ACOD1 deficiency offers protection in a mouse model of diet-induced obesity by maintaining a healthy gut microbiota.

Aconitate decarboxylase 1 (ACOD1) is the enzyme synthesizing itaconate, an immuno-regulatory metabolite tuning host-pathogen interactions. Such functions are achieved by affecting metabolic pathways regulating inflammation and microbe survival. However, at the whole-body level, metabolic roles of itaconate remain largely unresolved. By using multiomics-integrated approaches, here we show that ACOD1 responds to high-fat diet consumption in mice by promoting gut microbiota alterations supporting metabolic disease. Genetic disruption of itaconate biosynthesis protects mice against obesity, alterations in glucose homeostasis and liver metabolic dysfunctions by decreasing meta-inflammatory responses to dietary lipid overload. Mechanistically, fecal metagenomics and microbiota transplantation experiments demonstrate such effects are dependent on an amelioration of the intestinal ecosystem composition, skewed by high-fat diet feeding towards obesogenic phenotype. In particular, unbiased fecal microbiota profiling and axenic culture experiments point towards a primary role for itaconate in inhibiting growth of Bacteroidaceae and Bacteroides, family and genus of Bacteroidetes phylum, the major gut microbial taxon associated with metabolic health. Specularly to the effects imposed by Acod1 deficiency on fecal microbiota, oral itaconate consumption enhances diet-induced gut dysbiosis and associated obesogenic responses in mice. Unveiling an unrecognized role of itaconate, either endogenously produced or exogenously administered, in supporting microbiota alterations underlying diet-induced obesity in mice, our study points ACOD1 as a target against inflammatory consequences of overnutrition.

Pyruvate transamination and NAD biosynthesis enable proliferation of succinate dehydrogenase-deficient cells by supporting aerobic glycolysis.

Succinate dehydrogenase (SDH) is the mitochondrial enzyme converting succinate to fumarate in the tricarboxylic acid (TCA) cycle. SDH acts as a tumor suppressor with germline loss-of-function mutations in its encoding genes predisposing to aggressive familial neuroendocrine and renal cancer syndromes. Lack of SDH activity disrupts the TCA cycle, imposes Warburg-like bioenergetic features, and commits cells to rely on pyruvate carboxylation for anabolic needs. However, the spectrum of metabolic adaptations enabling SDH-deficient tumors to cope with a dysfunctional TCA cycle remains largely unresolved. By using previously characterized Sdhb-deleted kidney mouse cells, here we found that SDH deficiency commits cells to rely on mitochondrial glutamate-pyruvate transaminase (GPT2) activity for proliferation. We showed that GPT2-dependent alanine biosynthesis is crucial to sustain reductive carboxylation of glutamine, thereby circumventing the TCA cycle truncation determined by SDH loss. By driving the reductive TCA cycle anaplerosis, GPT2 activity fuels a metabolic circuit maintaining a favorable intracellular NAD+ pool to enable glycolysis, thus meeting the energetic demands of SDH-deficient cells. As a metabolic syllogism, SDH deficiency confers sensitivity to NAD+ depletion achieved by pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of the NAD+ salvage pathway. Beyond identifying an epistatic functional relationship between two metabolic genes in the control of SDH-deficient cell fitness, this study disclosed a metabolic strategy to increase the sensitivity of tumors to interventions limiting NAD availability.

Linoleic acid potentiates CD8+ T cell metabolic fitness and antitumor immunity.

The metabolic state represents a major hurdle for an effective adoptive T cell therapy (ACT). Indeed, specific lipids can harm CD8+ T cell (CTL) mitochondrial integrity, leading to defective antitumor responses. However, the extent to which lipids can affect the CTL functions and fate remains unexplored. Here, we show that linoleic acid (LA) is a major positive regulator of CTL activity by improving metabolic fitness, preventing exhaustion, and stimulating a memory-like phenotype with superior effector functions. We report that LA treatment enhances the formation of ER-mitochondria contacts (MERC), which in turn promotes calcium (Ca2+) signaling, mitochondrial energetics, and CTL effector functions. As a direct consequence, the antitumor potency of LA-instructed CD8 T cells is superior in vitro and in vivo. We thus propose LA treatment as an ACT potentiator in tumor therapy.

The heme synthesis-export system regulates the tricarboxylic acid cycle flux and oxidative phosphorylation.

Heme is an iron-containing porphyrin of vital importance for cell energetic metabolism. High rates of heme synthesis are commonly observed in proliferating cells. Moreover, the cell-surface heme exporter feline leukemia virus subgroup C receptor 1a (FLVCR1a) is overexpressed in several tumor types. However, the reasons why heme synthesis and export are enhanced in highly proliferating cells remain unknown. Here, we illustrate a functional axis between heme synthesis and heme export: heme efflux through the plasma membrane sustains heme synthesis, and implementation of the two processes down-modulates the tricarboxylic acid (TCA) cycle flux and oxidative phosphorylation. Conversely, inhibition of heme export reduces heme synthesis and promotes the TCA cycle fueling and flux as well as oxidative phosphorylation. These data indicate that the heme synthesis-export system modulates the TCA cycle and oxidative metabolism and provide a mechanistic basis for the observation that both processes are enhanced in cells with high-energy demand.

Oncogene-induced maladaptive activation of trained immunity in the pathogenesis and treatment of Erdheim-Chester disease.

Trained immunity (TI) is a proinflammatory program induced in monocyte/macrophages upon sensing of specific pathogens and is characterized by immunometabolic and epigenetic changes that enhance cytokine production. Maladaptive activation of TI (ie, in the absence of infection) may result in detrimental inflammation and development of disease; however, the exact role and extent of inappropriate activation of TI in the pathogenesis of human diseases is undetermined. In this study, we uncovered the oncogene-induced, maladaptive induction of TI in the pathogenesis of a human inflammatory myeloid neoplasm (Erdheim-Chester disease, [ECD]), characterized by the BRAFV600E oncogenic mutation in monocyte/macrophages and excess cytokine production. Mechanistically, myeloid cells expressing BRAFV600E exhibit all molecular features of TI: activation of the AKT/mammalian target of rapamycin signaling axis; increased glycolysis, glutaminolysis, and cholesterol synthesis; epigenetic changes on promoters of genes encoding cytokines; and enhanced cytokine production leading to hyperinflammatory responses. In patients with ECD, effective therapeutic strategies combat this maladaptive TI phenotype; in addition, pharmacologic inhibition of immunometabolic changes underlying TI (ie, glycolysis) effectively dampens cytokine production by myeloid cells. This study revealed the deleterious potential of inappropriate activation of TI in the pathogenesis of human inflammatory myeloid neoplasms and the opportunity for inhibition of TI in conditions characterized by maladaptive myeloid-driven inflammation.

D-mannose suppresses macrophage IL-1ß production.

D-mannose is a monosaccharide approximately a hundred times less abundant than glucose in human blood. Previous studies demonstrated that supraphysiological levels of D-mannose inhibit tumour growth and stimulate regulatory T cell differentiation. It is not known whether D-mannose metabolism affects the function of non-proliferative cells, such as inflammatory macrophages. Here, we show that D-mannose suppresses LPS-induced macrophage activation by impairing IL-1ß production. In vivo, mannose administration improves survival in a mouse model of LPS-induced endotoxemia as well as decreases progression in a mouse model of DSS-induced colitis. Phosphomannose isomerase controls response of LPS-activated macrophages to D-mannose, which impairs glucose metabolism by raising intracellular mannose-6-phosphate levels. Such alterations result in the suppression of succinate-mediated HIF-1α activation, imposing a consequent reduction of LPS-induced Il1b expression. Disclosing an unrecognized metabolic hijack of macrophage activation, our study points towards safe D-mannose utilization as an effective intervention against inflammatory conditions.

The Tricarboxylic Acid Cycle at the Crossroad Between Cancer and Immunity.

Significance: The tricarboxylic acid (TCA) cycle is a housekeeping metabolic pathway essential for generation of energy and biosynthetic intermediates. Alterations of the TCA cycle play a pivotal role in oncogenesis and inflammation. As such, some metabolic vulnerabilities, imposed by TCA cycle dysfunction in cancer, have been identified. Similarly, the TCA cycle appeared as an actionable pathway in immunopathologies. Recent Advances: Metabolic changes accompanying cell transformation have been usually considered as adaptive mechanisms to malignant transformation. The identification of oncogenic mutations in some TCA cycle enzymes changed this view, indicating altered mitochondrial metabolism as an instrumental mechanism for cancer initiation. Similarly, the observation that TCA cycle-derived metabolites have multiple signaling roles in immune cells supports the idea of this pathway as a metabolic rheostat of immune responses. Critical Issues: This review summarizes the crucial role of the TCA cycle in pathophysiology describing the post-translational and epigenetic impact of oncometabolites accumulation in cancer and immune cells. Future Directions: Additional studies will be necessary to further explore the role of oncometabolites in paracrine signaling and to identify genuine metabolic and nutritional liabilities imposed by TCA cycle dysfunction in cancer, hardly to be escaped by resistance mechanisms.

Mannose impairs tumour growth and enhances chemotherapy.

It is now well established that tumours undergo changes in cellular metabolism1. As this can reveal tumour cell vulnerabilities and because many tumours exhibit enhanced glucose uptake2, we have been interested in how tumour cells respond to different forms of sugar. Here we report that the monosaccharide mannose causes growth retardation in several tumour types in vitro, and enhances cell death in response to major forms of chemotherapy. We then show that these effects also occur in vivo in mice following the oral administration of mannose, without significantly affecting the weight and health of the animals. Mechanistically, mannose is taken up by the same transporter(s) as glucose3 but accumulates as mannose-6-phosphate in cells, and this impairs the further metabolism of glucose in glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway and glycan synthesis. As a result, the administration of mannose in combination with conventional chemotherapy affects levels of anti-apoptotic proteins of the Bcl-2 family, leading to sensitization to cell death. Finally we show that susceptibility to mannose is dependent on the levels of phosphomannose isomerase (PMI). Cells with low levels of PMI are sensitive to mannose, whereas cells with high levels are resistant, but can be made sensitive by RNA-interference-mediated depletion of the enzyme. In addition, we use tissue microarrays to show that PMI levels also vary greatly between different patients and different tumour types, indicating that PMI levels could be used as a biomarker to direct the successful administration of mannose. We consider that the administration of mannose could be a simple, safe and selective therapy in the treatment of cancer, and could be applicable to multiple tumour types.

S-nitrosylation drives cell senescence and aging in mammals by controlling mitochondrial dynamics and mitophagy.

S-nitrosylation, a prototypic redox-based posttranslational modification, is frequently dysregulated in disease. S-nitrosoglutathione reductase (GSNOR) regulates protein S-nitrosylation by functioning as a protein denitrosylase. Deficiency of GSNOR results in tumorigenesis and disrupts cellular homeostasis broadly, including metabolic, cardiovascular, and immune function. Here, we demonstrate that GSNOR expression decreases in primary cells undergoing senescence, as well as in mice and humans during their life span. In stark contrast, exceptionally long-lived individuals maintain GSNOR levels. We also show that GSNOR deficiency promotes mitochondrial nitrosative stress, including excessive S-nitrosylation of Drp1 and Parkin, thereby impairing mitochondrial dynamics and mitophagy. Our findings implicate GSNOR in mammalian longevity, suggest a molecular link between protein S-nitrosylation and mitochondria quality control in aging, and provide a redox-based perspective on aging with direct therapeutic implications.

Fumarate is an epigenetic modifier that elicits epithelial-to-mesenchymal transition.

Mutations of the tricarboxylic acid cycle enzyme fumarate hydratase cause hereditary leiomyomatosis and renal cell cancer. Fumarate hydratase-deficient renal cancers are highly aggressive and metastasize even when small, leading to a very poor clinical outcome. Fumarate, a small molecule metabolite that accumulates in fumarate hydratase-deficient cells, plays a key role in cell transformation, making it a bona fide oncometabolite. Fumarate has been shown to inhibit α-ketoglutarate-dependent dioxygenases that are involved in DNA and histone demethylation. However, the link between fumarate accumulation, epigenetic changes, and tumorigenesis is unclear. Here we show that loss of fumarate hydratase and the subsequent accumulation of fumarate in mouse and human cells elicits an epithelial-to-mesenchymal-transition (EMT), a phenotypic switch associated with cancer initiation, invasion, and metastasis. We demonstrate that fumarate inhibits Tet-mediated demethylation of a regulatory region of the antimetastatic miRNA cluster mir-200ba429, leading to the expression of EMT-related transcription factors and enhanced migratory properties. These epigenetic and phenotypic changes are recapitulated by the incubation of fumarate hydratase-proficient cells with cell-permeable fumarate. Loss of fumarate hydratase is associated with suppression of miR-200 and the EMT signature in renal cancer and is associated with poor clinical outcome. These results imply that loss of fumarate hydratase and fumarate accumulation contribute to the aggressive features of fumarate hydratase-deficient tumours.

S-nitrosylation of the Mitochondrial Chaperone TRAP1 Sensitizes Hepatocellular Carcinoma Cells to Inhibitors of Succinate Dehydrogenase.

S-nitrosoglutathione reductase (GSNOR) represents the best-documented denitrosylase implicated in regulating the levels of proteins posttranslationally modified by nitric oxide on cysteine residues by S-nitrosylation. GSNOR controls a diverse array of physiologic functions, including cellular growth and differentiation, inflammation, and metabolism. Chromosomal deletion of GSNOR results in pathologic protein S-nitrosylation that is implicated in human hepatocellular carcinoma (HCC). Here we identify a metabolic hallmark of aberrant S-nitrosylation in HCC and exploit it for therapeutic gain. We find that hepatocyte GSNOR deficiency is characterized by mitochondrial alteration and by marked increases in succinate dehydrogenase (SDH) levels and activity. We find that this depends on the selective S-nitrosylation of Cys(501) in the mitochondrial chaperone TRAP1, which mediates its degradation. As a result, GSNOR-deficient cells and tumors are highly sensitive to SDH inhibition, namely to α-tocopheryl succinate, an SDH-targeting molecule that induced RIP1/PARP1-mediated necroptosis and inhibited tumor growth. Our work provides a specific molecular signature of aberrant S-nitrosylation in HCC, a novel molecular target in SDH, and a first-in-class therapy to treat the disease. Cancer Res; 76(14); 4170-82. ©2016 AACR.

S-Nitrosoglutathione Reductase Plays Opposite Roles in SH-SY5Y Models of Parkinson's Disease and Amyotrophic Lateral Sclerosis.

Oxidative and nitrosative stresses have been reported as detrimental phenomena concurring to the onset of several neurodegenerative diseases. Here we reported that the ectopic modulation of the denitrosylating enzyme S-nitrosoglutathione reductase (GSNOR) differently impinges on the phenotype of two SH-SY5Y-based in vitro models of neurodegeneration, namely, Parkinson's disease (PD) and familial amyotrophic lateral sclerosis (fALS). In particular, we provide evidence that GSNOR-knocking down protects SH-SY5Y against PD toxins, while, by contrast, its upregulation is required for G93A-SOD1 expressing cells resistance to NO-releasing drugs. Although completely opposite, both conditions are characterized by Nrf2 localization in the nuclear compartment: in the first case induced by GSNOR silencing, while in the second one underlying the antinitrosative response. Overall, our results demonstrate that GSNOR expression has different effect on neuronal viability in dependence on the stimulus applied and suggest that GSNOR could be a responsive gene downstream of Nrf2 activation.

Pyruvate carboxylation enables growth of SDH-deficient cells by supporting aspartate biosynthesis.

Succinate dehydrogenase (SDH) is a heterotetrameric nuclear-encoded complex responsible for the oxidation of succinate to fumarate in the tricarboxylic acid cycle. Loss-of-function mutations in any of the SDH genes are associated with cancer formation. However, the impact of SDH loss on cell metabolism and the mechanisms enabling growth of SDH-defective cells are largely unknown. Here, we generated Sdhb-ablated kidney mouse cells and used comparative metabolomics and stable-isotope-labelling approaches to identify nutritional requirements and metabolic adaptations to SDH loss. We found that lack of SDH activity commits cells to consume extracellular pyruvate, which sustains Warburg-like bioenergetic features. We further demonstrated that pyruvate carboxylation diverts glucose-derived carbons into aspartate biosynthesis, thus sustaining cell growth. By identifying pyruvate carboxylase as essential for the proliferation and tumorigenic capacity of SDH-deficient cells, this study revealed a metabolic vulnerability for potential future treatment of SDH-associated malignancies.

Fumarate induces redox-dependent senescence by modifying glutathione metabolism.

Mutations in the tricarboxylic acid (TCA) cycle enzyme fumarate hydratase (FH) are associated with a highly malignant form of renal cancer. We combined analytical chemistry and metabolic computational modelling to investigate the metabolic implications of FH loss in immortalized and primary mouse kidney cells. Here, we show that the accumulation of fumarate caused by the inactivation of FH leads to oxidative stress that is mediated by the formation of succinicGSH, a covalent adduct between fumarate and glutathione. Chronic succination of GSH, caused by the loss of FH, or by exogenous fumarate, leads to persistent oxidative stress and cellular senescence in vitro and in vivo. Importantly, the ablation of p21, a key mediator of senescence, in Fh1-deficient mice resulted in the transformation of benign renal cysts into a hyperplastic lesion, suggesting that fumarate-induced senescence needs to be bypassed for the initiation of renal cancers.

MAPK14/p38α-dependent modulation of glucose metabolism affects ROS levels and autophagy during starvation.

Increased glycolytic flux is a common feature of many cancer cells, which have adapted their metabolism to maximize glucose incorporation and catabolism to generate ATP and substrates for biosynthetic reactions. Indeed, glycolysis allows a rapid production of ATP and provides metabolic intermediates required for cancer cells growth. Moreover, it makes cancer cells less sensitive to fluctuations of oxygen tension, a condition usually occurring in a newly established tumor environment. Here, we provide evidence for a dual role of MAPK14 in driving a rearrangement of glucose metabolism that contributes to limiting reactive oxygen species (ROS) production and autophagy activation in condition of nutrient deprivation. We demonstrate that MAPK14 is phosphoactivated during nutrient deprivation and affects glucose metabolism at 2 different levels: on the one hand, it increases SLC2A3 mRNA and protein levels, resulting in a higher incorporation of glucose within the cell. This event involves the MAPK14-mediated enhancement of HIF1A protein stability. On the other hand, MAPK14 mediates a metabolic shift from glycolysis to the pentose phosphate pathway (PPP) through the modulation of PFKFB3 (6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase 3) degradation by the proteasome. This event requires the presence of 2 distinct degradation sequences, KEN box and DSG motif Ser273, which are recognized by 2 different E3 ligase complexes. The mutation of either motif increases PFKFB3 resistance to starvation-induced degradation. The MAPK14-driven metabolic reprogramming sustains the production of NADPH, an important cofactor for many reduction reactions and for the maintenance of the proper intracellular redox environment, resulting in reduced levels of ROS. The final effect is a reduced activation of autophagy and an increased resistance to nutrient deprivation.

S-nitrosoglutathione reductase deficiency-induced S-nitrosylation results in neuromuscular dysfunction.

AIMS: Nitric oxide (NO) production is implicated in muscle contraction, growth and atrophy, and in the onset of neuropathy. However, many aspects of the mechanism of action of NO are not yet clarified, mainly regarding its role in muscle wasting. Notably, whether NO production-associated neuromuscular atrophy depends on tyrosine nitration or S-nitrosothiols (SNOs) formation is still a matter of debate. Here, we aim at assessing this issue by characterizing the neuromuscular phenotype of S-nitrosoglutathione reductase-null (GSNOR-KO) mice that maintain the capability to produce NO, but are unable to reduce SNOs. RESULTS: We demonstrate that, without any sign of protein nitration, young GSNOR-KO mice show neuromuscular atrophy due to loss of muscle mass, reduced fiber size, and neuropathic behavior. In particular, GSNOR-KO mice show a significant decrease in nerve axon number, with the myelin sheath appearing disorganized and reduced, leading to a dramatic development of a neuropathic phenotype. Mitochondria appear fragmented and depolarized in GSNOR-KO myofibers and myotubes, conditions that are reverted by N-acetylcysteine treatment. Nevertheless, although atrogene transcription is induced, and bulk autophagy activated, no removal of damaged mitochondria is observed. These events, alongside basal increase of apoptotic markers, contribute to persistence of a neuropathic and myopathic state. INNOVATION: Our study provides the first evidence that GSNOR deficiency, which affects exclusively SNOs reduction without altering nitrotyrosine levels, results in a clinically relevant neuromuscular phenotype. CONCLUSION: These findings provide novel insights into the involvement of GSNOR and S-nitrosylation in neuromuscular atrophy and neuropathic pain that are associated with pathological states; for example, diabetes and cancer.

TCA Cycle Defects and Cancer: When Metabolism Tunes Redox State.

Inborn defects of the tricarboxylic acid (TCA) cycle enzymes have been known for more than twenty years. Until recently, only recessive mutations were described which, although resulted in severe multisystem syndromes, did not predispose to cancer onset. In the last ten years, a causal role in carcinogenesis has been documented for inherited and acquired alterations in three TCA cycle enzymes, succinate dehydrogenase (SDH), fumarate hydratase (FH), and isocitrate dehydrogenase (IDH), pointing towards metabolic alterations as the underlying hallmark of cancer. This paper summarizes the neoplastic alterations of the TCA cycle enzymes focusing on the generation of pseudohypoxic phenotype and the alteration of epigenetic homeostasis as the main tumor-promoting effects of the TCA cycle affecting defects. Moreover, we debate on the ability of these mutations to affect cellular redox state and to promote carcinogenesis by impacting on redox biology.

Deprive to kill: glutamine closes the gate to anticancer monocarboxylic drugs.

Killing properties of antitumor drugs can be enhanced by strategies targeting biochemical adaptations of cancer cells. Recently, we reported that depriving cancer cells of glutamine is a feasible approach to enhance antitumor effects of the alkylating analog of pyruvic acid, 3-bromopyruvate, which rely on the induction of autophagic cell death by metabolic-oxidative stress. 3-bromopyruvate chemopotentiation is the result of its increased intracellular uptake mediated by the monocarboxylate transporter 1, whose expression is post-transcriptionally increased upon glutamine withdrawal. Overall, our results identified the metabolic condition able to increase the selectivity of 3-bromopyruvate targets in neoplastic tissues, thereby providing a stage for its use in clinical settings for targeting malignancies and represent a proof of principle that modulation of glutamine availability can influence the delivery of monocarboxylic drugs into tumors.

Glutamine deprivation enhances antitumor activity of 3-bromopyruvate through the stabilization of monocarboxylate transporter-1.

Anticancer drug efficacy might be leveraged by strategies to target certain biochemical adaptations of tumors. Here we show how depriving cancer cells of glutamine can enhance the anticancer properties of 3-bromopyruvate, a halogenated analog of pyruvic acid. Glutamine deprival potentiated 3-bromopyruvate chemotherapy by increasing the stability of the monocarboxylate transporter-1, an effect that sensitized cells to metabolic oxidative stress and autophagic cell death. We further elucidated mechanisms through which resistance to chemopotentiation by glutamine deprival could be circumvented. Overall, our findings offer a preclinical proof-of-concept for how to employ 3-bromopyruvate or other monocarboxylic-based drugs to sensitize tumors to chemotherapy.