacts hexosamine synthesis. Here, we identify that glutamine limitation in PDA cells suppresses de novo hexosamine synthesis but results in increased free GlcNAc abundance. GlcNAc salvage via N-acetylglucosamine kinase (NAGK) is engaged to feed UDP-GlcNAc pools. expression is elevated in human PDA, and deletion from PDA cells impairs tumor growth in mice. Together, these data identify an important role for NAGK-dependent hexosamine salvage in supporting PDA tumor growth.

The metabolic milieu of solid tumors provides a barrier to chimeric antigen receptor (CAR) T-cell therapies. Excessive lactate or hypoxia suppresses T-cell growth, through mechanisms including NADH buildup and the depletion of oxidized metabolites. NADH is converted into NAD by the enzyme NADH Oxidase (), which mimics the oxidative function of the electron transport chain without generating ATP. Here we determine if promotes human CAR T-cell metabolic activity and antitumor efficacy. CAR T-cells expressing have enhanced oxygen as well as lactate consumption and increased pyruvate production. renders CAR T-cells resilient to lactate dehydrogenase inhibition. But in vivo in a model of mesothelioma, CAR T-cell's expressing showed no increased antitumor efficacy over control CAR T-cells. We hypothesize that T cells in hostile environments face dual metabolic stressors of excessive NADH and insufficient ATP production. Accordingly, futile T-cell NADH oxidation by is insufficient to promote tumor clearance.

Cutaneous melanoma remains the most lethal skin cancer, and ranks third among all malignancies in terms of years of life lost. Despite the advent of immune checkpoint and targeted therapies, only roughly half of patients with advanced melanoma achieve a durable remission. Sirtuin 5 (SIRT5) is a member of the sirtuin family of protein deacylases that regulates metabolism and other biological processes. Germline Sirt5 deficiency is associated with mild phenotypes in mice. Here we showed that SIRT5 was required for proliferation and survival across all cutaneous melanoma genotypes tested, as well as uveal melanoma, a genetically distinct melanoma subtype that arises in the eye and is incurable once metastatic. Likewise, SIRT5 was required for efficient tumor formation by melanoma xenografts and in an autochthonous mouse Braf Pten-driven melanoma model. Via metabolite and transcriptomic analyses, we found that SIRT5 was required to maintain histone acetylation and methylation levels in melanoma cells, thereby promoting proper gene expression. SIRT5-dependent genes notably included MITF, a key lineage-specific survival oncogene in melanoma, and the c-MYC proto-oncogene. SIRT5 may represent a druggable genotype-independent addiction in melanoma.

Although eukaryotic messenger RNAs (mRNAs) normally possess a 5' end N-methyl guanosine (mG) cap, a non-canonical 5' nicotinamide adenine dinucleotide (NAD) cap can tag certain transcripts for degradation mediated by the NAD decapping enzyme DXO1. Despite this importance, whether NAD capping dynamically responds to specific stimuli to regulate eukaryotic transcriptomes remains unknown. Here, we reveal a link between NAD capping and tissue- and hormone response-specific mRNA stability. In the absence of DXO1 function, transcripts displaying a high proportion of NAD capping are instead processed into RNA-dependent RNA polymerase 6-dependent small RNAs, resulting in their continued turnover likely to free the NAD molecules. Additionally, the NAD-capped transcriptome is significantly remodeled in response to the essential plant hormone abscisic acid in a mechanism that is primarily independent of DXO1. Overall, our findings reveal a previously uncharacterized and essential role of NAD capping in dynamically regulating transcript stability during specific physiological responses.

Lysine lactoylation is a recently described protein post-translational modification (PTM). However, the biochemical pathways responsible for this acylation remain unclear. Two metabolite-dependent mechanisms have been proposed: enzymatic histone lysine lactoylation derived from lactoyl-coenzyme A (lactoyl-CoA, also termed lactyl-CoA), and non-enzymatic lysine lactoylation resulting from acyl-transfer via lactoyl-glutathione. While the former has precedent in the form of enzyme-catalysed lysine acylation, the lactoyl-CoA metabolite has not been previously quantified in mammalian systems. Here, we use liquid chromatography-high-resolution mass spectrometry (LC-HRMS) together with a synthetic standard to detect and validate the presence of lactoyl-CoA in cell and tissue samples. Conducting a retrospective analysis of data from previously analysed samples revealed the presence of lactoyl-CoA in diverse cell and tissue contexts. In addition, we describe a biosynthetic route to generate CN-isotopically labelled lactoyl-CoA, providing a co-eluting internal standard for analysis of this metabolite. We estimate lactoyl-CoA concentrations of 1.14 × 10 pmol per cell in cell culture and 0.0172 pmol mg tissue wet weight in mouse heart. These levels are similar to crotonyl-CoA, but between 20 and 350 times lower than predominant acyl-CoAs such as acetyl-, propionyl- and succinyl-CoA. Overall our studies provide the first quantitative measurements of lactoyl-CoA in metazoans, and provide a methodological foundation for the interrogation of this novel metabolite in biology and disease.

Consumption of fructose has risen markedly in recent decades owing to the use of sucrose and high-fructose corn syrup in beverages and processed foods, and this has contributed to increasing rates of obesity and non-alcoholic fatty liver disease. Fructose intake triggers de novo lipogenesis in the liver, in which carbon precursors of acetyl-CoA are converted into fatty acids. The ATP citrate lyase (ACLY) enzyme cleaves cytosolic citrate to generate acetyl-CoA, and is upregulated after consumption of carbohydrates. Clinical trials are currently pursuing the inhibition of ACLY as a treatment for metabolic diseases. However, the route from dietary fructose to hepatic acetyl-CoA and lipids remains unknown. Here, using in vivo isotope tracing, we show that liver-specific deletion of Acly in mice is unable to suppress fructose-induced lipogenesis. Dietary fructose is converted to acetate by the gut microbiota, and this supplies lipogenic acetyl-CoA independently of ACLY. Depletion of the microbiota or silencing of hepatic ACSS2, which generates acetyl-CoA from acetate, potently suppresses the conversion of bolus fructose into hepatic acetyl-CoA and fatty acids. When fructose is consumed more gradually to facilitate its absorption in the small intestine, both citrate cleavage in hepatocytes and microorganism-derived acetate contribute to lipogenesis. By contrast, the lipogenic transcriptional program is activated in response to fructose in a manner that is independent of acetyl-CoA metabolism. These data reveal a two-pronged mechanism that regulates hepatic lipogenesis, in which fructolysis within hepatocytes provides a signal to promote the expression of lipogenic genes, and the generation of microbial acetate feeds lipogenic pools of acetyl-CoA.

Many metabolites serve as important signalling molecules to adjust cellular activities and functions based on nutrient availability. Links between acetyl-CoA metabolism, histone lysine acetylation, and gene expression have been documented and studied over the past decade. In recent years, several additional acyl modifications to histone lysine residues have been identified, which depend on acyl-coenzyme A thioesters (acyl-CoAs) as acyl donors. Acyl-CoAs are intermediates of multiple distinct metabolic pathways, and substantial evidence has emerged that histone acylation is metabolically sensitive. Nevertheless, the metabolic sources of acyl-CoAs used for chromatin modification in most cases remain poorly understood. Elucidating how these diverse chemical modifications are coupled to and regulated by cellular metabolism is important in deciphering their functional significance.

The dynamic regulation of metabolic pathways can be monitored by stable isotope tracing. Yet, many metabolites are part of distinct processes within different subcellular compartments. Standard isotope tracing experiments relying on analyses in whole cells may not accurately reflect compartmentalized metabolic processes. Analysis of compartmentalized metabolism and the dynamic interplay between compartments can potentially be achieved by stable isotope tracing followed by subcellular fractionation. Although it is recognized that metabolism can take place during biochemical fractionation of cells, a clear understanding of how such post-harvest metabolism impacts the interpretation of subcellular isotope tracing data and methods to correct for this are lacking. We set out to directly assess artifactual metabolism, enabling us to develop and test strategies to correct for it. We apply these techniques to examine the compartment-specific metabolic kinetics of C-labeled substrates targeting central metabolic pathways.

Dynamic interplay between cellular metabolism and histone acetylation is a key mechanism underlying metabolic control of epigenetics. In particular, the central metabolite acetyl-coenzyme A (acetyl-CoA) acts as the acetyl-donor for histone acetylation in both an enzymatic and non-enzymatic manner. Since members of the family of histone acetyl transferases (HATs) that catalyze the acetylation of histone tails possess a Michaelis constant (Km) within the range of physiological cellular acetyl-CoA concentrations, changing concentrations of acetyl-CoA can restrict or promote enzymatic histone acetylation. Likewise, non-enzymatic histone acetylation occurs at physiological concentrations. These concepts implicate acetyl-CoA as a rheostat for nutrient availability acting, in part, by controlling histone acetylation. Histone acetylation is an important epigenetic modification that controls gene expression and acetyl-CoA dependent changes in both histone acetylation and gene expression have been shown in yeast and mammalian systems. However, quantifying the metabolic conditions required to achieve specific changes in histone acetylation is a major challenge. The relationship between acetyl-CoA and histone acetylation may be influenced by a variety of factors including sub-cellular location of metabolites and enzymes, relative quantities of metabolites, and substrate availability/preference. A diversity of substrates can contribute the two-carbon acyl-chain to acetyl-CoA, a number of pathways can create or degrade acetyl-CoA, and only a handful of potential mechanisms for the crosstalk between metabolism and histone acetylation have been explored. The centrality of acetyl-CoA in intermediary metabolism means that acetyl-CoA levels may change, or be resistant to change, in unexpected ways. Thus, quantification of relevant metabolites is critical evidence in understanding how the nutrient rheostat is set in normal and pathological contexts. Coupling metabolite quantitation with isotope tracing to examine fate of specific metabolites is critical to the crosstalk between metabolism and histone acetylation, including but not limited to acetyl-CoA provides necessary context. This chapter provides guidance on experimental design of quantification with isotope dilution and/or tracing of acetyl-CoA within a targeted or highly multiplexed multi-analyte workflow.

Diseases associated with mitochondrial DNA (mtDNA) mutations are highly variable in phenotype, in large part because of differences in the percentage of normal and mutant mtDNAs (heteroplasmy) present within the cell. For example, increasing heteroplasmy levels of the mtDNA tRNA nucleotide (nt) 3243A > G mutation result successively in diabetes, neuromuscular degenerative disease, and perinatal lethality. These phenotypes are associated with differences in mitochondrial function and nuclear DNA (nDNA) gene expression, which are recapitulated in cybrid cell lines with different percentages of m.3243G mutant mtDNAs. Using metabolic tracing, histone mass spectrometry, and NADH fluorescence lifetime imaging microscopy in these cells, we now show that increasing levels of this single mtDNA mutation cause profound changes in the nuclear epigenome. At high heteroplasmy, mitochondrially derived acetyl-CoA levels decrease causing decreased histone H4 acetylation, with glutamine-derived acetyl-CoA compensating when glucose-derived acetyl-CoA is limiting. In contrast, α-ketoglutarate levels increase at midlevel heteroplasmy and are inversely correlated with histone H3 methylation. Inhibition of mitochondrial protein synthesis induces acetylation and methylation changes, and restoration of mitochondrial function reverses these effects. mtDNA heteroplasmy also affects mitochondrial NAD/NADH ratio, which correlates with nuclear histone acetylation, whereas nuclear NAD/NADH ratio correlates with changes in nDNA and mtDNA transcription. Thus, mutations in the mtDNA cause distinct metabolic and epigenomic changes at different heteroplasmy levels, potentially explaining transcriptional and phenotypic variability of mitochondrial disease.

The precursor cells for metabolically beneficial beige adipocytes can alternatively become fibrogenic and contribute to adipose fibrosis. We found that cold exposure or β3-adrenergic agonist treatment of mice decreased the fibrogenic profile of precursor cells and stimulated beige adipocyte differentiation. This fibrogenic-to-adipogenic transition was impaired in aged animals, correlating with reduced adipocyte expression of the transcription factor PRDM16. Genetic loss of Prdm16 mimicked the effect of aging in promoting fibrosis, whereas increasing PRDM16 in aged mice decreased fibrosis and restored beige adipose development. PRDM16-expressing adipose cells secreted the metabolite β-hydroxybutyrate (BHB), which blocked precursor fibrogenesis and facilitated beige adipogenesis. BHB catabolism in precursor cells, mediated by BDH1, was required for beige fat differentiation in vivo. Finally, dietary BHB supplementation in aged animals reduced adipose fibrosis and promoted beige fat formation. Together, our results demonstrate that adipocytes secrete a metabolite signal that controls beige fat remodeling.

Sugars and refined carbohydrates are major components of the modern diet. ATP-citrate lyase (ACLY) is upregulated in adipocytes in response to carbohydrate consumption and generates acetyl-coenzyme A (CoA) for both lipid synthesis and acetylation reactions. Here, we investigate the role of ACLY in the metabolic and transcriptional responses to carbohydrates in adipocytes and unexpectedly uncover a sexually dimorphic function in maintaining systemic metabolic homeostasis. When fed a high-sucrose diet, Acly females exhibit a lipodystrophy-like phenotype, with minimal fat accumulation, insulin resistance, and hepatic lipid accumulation, whereas Acly males have only mild metabolic phenotypes. We find that ACLY is crucial for nutrient-dependent carbohydrate response element-binding protein (ChREBP) activation in adipocytes and plays a key role, particularly in females, in the storage of newly synthesized fatty acids in adipose tissue. The data indicate that adipocyte ACLY is important in females for the systemic handling of dietary carbohydrates and for the preservation of metabolic homeostasis.

Acetylation is a highly abundant and dynamic post-translational modification (PTM) on histone proteins which, when present on chromatin-bound histones, facilitates the accessibility of DNA for gene transcription. The central metabolite, acetyl-CoA, is a substrate for acetyltransferases, which catalyze protein acetylation. Acetyl-CoA is an essential intermediate in diverse metabolic pathways, and cellular acetyl-CoA levels fluctuate according to extracellular nutrient availability and the metabolic state of the cell. The Michaelis constant (Km) of most histone acetyltransferases (HATs), which specifically target histone proteins, falls within the range of cellular acetyl-CoA concentrations. As a consequence, global levels of histone acetylation are often restricted by availability of acetyl-CoA. Such metabolic regulation of histone acetylation is important for cell proliferation, differentiation, and a variety of cellular functions. In cancer, numerous oncogenic signaling events hijack cellular metabolism, ultimately inducing an extensive rearrangement of the epigenetic state of the cell. Understanding metabolic control of the epigenome through histone acetylation is essential to illuminate the molecular mechanisms by which cells sense, adapt, and occasionally disengage nutrient fluctuations and environmental cues from gene expression. In particular, targeting metabolic regulators or even dietary interventions to impact acetyl-CoA availability and histone acetylation is a promising target in cancer therapy. Liquid chromatography coupled to mass spectrometry (LC-MS) is the most accurate methodology to quantify protein PTMs and metabolites. In this chapter, we present state-of-the-art protocols to analyze histone acetylation and acetyl-CoA. Histones are extracted and digested into short peptides (4-20 aa) prior to LC-MS. Acetyl-CoA is extracted from cells and analyzed using an analogous mass spectrometry-based procedure. Model systems can be fed with isotopically labeled substrates to investigate the metabolic preference for acetyl-CoA production and the metabolic dependence and turnover of histone acetylation. We also present an example of data integration to correlate changes in acetyl-CoA production with histone acetylation.

Macrophages enforce antitumor immunity by engulfing and killing tumor cells. Although these functions are determined by a balance of stimulatory and inhibitory signals, the role of macrophage metabolism is unknown. Here, we study the capacity of macrophages to circumvent inhibitory activity mediated by CD47 on cancer cells. We show that stimulation with a CpG oligodeoxynucleotide, a Toll-like receptor 9 agonist, evokes changes in the central carbon metabolism of macrophages that enable antitumor activity, including engulfment of CD47 cancer cells. CpG activation engenders a metabolic state that requires fatty acid oxidation and shunting of tricarboxylic acid cycle intermediates for de novo lipid biosynthesis. This integration of metabolic inputs is underpinned by carnitine palmitoyltransferase 1A and adenosine tri-phosphate citrate lyase, which, together, impart macrophages with antitumor potential capable of overcoming inhibitory CD47 on cancer cells. Our findings identify central carbon metabolism to be a novel determinant and potential therapeutic target for stimulating antitumor activity by macrophages.

Pancreatic ductal adenocarcinoma (PDA) has a poor prognosis, and new strategies for prevention and treatment are urgently needed. We previously reported that histone H4 acetylation is elevated in pancreatic acinar cells harboring mutations prior to the appearance of premalignant lesions. Because acetyl-CoA abundance regulates global histone acetylation, we hypothesized that altered acetyl-CoA metabolism might contribute to metabolic or epigenetic alterations that promote tumorigenesis. We found that acetyl-CoA abundance is elevated in -mutant acinar cells and that its use in the mevalonate pathway supports acinar-to-ductal metaplasia (ADM). Pancreas-specific loss of the acetyl-CoA-producing enzyme ATP-citrate lyase (ACLY) accordingly suppresses ADM and tumor formation. In PDA cells, growth factors promote AKT-ACLY signaling and histone acetylation, and both cell proliferation and tumor growth can be suppressed by concurrent BET inhibition and statin treatment. Thus, KRAS-driven metabolic alterations promote acinar cell plasticity and tumor development, and targeting acetyl-CoA-dependent processes exerts anticancer effects. SIGNIFICANCE: Pancreatic cancer is among the deadliest of human malignancies. We identify a key role for the metabolic enzyme ACLY, which produces acetyl-CoA, in pancreatic carcinogenesis. The data suggest that acetyl-CoA use for histone acetylation and in the mevalonate pathway facilitates cell plasticity and proliferation, suggesting potential to target these pathways...

The discovery of significant amounts of metabolically active brown adipose tissue (BAT) in adult humans renders it a promising target for anti-obesity therapies by inducing weight loss through increased energy expenditure. The components of the N-acetylaspartate (NAA) pathway are highly abundant in BAT. Aspartate N-acetyltransferase (Asp-NAT, encoded by Nat8l) synthesizes NAA from acetyl-CoA and aspartate and increases energy expenditure in brown adipocytes. However, the exact mechanism how the NAA pathway contributes to accelerated mobilization and oxidation of lipids and the physiological regulation of the NAA pathway remained elusive. Here, we demonstrate that the expression of NAA pathway genes corresponds to nutrient availability and specifically responds to changes in exogenous glucose. NAA is preferentially produced from glucose-derived acetyl-CoA and aspartate and its concentration increases during adipogenesis. Overexpression of Nat8l drains glucose-derived acetyl-CoA into the NAA pool at the expense of cellular lipids and certain amino acids. Mechanistically, we elucidated that a combined activation of neutral and lysosomal (acid) lipolysis is responsible for the increased lipid degradation. Specifically, translocation of the transcription factor EB to the nucleus activates the biosynthesis of autophagosomes and lysosomes. Lipid degradation within lysosomes accompanied by adipose triglyceride lipase-mediated lipolysis delivers fatty acids for the support of elevated mitochondrial respiration. Together, our data suggest a crucial role of the NAA pathway in energy metabolism and metabolic adaptation in BAT.

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Lactate and pyruvate are high abundance products of glucose metabolism. Analysis of both molecules as part of metabolomics studies in cellular metabolism and physiology have been aided by advances in liquid chromatography-mass spectrometry (LC-MS).

Etomoxir (ETO) is a widely used small-molecule inhibitor of fatty acid oxidation (FAO) through its irreversible inhibitory effects on the carnitine palmitoyl-transferase 1a (CPT1a). We used this compound to evaluate the role of fatty acid oxidation in rapidly proliferating T cells following costimulation through the CD28 receptor. We show that ETO has a moderate effect on T cell proliferation with no observable effect on memory differentiation, but a marked effect on oxidative metabolism. We show that this oxidative metabolism is primarily dependent upon glutamine rather than FAO. Using an shRNA approach to reduce CPT1a in T cells, we further demonstrate that the inhibition of oxidative metabolism in T cells by ETO is independent of its effects on FAO at concentrations exceeding 5 μM. Concentrations of ETO above 5 μM induce acute production of ROS with associated evidence of severe oxidative stress in proliferating T cells. In aggregate, these data indicate that ETO lacks specificity for CTP1a above 5 μM, and caution should be used when employing this compound for studies in cells due to its non-specific effects on oxidative metabolism and cellular redox.

The metabolite acetyl-coenzyme A (acetyl-CoA) is the required acetyl donor for lysine acetylation and thereby links metabolism, signaling, and epigenetics. Nutrient availability alters acetyl-CoA levels in cancer cells, correlating with changes in global histone acetylation and gene expression. However, the specific molecular mechanisms through which acetyl-CoA production impacts gene expression and its functional roles in promoting malignant phenotypes are poorly understood. Here, using histone H3 Lys27 acetylation (H3K27ac) ChIP-seq (chromatin immunoprecipitation [ChIP] coupled with next-generation sequencing) with normalization to an exogenous reference genome (ChIP-Rx), we found that changes in acetyl-CoA abundance trigger site-specific regulation of H3K27ac, correlating with gene expression as opposed to uniformly modulating this mark at all genes. Genes involved in integrin signaling and cell adhesion were identified as acetyl-CoA-responsive in glioblastoma cells, and we demonstrate that ATP citrate lyase (ACLY)-dependent acetyl-CoA production promotes cell migration and adhesion to the extracellular matrix. Mechanistically, the transcription factor NFAT1 (nuclear factor of activated T cells 1) was found to mediate acetyl-CoA-dependent gene regulation and cell adhesion. This occurs through modulation of Ca signals, triggering NFAT1 nuclear translocation when acetyl-CoA is abundant. The findings of this study thus establish that acetyl-CoA impacts H3K27ac at specific loci, correlating with gene expression, and that expression of cell adhesion genes are driven by acetyl-CoA in part through activation of Ca-NFAT signaling.

Nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) are conserved metabolic cofactors that mediate reduction-oxidation (redox) reactions throughout all domains of life. The diversity of synthetic routes and cellular processes involving the transfer of reducing equivalents to and from these cofactors makes the accurate quantitation and metabolic tracing of NAD(H) and NADP(H) of broad interest. However, current analytical techniques typically rely on standard curves that do not incorporate confounding effects of the sample matrix. We utilized the essential requirement of niacin and tryptophan for NAD synthesis in mammalian cells to devise a stable isotope labeling by essential nutrients in cell culture (SILEC) method for efficient labeling of intracellular NAD(H) and NADP(H) pools. Coupling this approach with detection by liquid chromatography-high resolution mass spectrometry (LC-HRMS), we demonstrate the utility of incorporating a [CN]-nicotinamide moiety into a library of NAD-derived metabolites for use as internal standards in matrixed samples. Using a two-label system incorporating [CN]-nicotinamide and [C]-tryptophan, we quantify the relative contribution of salvage and de novo NAD synthesis, respectively, in S. cerevisiae and HepG2 human hepatocellular carcinoma cells under basal conditions. As a further proof-of-principle, we demonstrate an improvement in the linear range for quantification of NAD and apply this method to analysis of NAD(H) in mouse liver. This method demonstrates the generalizability of SILEC, and provides a simple method for generating a library of stable isotope labeled internal standards for quantifying and tracing the metabolism of cellular and tissue NAD(H) and NADP(H).

Cellular metabolism dynamically regulates the epigenome via availability of the metabolite substrates of chromatin-modifying enzymes. The impact of diet on the metabolism-epigenome axis is poorly understood but could alter gene expression and influence metabolic health. ATP citrate-lyase produces acetyl-CoA in the nucleus and cytosol and regulates histone acetylation levels in many cell types. Consumption of a high-fat diet (HFD) results in suppression of ATP citrate-lyase levels in tissues such as adipose and liver, but the impact of diet on acetyl-CoA and histone acetylation in these tissues remains unknown. Here we examined the effects of HFD on levels of acyl-CoAs and histone acetylation in mouse white adipose tissue (WAT), liver, and pancreas. We report that mice consuming a HFD have reduced levels of acetyl-CoA and/or acetyl-CoA:CoA ratio in these tissues. In WAT and the pancreas, HFD also impacted the levels of histone acetylation; in particular, histone H3 lysine 23 acetylation was lower in HFD-fed mice. Genetic deletion of in cultured adipocytes also suppressed acetyl-CoA and histone acetylation levels. In the liver, no significant effects on histone acetylation were observed with a HFD despite lower acetyl-CoA levels. Intriguingly, acetylation of several histone lysines correlated with the acetyl-CoA: (iso)butyryl-CoA ratio in liver. Butyryl-CoA and isobutyryl-CoA interacted with the acetyltransferase P300/CBP-associated factor (PCAF) in liver lysates and inhibited its activity This study thus provides evidence that diet can impact tissue acyl-CoA and histone acetylation levels and that acetyl-CoA abundance correlates with acetylation of specific histone lysines in WAT but not in the liver.

Isotopic tracer analysis by mass spectrometry is a core technique for the study of metabolism. Isotopically labeled atoms from substrates, such as [C]-labeled glucose, can be traced by their incorporation over time into specific metabolic products. Mass spectrometry is often used for the detection and differentiation of the isotopologues of each metabolite of interest. For meaningful interpretation, mass spectrometry data from metabolic tracer experiments must be corrected to account for the naturally occurring isotopologue distribution. The calculations required for this correction are time consuming and error prone and existing programs are often platform specific, non-intuitive, commercially licensed and/or limited in accuracy by using theoretical isotopologue distributions, which are prone to artifacts from noise or unresolved interfering signals.

Mechanisms of metabolic flexibility enable cells to survive under stressful conditions and can thwart therapeutic responses. Acetyl-coenzyme A (CoA) plays central roles in energy production, lipid metabolism, and epigenomic modifications. Here, we show that, upon genetic deletion of Acly, the gene coding for ATP-citrate lyase (ACLY), cells remain viable and proliferate, although at an impaired rate. In the absence of ACLY, cells upregulate ACSS2 and utilize exogenous acetate to provide acetyl-CoA for de novo lipogenesis (DNL) and histone acetylation. A physiological level of acetate is sufficient for cell viability and abundant acetyl-CoA production, although histone acetylation levels remain low in ACLY-deficient cells unless supplemented with high levels of acetate. ACLY-deficient adipocytes accumulate lipid in vivo, exhibit increased acetyl-CoA and malonyl-CoA production from acetate, and display some differences in fatty acid content and synthesis. Together, these data indicate that engagement of acetate metabolism is a crucial, although partial, mechanism of compensation for ACLY deficiency.

The androgen receptor (AR) plays a central role in prostate tumor growth. Inappropriate reactivation of the AR after androgen deprivation therapy promotes development of incurable castration-resistant prostate cancer (CRPC). In this study, we provide evidence that metabolic features of prostate cancer cells can be exploited to sensitize CRPC cells to AR antagonism. We identify a feedback loop between ATP-citrate lyase (ACLY)-dependent fatty acid synthesis, AMPK, and the AR in prostate cancer cells that could contribute to therapeutic resistance by maintaining AR levels. When combined with an AR antagonist, ACLY inhibition in CRPC cells promotes energetic stress and AMPK activation, resulting in further suppression of AR levels and target gene expression, inhibition of proliferation, and apoptosis. Supplying exogenous fatty acids can restore energetic homeostasis; however, this rescue does not occur through increased β-oxidation to support mitochondrial ATP production. Instead, concurrent inhibition of ACLY and AR may drive excess ATP consumption as cells attempt to cope with endoplasmic reticulum (ER) stress, which is prevented by fatty acid supplementation. Thus, fatty acid metabolism plays a key role in coordinating ER and energetic homeostasis in CRPC cells, thereby sustaining AR action and promoting proliferation. Consistent with a role for fatty acid metabolism in sustaining AR levels in prostate cancer in vivo, AR mRNA levels in human prostate tumors correlate positively with expression of ACLY and other fatty acid synthesis genes. The ACLY-AMPK-AR network can be exploited to sensitize CRPC cells to AR antagonism, suggesting novel therapeutic opportunities for prostate cancer.

Acyl-coenzyme A (acyl-CoA) thioesters are evolutionarily conserved, compartmentalized, and energetically activated substrates for biochemical reactions. The ubiquitous involvement of acyl-CoA thioesters in metabolism, including the tricarboxylic acid cycle, fatty acid metabolism, amino acid degradation, and cholesterol metabolism highlights the broad applicability of applied measurements of acyl-CoA thioesters. However, quantitation of acyl-CoA levels provides only one dimension of metabolic information and a more complete description of metabolism requires the relative contribution of different precursors to individual substrates and pathways. Using two distinct stable isotope labeling approaches, acyl-CoA thioesters can be labeled with either a fixed [(13)C3(15)N1] label derived from pantothenate into the CoA moiety or via variable [(13)C] labeling into the acyl chain from metabolic precursors. Liquid chromatography-hybrid quadrupole/Orbitrap high-resolution mass spectrometry using parallel reaction monitoring, but not single ion monitoring, allowed the simultaneous quantitation of acyl-CoA thioesters by stable isotope dilution using the [(13)C3(15)N1] label and measurement of the incorporation of labeled carbon atoms derived from [(13)C6]-glucose, [(13)C5(15)N2]-glutamine, and [(13)C3]-propionate. As a proof of principle, we applied this method to human B cell lymphoma (WSU-DLCL2) cells in culture to precisely describe the relative pool size and enrichment of isotopic tracers into acetyl-, succinyl-, and propionyl-CoA. This method will allow highly precise, multiplexed, and stable isotope-resolved determination of metabolism to refine metabolic models, characterize novel metabolism, and test modulators of metabolic pathways involving acyl-CoA thioesters.

Exercise is essential in regulating energy metabolism and whole-body insulin sensitivity. To explore the exercise signaling network, we undertook a global analysis of protein phosphorylation in human skeletal muscle biopsies from untrained healthy males before and after a single high-intensity exercise bout, revealing 1,004 unique exercise-regulated phosphosites on 562 proteins. These included substrates of known exercise-regulated kinases (AMPK, PKA, CaMK, MAPK, mTOR), yet the majority of kinases and substrate phosphosites have not previously been implicated in exercise signaling. Given the importance of AMPK in exercise-regulated metabolism, we performed a targeted in vitro AMPK screen and employed machine learning to predict exercise-regulated AMPK substrates. We validated eight predicted AMPK substrates, including AKAP1, using targeted phosphoproteomics. Functional characterization revealed an undescribed role for AMPK-dependent phosphorylation of AKAP1 in mitochondrial respiration. These data expose the unexplored complexity of acute exercise signaling and provide insights into the role of AMPK in mitochondrial biochemistry.

The insulin/insulin-like growth factor (IGF)-1 signaling pathway (ISP) plays a fundamental role in long term health in a range of organisms. Protein kinases including Akt and ERK are intimately involved in the ISP. To identify other kinases that may participate in this pathway or intersect with it in a regulatory manner, we performed a whole kinome (779 kinases) siRNA screen for positive or negative regulators of the ISP, using GLUT4 translocation to the cell surface as an output for pathway activity. We identified PFKFB3, a positive regulator of glycolysis that is highly expressed in cancer cells and adipocytes, as a positive ISP regulator. Pharmacological inhibition of PFKFB3 suppressed insulin-stimulated glucose uptake, GLUT4 translocation, and Akt signaling in 3T3-L1 adipocytes. In contrast, overexpression of PFKFB3 in HEK293 cells potentiated insulin-dependent phosphorylation of Akt and Akt substrates. Furthermore, pharmacological modulation of glycolysis in 3T3-L1 adipocytes affected Akt phosphorylation. These data add to an emerging body of evidence that metabolism plays a central role in regulating numerous biological processes including the ISP. Our findings have important implications for diseases such as type 2 diabetes and cancer that are characterized by marked disruption of both metabolism and growth factor signaling.

Myocardial ischemia and cardioprotection by ischemic pre-conditioning induce signal networks aimed at survival or cell death if the ischemic period is prolonged. These pathways are mediated by protein post-translational modifications that are hypothesized to cross-talk with and regulate each other. Phosphopeptides and lysine-acetylated peptides were quantified in isolated rat hearts subjected to ischemia or ischemic pre-conditioning, with and without splitomicin inhibition of lysine deacetylation. We show lysine acetylation (acetyl-Lys)-dependent activation of AMP-activated protein kinase, AKT, and PKA kinases during ischemia. Phosphorylation and acetyl-Lys sites mapped onto tertiary structures were proximal in >50% of proteins investigated, yet they were mutually exclusive in 50 ischemic pre-conditioning- and/or ischemia-associated peptides containing the KXXS basophilic protein kinase consensus motif. Modifications in this motif were modeled in the C terminus of muscle-type creatine kinase. Acetyl-Lys increased proximal dephosphorylation by 10-fold. Structural analysis of modified muscle-type creatine kinase peptide variants by two-dimensional NMR revealed stabilization via a lysine-phosphate salt bridge, which was disrupted by acetyl-Lys resulting in backbone flexibility and increased phosphatase accessibility.

Grb10 is an intracellular adaptor protein that acts as a negative regulator of insulin and insulin-like growth factor 1 (IGF1) receptors. Since global deletion of Grb10 in mice causes hypermuscularity, we have characterized the skeletal muscle physiology underlying this phenotype. Compared to wild-type (WT) controls, adult mice deficient in Grb10 have elevated body mass and muscle mass throughout adulthood, up to 12 mo of age. The muscle enlargement is not due to increased myofiber size, but rather an increase in myofiber number (142% of WT, P<0.01). There is no change in myofiber type proportions between WT and Grb10-deficient muscles, nor are the metabolic properties of the muscles altered on Grb10 deletion. Notably, the weight and cross-sectional area of hindlimbs from neonatal mice are increased in Grb10-deficient animals (198 and 137% of WT, respectively, both P<0.001). Functional gene signatures for myogenic signaling and proliferation are up-regulated in Grb10-deficient neonatal muscle. Our findings indicate that Grb10 plays a previously unrecognized role in regulating the development of fiber number during murine embryonic growth. In addition, Grb10-ablated muscle from adult mice shows coordinate gene changes that oppose those of muscle wasting pathologies, highlighting Grb10 as a potential therapeutic target for these conditions.