Obesity Shapes Metabolism in the Tumor Microenvironment to Suppress Anti-Tumor Immunity.

Obesity is a major cancer risk factor, but how differences in systemic metabolism change the tumor microenvironment (TME) and impact anti-tumor immunity is not understood. Here, we demonstrate that high-fat diet (HFD)-induced obesity impairs CD8+ T cell function in the murine TME, accelerating tumor growth. We generate a single-cell resolution atlas of cellular metabolism in the TME, detailing how it changes with diet-induced obesity. We find that tumor and CD8+ T cells display distinct metabolic adaptations to obesity. Tumor cells increase fat uptake with HFD, whereas tumor-infiltrating CD8+ T cells do not. These differential adaptations lead to altered fatty acid partitioning in HFD tumors, impairing CD8+ T cell infiltration and function. Blocking metabolic reprogramming by tumor cells in obese mice improves anti-tumor immunity. Analysis of human cancers reveals similar transcriptional changes in CD8+ T cell markers, suggesting interventions that exploit metabolism to improve cancer immunotherapy.

Lipid metabolism in sickness and in health: Emerging regulators of lipotoxicity.

Lipids play crucial roles in signal transduction, contribute to the structural integrity of cellular membranes, and regulate energy metabolism. Questions remain as to which lipid species maintain metabolic homeostasis and which disrupt essential cellular functions, leading to metabolic disorders. Here, we discuss recent advances in understanding lipid metabolism with a focus on catabolism, synthesis, and signaling. Technical advances, including functional genomics, metabolomics, lipidomics, lipid-protein interaction maps, and advances in mass spectrometry, have uncovered new ways to prioritize molecular mechanisms mediating lipid function. By reviewing what is known about the distinct effects of specific lipid species in physiological pathways, we provide a framework for understanding newly identified targets regulating lipid homeostasis with implications for ameliorating metabolic diseases.

PHD3 Loss in Cancer Enables Metabolic Reliance on Fatty Acid Oxidation via Deactivation of ACC2.

While much research has examined the use of glucose and glutamine by tumor cells, many cancers instead prefer to metabolize fats. Despite the pervasiveness of this phenotype, knowledge of pathways that drive fatty acid oxidation (FAO) in cancer is limited. Prolyl hydroxylase domain proteins hydroxylate substrate proline residues and have been linked to fuel switching. Here, we reveal that PHD3 rapidly triggers repression of FAO in response to nutrient abundance via hydroxylation of acetyl-coA carboxylase 2 (ACC2). We find that PHD3 expression is strongly decreased in subsets of cancer including acute myeloid leukemia (AML) and is linked to a reliance on fat catabolism regardless of external nutrient cues. Overexpressing PHD3 limits FAO via regulation of ACC2 and consequently impedes leukemia cell proliferation. Thus, loss of PHD3 enables greater utilization of fatty acids but may also serve as a metabolic and therapeutic liability by indicating cancer cell susceptibility to FAO inhibition.

Gamma-Aminobutyric Acid Signaling in Damage Response, Metabolism, and Disease.

Gamma-aminobutyric acid (GABA) plays a crucial role in signal transduction and can function as a neurotransmitter. Although many studies have been conducted on GABA in brain biology, the cellular function and physiological relevance of GABA in other metabolic organs remain unclear. Here, we will discuss recent advances in understanding GABA metabolism with a focus on its biosynthesis and cellular functions in other organs. The mechanisms of GABA in liver biology and disease have revealed new ways to link the biosynthesis of GABA to its cellular function. By reviewing what is known about the distinct effects of GABA and GABA-mediated metabolites in physiological pathways, we provide a framework for understanding newly identified targets regulating the damage response, with implications for ameliorating metabolic diseases. With this review, we suggest that further research is necessary to develop GABA's beneficial and toxic effects on metabolic disease progression.

Development of a colorimetric α-ketoglutarate detection assay for prolyl hydroxylase domain (PHD) proteins.

Since the discovery of the prolyl hydroxylases domain (PHD) proteins and their canonical hypoxia-inducible factor (HIF) substrate two decades ago, a number of in vitro hydroxylation (IVH) assays for PHD activity have been developed to measure the PHD-HIF interaction. However, most of these assays either require complex proteomics mass spectrometry methods that rely on the specific PHD-HIF interaction or require the handling of radioactive material, as seen in the most commonly used assay measuring [14C]O2 release from labeled [14C]α-ketoglutarate. Here, we report an alternative rapid, cost-effective assay in which the consumption of α-ketoglutarate is monitored by its derivatization with 2,4-dinitrophenylhydrazine (2,4-DNPH) followed by treatment with concentrated base. We extensively optimized this 2,4-DNPH α-ketoglutarate assay to maximize the signal-to-noise ratio and demonstrated that it is robust enough to obtain kinetic parameters of the well-characterized PHD2 isoform comparable with those in published literature. We further showed that it is also sensitive enough to detect and measure the IC50 values of pan-PHD inhibitors and several PHD2 inhibitors in clinical trials for chronic kidney disease (CKD)-induced anemia. Given the efficiency of this assay coupled with its multiwell format, the 2,4-DNPH α-KG assay may be adaptable to explore non-HIF substrates of PHDs and potentially to high-throughput assays.

Sirtuin 1 attenuates nasal polypogenesis by suppressing epithelial-to-mesenchymal transition.

BACKGROUND: Nasal polyps (NPs) imply a refractory clinical course in patients with chronic rhinosinusitis (CRS). Previously, we showed that hypoxia-inducible factor (HIF) 1 could mediate nasal polypogenesis through epithelial-to-mesenchymal transition (EMT). Sirtuin 1 (SIRT1), a histone deacetylase, reportedly suppresses the transcriptional activity of HIF-1. Thus we hypothesized that SIRT1 attenuates nasal polyposis by inhibiting HIF-1-induced EMT. OBJECTIVE: We sought to determine the role of SIRT1 in patients with nasal polyposis. METHODS: The effects of SIRT1 on nasal polypogenesis were investigated in previously developed murine models. Immunohistochemistry, immunoblotting, and immunoprecipitation were done to evaluate SIRT1, EMT, and hypoxic markers in human nasal epithelial cells or sinonasal tissues from the mice and the patients with CRS with or without NPs. RESULTS: SIRT1 transgenic mice had significantly fewer mucosal lesions with epithelial disruption and fewer NPs than wild-type (WT) mice. In addition, resveratrol (a SIRT1 activator) treatment suppressed nasal polypogenesis in WT mice; however, sirtinol (a SIRT1 inhibitor) administration increased the polyp burden in SIRT1 transgenic mice. In sinonasal specimens from patients with CRS, SIRT1 was downregulated in the mucosa from patients with polyps compared with levels seen in patients without polyps. SIRT1 overexpression or activation reversed hypoxia-induced EMT in human nasal epithelial cells. The intranasal transfection of a small hairpin SIRT1 lentiviral vector induced more nasal polypoid lesions in SIRT1 transgenic mice. Finally, mucosal extracts from patients with CRS without NPs increased SIRT1 expression in nasal epithelial cells, whereas those from patients with CRS with NPs did not. CONCLUSION: SIRT1 suppressed NP formation, possibly because of inhibition of HIF-1-induced EMT. Thus nasal epithelium SIRT1 might be a therapeutic target for NPs.

Differential roles of Sirt1 in HIF-1α and HIF-2α mediated hypoxic responses.

Hypoxia-inducible factors 1α and 2α (HIF-1α and HIF-2α) determine cancer cell fate under hypoxia. Despite the similarities of their structures, HIF-1α and HIF-2α have distinct roles in cancer growth under hypoxia, that is, HIF-1α induces growth arrest whereas HIF-2α promotes cell growth. Recently, sirtuin 1 (Sirt1) was reported to fine-tune cellular responses to hypoxia by deacetylating HIF-1α and HIF-2α. Yet, the roles of Sirt1 in HIF-1α and HIF-2α functions have been controversial. We here investigated the precise roles of Sirt1 in HIF-1α and HIF-2α regulations. Immunological analyses revealed that HIF-1α K674 and HIF-2α K741 are acetylated by PCAF and CBP, respectively, but are deacetylated commonly by Sirt1. In the Gal4 reporter systems, Sirt1 was found to repress HIF-1α activity constantly in ten cancer cell-lines but to regulate HIF-2α activity cell type-dependently. Moreover, Sirt1 determined cell growth under hypoxia depending on HIF-1α and HIF-2α. Under hypoxia, Sirt1 promoted cell proliferation of HepG2, in which Sirt1 differentially regulates HIF-1α and HIF-2α. In contrast, such an effect of Sirt1 was not shown in HCT116, in which Sirt1 inactivates both HIF-1α and HIF-2α because conflicting actions of HIF-1α and HIF-2α on cell growth may be offset. Our results provide a better understanding of the roles of Sirt1 in HIF-mediated hypoxic responses and also a basic concept for developing anticancer strategy targeting Sirt1.

Mitochondrial sirtuins: Energy dynamics and cancer metabolism.

Mitochondria are pivotal for energy regulation and are linked to cancer. Mitochondrial sirtuins, (Sirtuin) SIRT3, SIRT4, and SIRT5, play crucial roles in cancer metabolism. This review explores their impact on cellular processes, with a focus on the NAD+ interplay and the modulation of their enzymatic activities. The varied roles of SIRT3, SIRT4, and SIRT5 in metabolic adaptation and cancer are outlined, emphasizing their tumor suppressor or oncogenic nature. We propose new insights into sirtuin biology, and cancer therapeutics, suggesting an integrated proteomics and metabolomics approach for a comprehensive understanding of mitochondrial sirtuins in cancer.

Metabolic Adaptation and Cellular Stress Response As Targets for Cancer Therapy.

Cancer cells, which divide indefinitely and without control, are frequently exposed to various stress factors but manage to adapt and survive. The mechanisms by which cancer cells maintain cellular homeostasis and exploit stress conditions are not yet clear. Here, we elucidate the roles of diverse cellular metabolism and its regulatory mechanisms, highlighting the essential role of metabolism in cellular composition and signal transduction. Cells respond to various stresses, including DNA damage, energy stress, and oxidative stress, thereby causing metabolic alteration. We provide profound insight into the adaptive mechanisms employed by cancer cells to ensure their survival among internal and external stressors through a comprehensive analysis of the correlation between metabolic alterations and cellular stress. Furthermore, this research establishes a robust framework for the development of innovative therapeutic strategies that specifically target the cellular adaptations of cancer cells.

Mebendazole preferentially inhibits cilia formation and exerts anticancer activity by synergistically augmenting DNA damage.

The cilium is a microtubule-based organelle that plays a pivotal role in embryonic development and maintenance of physiological functions in the human body. In addition to their function as sensors that transduce diverse extracellular signals, including growth factors, fluid flow, and physical forces, cilia are intricately involved in cell cycle regulation and preservation of DNA integrity, as their formation and resorption dynamics are tightly linked to cell cycle progression. Recently, several studies have linked defects in specific ciliary proteins to the DNA damage response. However, it remains unclear whether and how primary cilia contribute to cancer development. Mebendazole (MBZ) is an anthelmintic drug with anticancer properties in some cancer cells. MBZ is continuously being tested for clinical studies, but the precise mechanism of its anticancer activities remains unknown. Here, using Xenopus laevis embryos as a model system, we discovered that MBZ significantly hinders cilia formation and induces DNA damage. Remarkably, primary cilium-bearing cancer cells exhibited heightened vulnerability to combined treatment with MBZ and conventional anticancer drugs. Our findings shed light on the specific influence of MBZ on cilia, rather than cytosolic microtubules, in triggering DNA damage, elucidating a previously unidentified mechanism underlying potential MBZ-mediated cancer therapy.

Lactate oxidase/vSIRPα conjugates efficiently consume tumor-produced lactates and locally produce tumor-necrotic H2O2 to suppress tumor growth.

Aggressive tumor formation often leads to excessive anaerobic glycolysis and massive production and accumulation of lactate in the tumor microenvironment (TME). To significantly curb lactate accumulation in TME, in this study, lactate oxidase (LOX) was used as a potential therapeutic enzyme and signal regulatory protein α variant (vSIRPα) as a tumor cell targeting ligand. SpyCatcher protein and SpyTag peptide were genetically fused to LOX and vSIRPα, respectively, to form SC-LOX and ST-vSIRPα and tumor-targeting LOX/vSIRPα conjugates were constructed via a SpyCatcher/SpyTag protein ligation system. LOX/vSIRPα conjugates selectively bound to the CD47-overexpressing mouse melanoma B16-F10 cells and effectively consumed lactate produced by the B16-F10 cells, generating adequate amounts of hydrogen peroxide (H2O2), which induces drastic necrotic tumor cell death. Local treatments of B16-F10 tumor-bearing mice with LOX/vSIRPα conjugates significantly suppressed B16-F10 tumor growth in vivo without any severe side effects. Tumor-targeting vSIRPα may allow longer retention of LOX in tumor sites, effectively consuming surrounding lactate in TME and locally generating adequate amounts of cytotoxic H2O2 to suppress tumor growth. The approach restraining the local lactate concentration and H2O2 in TME using LOX and vSIRPα could offer new opportunities for developing enzyme/targeting ligand conjugate-based therapeutic tools for tumor treatment.

FALCON systematically interrogates free fatty acid biology and identifies a novel mediator of lipotoxicity.

Cellular exposure to free fatty acids (FFA) is implicated in the pathogenesis of obesity-associated diseases. However, studies to date have assumed that a few select FFAs are representative of broad structural categories, and there are no scalable approaches to comprehensively assess the biological processes induced by exposure to diverse FFAs circulating in human plasma. Furthermore, assessing how these FFA- mediated processes interact with genetic risk for disease remains elusive. Here we report the design and implementation of FALCON (Fatty Acid Library for Comprehensive ONtologies) as an unbiased, scalable and multimodal interrogation of 61 structurally diverse FFAs. We identified a subset of lipotoxic monounsaturated fatty acids (MUFAs) with a distinct lipidomic profile associated with decreased membrane fluidity. Furthermore, we developed a new approach to prioritize genes that reflect the combined effects of exposure to harmful FFAs and genetic risk for type 2 diabetes (T2D). Importantly, we found that c-MAF inducing protein (CMIP) protects cells from exposure to FFAs by modulating Akt signaling and we validated the role of CMIP in human pancreatic beta cells. In sum, FALCON empowers the study of fundamental FFA biology and offers an integrative approach to identify much needed targets for diverse diseases associated with disordered FFA metabolism. Highlights: FALCON (Fatty Acid Library for Comprehensive ONtologies) enables multimodal profiling of 61 free fatty acids (FFAs) to reveal 5 FFA clusters with distinct biological effectsFALCON is applicable to many and diverse cell typesA subset of monounsaturated FAs (MUFAs) equally or more toxic than canonical lipotoxic saturated FAs (SFAs) leads to decreased membrane fluidityNew approach prioritizes genes that represent the combined effects of environmental (FFA) exposure and genetic risk for diseaseC-Maf inducing protein (CMIP) is identified as a suppressor of FFA-induced lipotoxicity via Akt-mediated signaling.

FALCON systematically interrogates free fatty acid biology and identifies a novel mediator of lipotoxicity.

Cellular exposure to free fatty acids (FFAs) is implicated in the pathogenesis of obesity-associated diseases. However, there are no scalable approaches to comprehensively assess the diverse FFAs circulating in human plasma. Furthermore, assessing how FFA-mediated processes interact with genetic risk for disease remains elusive. Here, we report the design and implementation of fatty acid library for comprehensive ontologies (FALCON), an unbiased, scalable, and multimodal interrogation of 61 structurally diverse FFAs. We identified a subset of lipotoxic monounsaturated fatty acids associated with decreased membrane fluidity. Furthermore, we prioritized genes that reflect the combined effects of harmful FFA exposure and genetic risk for type 2 diabetes (T2D). We found that c-MAF-inducing protein (CMIP) protects cells from FFA exposure by modulating Akt signaling. In sum, FALCON empowers the study of fundamental FFA biology and offers an integrative approach to identify much needed targets for diverse diseases associated with disordered FFA metabolism.

CITED2 controls the hypoxic signaling by snatching p300 from the two distinct activation domains of HIF-1α.

HIF-1α plays a central role in cellular adaptation to hypoxia, and is closely related to the pathogeneses of life-threatening disorders. HIF-1α induces the expressions of numerous hypoxia-induced genes through two transactivation domains; N-terminal TAD (NAD) and C-terminal TAD (CAD). Furthermore, p300 is known to boost CAD-dependent transactivation, and CBP/p300-interacting transactivator with an ED-rich tail 2 (CITED2) inhibits HIF-1α-driven gene expression by interfering with the interaction between CAD and p300. However, few researches have focused on the role of CITED2 in the regulation of NAD activity, and thus, we addressed this point. CITED2 was found to attenuate the hypoxic activations of NAD-dependent and CAD-dependent genes, suggesting that CITED2 negatively regulates both CAD and NAD. Immunoprecipitation analyses showed that NAD interacts with the Cystein/Histidine region (CH) 1 and CH3 domains of p300. Moreover, CH1 and CH3 both were required for NAD-dependent transactivation. Furthermore, CITED2 was found to inactivate NAD by interfering with NAD binding to CH1, but not to CH3. These results indicate that CITED2 inactivates HIF-1α by blocking p300 recruitment by both NAD and CAD. We also found that pVHL inhibits NAD activity regardless of NAD degradation by blocking the interaction between p300 and NAD. Summarizing, NAD was activated by binding to p300, and this was blocked by either CITED2 or pVHL. We propose that pVHL controls NAD during normoxia and that CITED2 controls NAD during hypoxia. Our results provide a new strategy for controlling HIF-1α.

Antihepatoma activity of chaetocin due to deregulated splicing of hypoxia-inducible factor 1α pre-mRNA in mice and in vitro.

UNLABELLED: Chaetocin, an antibiotic produced by Chaetomium species fungi, was recently found to have antimyeloma activity. Here we examined whether chaetocin has anticancer activities against solid tumors. Chaetocin inhibited the growth of mouse and human hepatoma grafts in nude mice. Immunohistochemical analyses revealed that chaetocin inhibits hypoxia-inducible factor-1α (HIF-1α) expression and vessel formation in the tumors. Chaetocin also showed antiangiogenic anticancer activities in HIF-1α(+/+) fibrosarcoma grafted in mice, but not in HIF-1α(-/-) fibrosarcoma. Biochemical analyses showed that chaetocin down-regulated HIF-1α and the transcripts of HIF-1 target genes including vascular endothelial growth factor in hepatoma tissues and in various hepatoma cell lines. Based on the reported literature, unsuccessful efforts were made to determine the mechanism underlying the action of chaetocin. Unexpectedly, chaetocin was found to cause the accumulation of HIF-1α premessenger RNA (pre-mRNA) but to reduce mature mRNA levels in hepatoma cells and tissues. Such an effect of chaetocin was not observed in cell lines derived from normal cells, and was cell type-dependent even among cancer cell lines. CONCLUSIONS: Our results suggest that chaetocin could be developed as an anticancer agent to target HIF-1 in some cancers including hepatoma. It is also suggested that the HIF-1α pre-mRNA splicing is a novel therapeutic target for controlling HIF-1-mediated pathological processes.

A general toxicity and biodistribution study of human natural killer cells by single or repeated intravenous dose in severe combined immune deficient mice.

Natural killer (NK) cells are a part of the innate immune system and represent the first line of defense against infections and tumors. NK cells can eliminate tumor cells without major histocompatibility restriction and are independent of the expression of tumor-associated antigens. Therefore, they are considered an emerging tool for cancer immunotherapy. However, the general toxicity and biodistribution of NK cells after transplantation remain to be understood. This study was conducted to evaluate the general toxicity and biodistribution of human NK cells after single or repeated intravenous dosing in severely combined immunodeficient (SCID) mice. There were no test item-related toxicological changes in single and repeated administration groups. The no observed adverse effect level of human NK cells was 2 × 107 cells/head for both male and female SCID mice. Results from the biodistribution study showed that human NK cells were mainly distributed in the lungs, and a small number of the cells were detected in the liver, heart, spleen, and kidney of SCID mice, in both the single and repeated dose groups. Additionally, human NK cells were completely eliminated from all organs of the mice in the single dose group on day 7, while the cells persisted in mice in the repeated dose group until day 64. In conclusion, transplantation of human NK cells in SCID mice had no toxic effects. The cells were mainly distributed in the lungs and completely disappeared from the body over time after single or repeated intravenous administration.

Intrinsic myocardial defects underlie an Rbfox-deficient zebrafish model of hypoplastic left heart syndrome.

Hypoplastic left heart syndrome (HLHS) is characterized by underdevelopment of left sided structures including the ventricle, valves, and aorta. Prevailing paradigm suggests that HLHS is a multigenic disease of co-occurring phenotypes. Here, we report that zebrafish lacking two orthologs of the RNA binding protein RBFOX2, a gene linked to HLHS in humans, display cardiovascular defects overlapping those in HLHS patients including ventricular, valve, and aortic deficiencies. In contrast to current models, we demonstrate that these structural deficits arise secondary to impaired pump function as these phenotypes are rescued when Rbfox is specifically expressed in the myocardium. Mechanistically, we find diminished expression and alternative splicing of sarcomere and mitochondrial components that compromise sarcomere assembly and mitochondrial respiration, respectively. Injection of human RBFOX2 mRNA restores cardiovascular development in rbfox mutant zebrafish, while HLHS-linked RBFOX2 variants fail to rescue. This work supports an emerging paradigm for HLHS pathogenesis that centers on myocardial intrinsic defects.

Extracellular Vesicle Proteomes Shed Light on the Evolutionary, Interactive, and Functional Divergence of Their Biogenesis Mechanisms.

Extracellular vesicles (EVs) are membranous structures containing bioactive molecules, secreted by most cells into the extracellular environment. EVs are classified by their biogenesis mechanisms into two major subtypes: ectosomes (enriched in large EVs; lEVs), budding directly from the plasma membrane, which is common in both prokaryotes and eukaryotes, and exosomes (enriched in small EVs; sEVs) generated through the multivesicular bodies via the endomembrane system, which is unique to eukaryotes. Even though recent proteomic analyses have identified key proteins associated with EV subtypes, there has been no systematic analysis, thus far, to support the general validity and utility of current EV subtype separation methods, still largely dependent on physical properties, such as vesicular size and sedimentation. Here, we classified human EV proteomic datasets into two main categories based on distinct centrifugation protocols commonly used for isolating sEV or lEV fractions. We found characteristic, evolutionarily conserved profiles of sEV and lEV proteins linked to their respective biogenetic origins. This may suggest that the evolutionary trajectory of vesicular proteins may result in a membership bias toward specific EV subtypes. Protein-protein interaction (PPI) network analysis showed that vesicular proteins formed distinct clusters with proteins in the same EV fraction, providing evidence for the existence of EV subtype-specific protein recruiters. Moreover, we identified functional modules enriched in each fraction, including multivesicular body sorting for sEV, and mitochondria cellular respiration for lEV proteins. Our analysis successfully captured novel features of EVs embedded in heterogeneous proteomics studies and suggests specific protein markers and signatures to be used as quality controllers in the isolation procedure for subtype-enriched EV fractions.

SIRT4 is an early regulator of branched-chain amino acid catabolism that promotes adipogenesis.

Upon nutrient stimulation, pre-adipocytes undergo differentiation to transform into mature adipocytes capable of storing nutrients as fat. We profiled cellular metabolite consumption to identify early metabolic drivers of adipocyte differentiation. We find that adipocyte differentiation raises the uptake and consumption of numerous amino acids. In particular, branched-chain amino acid (BCAA) catabolism precedes and promotes peroxisome proliferator-activated receptor gamma (PPARγ), a key regulator of adipogenesis. In early adipogenesis, the mitochondrial sirtuin SIRT4 elevates BCAA catabolism through the activation of methylcrotonyl-coenzyme A (CoA) carboxylase (MCCC). MCCC supports leucine oxidation by catalyzing the carboxylation of 3-methylcrotonyl-CoA to 3-methylglutaconyl-CoA. Sirtuin 4 (SIRT4) expression is decreased in adipose tissue of numerous diabetic mouse models, and its expression is most correlated with BCAA enzymes, suggesting a potential role for SIRT4 in adipose pathology through the alteration of BCAA metabolism. In summary, this work provides a temporal analysis of adipocyte differentiation and uncovers early metabolic events that stimulate transcriptional reprogramming.

PHD3 Loss Promotes Exercise Capacity and Fat Oxidation in Skeletal Muscle.

Rapid alterations in cellular metabolism allow tissues to maintain homeostasis during changes in energy availability. The central metabolic regulator acetyl-CoA carboxylase 2 (ACC2) is robustly phosphorylated during cellular energy stress by AMP-activated protein kinase (AMPK) to relieve its suppression of fat oxidation. While ACC2 can also be hydroxylated by prolyl hydroxylase 3 (PHD3), the physiological consequence thereof is poorly understood. We find that ACC2 phosphorylation and hydroxylation occur in an inverse fashion. ACC2 hydroxylation occurs in conditions of high energy and represses fatty acid oxidation. PHD3-null mice demonstrate loss of ACC2 hydroxylation in heart and skeletal muscle and display elevated fatty acid oxidation. Whole body or skeletal muscle-specific PHD3 loss enhances exercise capacity during an endurance exercise challenge. In sum, these data identify an unexpected link between AMPK and PHD3, and a role for PHD3 in acute exercise endurance capacity and skeletal muscle metabolism.