Acetate Production from Glucose and Coupling to Mitochondrial Metabolism in Mammals.

Acetate is a major nutrient that supports acetyl-coenzyme A (Ac-CoA) metabolism and thus lipogenesis and protein acetylation. However, its source is unclear. Here, we report that pyruvate, the end product of glycolysis and key node in central carbon metabolism, quantitatively generates acetate in mammals. This phenomenon becomes more pronounced in the context of nutritional excess, such as during hyperactive glucose metabolism. Conversion of pyruvate to acetate occurs through two mechanisms: (1) coupling to reactive oxygen species (ROS) and (2) neomorphic enzyme activity from keto acid dehydrogenases that enable function as pyruvate decarboxylases. Further, we demonstrate that de novo acetate production sustains Ac-CoA pools and cell proliferation in limited metabolic environments, such as during mitochondrial dysfunction or ATP citrate lyase (ACLY) deficiency. By virtue of de novo acetate production being coupled to mitochondrial metabolism, there are numerous possible regulatory mechanisms and links to pathophysiology.

Quantitative subcellular acyl-CoA analysis reveals distinct nuclear metabolism and isoleucine-dependent histone propionylation.

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.

The ß1-adrenergic receptor links sympathetic nerves to T cell exhaustion.

CD8+ T cells are essential components of the immune response against viral infections and tumours, and are capable of eliminating infected and cancerous cells. However, when the antigen cannot be cleared, T cells enter a state known as exhaustion1. Although it is clear that chronic antigen contributes to CD8+ T cell exhaustion, less is known about how stress responses in tissues regulate T cell function. Here we show a new link between the stress-associated catecholamines and the progression of T cell exhaustion through the ß1-adrenergic receptor ADRB1. We identify that exhausted CD8+ T cells increase ADRB1 expression and that exposure of ADRB1+ T cells to catecholamines suppresses their cytokine production and proliferation. Exhausted CD8+ T cells cluster around sympathetic nerves in an ADRB1-dependent manner. Ablation of ß1-adrenergic signalling limits the progression of T cells towards the exhausted state in chronic infection and improves effector functions when combined with immune checkpoint blockade (ICB) in melanoma. In a pancreatic cancer model resistant to ICB, ß-blockers and ICB synergize to boost CD8+ T cell responses and induce the development of tissue-resident memory-like T cells. Malignant disease is associated with increased catecholamine levels in patients2,3, and our results establish a connection between the sympathetic stress response, tissue innervation and T cell exhaustion. Here, we uncover a new mechanism by which blocking ß-adrenergic signalling in CD8+ T cells rejuvenates anti-tumour functions.

Dietary fructose feeds hepatic lipogenesis via microbiota-derived acetate.

Consumption of fructose has risen markedly in recent decades owing to the use of sucrose and high-fructose corn syrup in beverages and processed foods1, and this has contributed to increasing rates of obesity and non-alcoholic fatty liver disease2-4. Fructose intake triggers de novo lipogenesis in the liver4-6, 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 carbohydrates7. Clinical trials are currently pursuing the inhibition of ACLY as a treatment for metabolic diseases8. 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 microbiota9, and this supplies lipogenic acetyl-CoA independently of ACLY10. 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.

Acetyl-CoA promotes glioblastoma cell adhesion and migration through Ca2+-NFAT signaling.

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 Ca2+ 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 Ca2+-NFAT signaling.

Nuclear Acetyl-CoA Production by ACLY Promotes Homologous Recombination.

While maintaining the integrity of the genome and sustaining bioenergetics are both fundamental functions of the cell, potential crosstalk between metabolic and DNA repair pathways is poorly understood. Since histone acetylation plays important roles in DNA repair and is sensitive to the availability of acetyl coenzyme A (acetyl-CoA), we investigated a role for metabolic regulation of histone acetylation during the DNA damage response. In this study, we report that nuclear ATP-citrate lyase (ACLY) is phosphorylated at S455 downstream of ataxia telangiectasia mutated (ATM) and AKT following DNA damage. ACLY facilitates histone acetylation at double-strand break (DSB) sites, impairing 53BP1 localization and enabling BRCA1 recruitment and DNA repair by homologous recombination. ACLY phosphorylation and nuclear localization are necessary for its role in promoting BRCA1 recruitment. Upon PARP inhibition, ACLY silencing promotes genomic instability and cell death. Thus, the spatial and temporal control of acetyl-CoA production by ACLY participates in the mechanism of DNA repair pathway choice.

Metabolic regulation of T cells in the tumor microenvironment by nutrient availability and diet.

Recent advances in immunotherapies such as immune checkpoint blockade (ICB) and chimeric antigen receptor T cells (CAR-T) for the treatment of cancer have generated excitement over their ability to yield durable, and potentially curative, responses in a multitude of cancers. These findings have established that the immune system is capable of eliminating tumors and led us to a better, albeit still incomplete, understanding of the mechanisms by which tumors interact with and evade destruction by the immune system. Given the central role of T cells in immunotherapy, elucidating the cell intrinsic and extrinsic factors that govern T cell function in tumors will facilitate the development of immunotherapies that establish durable responses in a greater number of patients. One such factor is metabolism, a set of fundamental cellular processes that not only sustains cell survival and proliferation, but also serves as a means for cells to interpret their local environment. Nutrient sensing is critical for T cells that must infiltrate into a metabolically challenging tumor microenvironment and expand under these harsh conditions to eliminate cancerous cells. Here we introduce T cell exhaustion with respect to cellular metabolism, followed by a discussion of nutrient availability at the tumor and organismal level in relation to T cell metabolism and function to provide rationale for the study and targeting of metabolism in anti-tumor immune responses.

Bempedoic acid suppresses diet-induced hepatic steatosis independently of ATP-citrate lyase.

ATP citrate lyase (ACLY) synthesizes acetyl-CoA for de novo lipogenesis (DNL), which is elevated in metabolic dysfunction-associated steatotic liver disease. Hepatic ACLY is inhibited by the LDL-cholesterol-lowering drug bempedoic acid (BPA), which also improves steatosis in mice. While BPA potently suppresses hepatic DNL and increases fat catabolism, it is unclear if ACLY is its primary molecular target in reducing liver triglyceride. We show that on a Western diet, loss of hepatic ACLY alone or together with the acetyl-CoA synthetase ACSS2 unexpectedly exacerbates steatosis, linked to reduced PPARα target gene expression and fatty acid oxidation. Importantly, BPA treatment ameliorates Western diet-mediated triacylglyceride accumulation in both WT and liver ACLY knockout mice, indicating that its primary effects on hepatic steatosis are ACLY independent. Together, these data indicate that hepatic ACLY plays an unexpected role in restraining diet-dependent lipid accumulation and that BPA exerts substantial effects on hepatic lipid metabolism independently of ACLY.

Acetate acts as a metabolic immunomodulator by bolstering T-cell effector function and potentiating antitumor immunity in breast cancer.

Acetate metabolism is an important metabolic pathway in many cancers and is controlled by acetyl-CoA synthetase 2 (ACSS2), an enzyme that catalyzes the conversion of acetate to acetyl-CoA. While the metabolic role of ACSS2 in cancer is well described, the consequences of blocking tumor acetate metabolism on the tumor microenvironment and antitumor immunity are unknown. We demonstrate that blocking ACSS2, switches cancer cells from acetate consumers to producers of acetate thereby freeing acetate for tumor-infiltrating lymphocytes to use as a fuel source. We show that acetate supplementation metabolically bolsters T-cell effector functions and proliferation. Targeting ACSS2 with CRISPR-Cas9 guides or a small-molecule inhibitor promotes an antitumor immune response and enhances the efficacy of chemotherapy in preclinical breast cancer models. We propose a paradigm for targeting acetate metabolism in cancer in which inhibition of ACSS2 dually acts to impair tumor cell metabolism and potentiate antitumor immunity.

Eicosanoids in the pancreatic tumor microenvironment - a multicellular, multifaceted progression.

Background and Aims: Eicosanoids, oxidized fatty acids that serve as cell-signaling molecules, have been broadly implicated in tumorigenesis. Here, we aimed to identify eicosanoids associated with pancreatic tumorigenesis and the cell types responsible for their synthesis. Methods: We profiled normal pancreas and pancreatic ductal adenocarcinoma (PDAC) in mouse models and patient samples using mass spectrometry. We interrogated RNA sequencing datasets for eicosanoid synthase or receptor expression. Findings were confirmed by immunostaining. Results: In murine models, we identified elevated levels of PGD2, prostacyclin, and thromboxanes in neoplasia while PGE2, 12-HHTre, HETEs, and HDoHEs are elevated specifically in tumors. Analysis of scRNA-seq datasets suggests that PGE2 and prostacyclins are derived from fibroblasts, PGD2 and thromboxanes from myeloid cells, and PGD2 and 5-HETE from tuft cells. In patient samples, we identified a transition from PGD2 to PGE2-producing enzymes in the epithelium during the transition to PDAC, fibroblast/tumor expression of PTGIS, and myeloid/tumor cell expression of TBXAS1. Conclusions: Our analyses identify key changes in eicosanoid species during pancreatic tumorigenesis and the cell types that contribute to their synthesis. Thromboxane and prostacyclin expression is conserved between animal models and human disease and may represent new druggable targets.

Management of Right Ventricular Failure in Pulmonary Embolism.

Acute right ventricular failure remains the leading cause of mortality associated with acute pulmonary embolism (PE). This article reviews the pathophysiology behind acute right ventricular failure and strategies for managing right ventricular failure in acute PE. Immediate clot reduction via systemic thrombolytics, catheter based procedures, or surgery is always advocated for unstable patients. While waiting to mobilize these resources, it often becomes necessary to support the RV with vasoactive medications. Clinicians should carefully assess volume status and use caution with volume resuscitation. Right ventricular assist devices may have an expanding role in the future.

Pre-existing traits associated with Covid-19 illness severity.

IMPORTANCE: Certain individuals, when infected by SARS-CoV-2, tend to develop the more severe forms of Covid-19 illness for reasons that remain unclear. OBJECTIVE: To determine the demographic and clinical characteristics associated with increased severity of Covid-19 infection. DESIGN: Retrospective observational study. We curated data from the electronic health record, and used multivariable logistic regression to examine the association of pre-existing traits with a Covid-19 illness severity defined by level of required care: need for hospital admission, need for intensive care, and need for intubation. SETTING: A large, multihospital healthcare system in Southern California. PARTICIPANTS: All patients with confirmed Covid-19 infection (N = 442). RESULTS: Of all patients studied, 48% required hospitalization, 17% required intensive care, and 12% required intubation. In multivariable-adjusted analyses, patients requiring a higher levels of care were more likely to be older (OR 1.5 per 10 years, P<0.001), male (OR 2.0, P = 0.001), African American (OR 2.1, P = 0.011), obese (OR 2.0, P = 0.021), with diabetes mellitus (OR 1.8, P = 0.037), and with a higher comorbidity index (OR 1.8 per SD, P<0.001). Several clinical associations were more pronounced in younger compared to older patients (Pinteraction<0.05). Of all hospitalized patients, males required higher levels of care (OR 2.5, P = 0.003) irrespective of age, race, or morbidity profile. CONCLUSIONS AND RELEVANCE: In our healthcare system, greater Covid-19 illness severity is seen in patients who are older, male, African American, obese, with diabetes, and with greater overall comorbidity burden. Certain comorbidities paradoxically augment risk to a greater extent in younger patients. In hospitalized patients, male sex is the main determinant of needing more intensive care. Further investigation is needed to understand the mechanisms underlying these findings.

Unripe peach (Prunus persica) extract ameliorates damage from UV irradiation and improved collagen XVIII expression in 3D skin model.

INTRODUCTION: Collagen type XVIII regulates cellular activities of adjacent cells at the dermal-epidermal junction (DEJ). To investigate its possible changes during aging, we compared its mRNA levels and protein localization in skin samples from female participants aged 20-70 years old. In addition, we evaluated the beneficial effects of unripe peach extracts in a 3D skin model. METHODS: Sun-exposed or sun-protected female skin samples were compared by DNA array or by immunohistochemistry for basement membrane components. To evaluate protective effects of fresh unripe peach extract, UV-B irradiated human 3D skin models were incubated in the presence or absence of the extract, followed by measurements of mRNA levels by real-time PCR, or by immunohistochemistry. RESULTS: In aged skin samples, COL18A1 mRNA levels were lower and the protein localization exhibited less intensive signal by anti-collagen type XVIII immunostaining. As observed in the skin tissues, collagen type XVIII exists at the DEJ in the 3D skin model. Fresh unripe peach extract significantly improved mRNA levels and partially localizations of collagen type XVIII, suggesting that fresh unripe peach extract ameliorates DEJ damages caused by UV-B irradiation. CONCLUSION: Collagen type XVIII and fresh unripe peach extract can be promising protective cosmetic strategies against skin aging.

Adipocyte ACLY Facilitates Dietary Carbohydrate Handling to Maintain Metabolic Homeostasis in Females.

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, AclyFAT-/- females exhibit a lipodystrophy-like phenotype, with minimal fat accumulation, insulin resistance, and hepatic lipid accumulation, whereas AclyFAT-/- 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.

Acetyl-CoA Metabolism Supports Multistep Pancreatic Tumorigenesis.

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 Kras 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 KRAS-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.See related commentary by Halbrook et al., p. 326.This article is highlighted in the In This Issue feature, p. 305.

Metabolic control of epigenetics in cancer.

Alterations in the epigenome and metabolism both affect molecular rewiring in cancer cells and facilitate cancer development and progression. However, recent evidence suggests the existence of important bidirectional regulatory mechanisms between metabolic remodelling and the epigenome (specifically methylation and acetylation of histones) in cancer. Most chromatin-modifying enzymes require substrates or cofactors that are intermediates of cell metabolism. Such metabolites, and often the enzymes that produce them, can transfer into the nucleus, directly linking metabolism to nuclear transcription. We discuss how metabolic remodelling can contribute to tumour epigenetic alterations, thereby affecting cancer cell differentiation, proliferation and/or apoptosis, as well as therapeutic responses.

ATP-Citrate Lyase Controls a Glucose-to-Acetate Metabolic Switch.

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.

U1 Adaptor Oligonucleotides Targeting BCL2 and GRM1 Suppress Growth of Human Melanoma Xenografts In Vivo.

U1 Adaptor is a recently discovered oligonucleotide-based gene-silencing technology with a unique mechanism of action that targets nuclear pre-mRNA processing. U1 Adaptors have two distinct functional domains, both of which must be present on the same oligonucleotide to exert their gene-silencing function. Here, we present the first in vivo use of U1 Adaptors by targeting two different human genes implicated in melanomagenesis, B-cell lymphoma 2 (BCL2) and metabotropic glutamate receptor 1 (GRM1), in a human melanoma cell xenograft mouse model system. Using a newly developed dendrimer delivery system, anti-BCL2 U1 Adaptors were very potent and suppressed tumor growth at doses as low as 34 µg/kg with twice weekly intravenous (iv) administration. Anti-GRM1 U1 Adaptors suppressed tumor xenograft growth with similar potency. Mechanism of action was demonstrated by showing target gene suppression in tumors and by observing that negative control U1 Adaptors with just one functional domain show no tumor suppression activity. The anti-BCL2 and anti-GRM1 treatments were equally effective against cell lines harboring either wild-type or a mutant V600E B-RAF allele, the most common mutation in melanoma. Treatment of normal immune-competent mice (C57BL6) indicated no organ toxicity or immune stimulation. These proof-of-concept studies represent an in-depth (over 800 mice in ~108 treatment groups) validation that U1 Adaptors are a highly potent gene-silencing therapeutic and open the way for their further development to treat other human diseases.Molecular Therapy - Nucleic Acids (2013) 2, e92; doi:10.1038/mtna.2013.24; published online 14 May 2013.