Cell Metabolism

Mitochondrial dysfunction has long been associated with insulin resistance, yet the causal relationship in humans remains unresolved. Here, we leveraged individuals carrying the diabetogenic m.3243A>G mtDNA mutation as a human genetic model to probe the causal contribution of mitochondrial defects to insulin resistance and delineate the underlying molecular and bioenergetic mechanisms. In vivo metabolic phenotyping revealed selective skeletal muscle insulin resistance with preserved liver and adipose insulin sensitivity, accompanied by impaired glucose tolerance, {beta}-cell dysfunction, and elevated circulating levels of the mitochondrial stress-responsive cytokine GDF15. At the molecular level, this muscle-specific insulin-resistant phenotype featured preserved insulin-stimulated Akt-TBC1D4 signaling but blunted mTORC1 activation. Integrated muscle proteomic and bioenergetic profiling demonstrated reduced mitochondrial protein abundance and complex I-specific molecular and functional impairments, alongside downregulation of non-mitochondrial metabolic proteins including AMPK{gamma}2. In summary, our study establishes a mitochondrial basis for insulin resistance in humans, linking reduced mitochondrial content and complex I-related defects to disrupted mTORC1 signaling and impaired muscle insulin action. These findings highlight mitochondria-dependent molecular signatures of insulin resistance that may hold translational relevance for improving glucose regulation in common metabolic diseases. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/25342274v1_ufig1.gif" ALT="Figure 1"> View larger version (75K): org.highwire.dtl.DTLVardef@7b7f35org.highwire.dtl.DTLVardef@1dc7a0dorg.highwire.dtl.DTLVardef@1d121c7org.highwire.dtl.DTLVardef@10094da_HPS_FORMAT_FIGEXP M_FIG GRAPHICAL ABSTRACT C_FIG

Pyruvate is a critical intermediary metabolite in gluconeogenesis, lipogenesis, as well as NADH production. As a result, there is growing interest in targeting the mitochondrial pyruvate carrier (MPC) complex in liver and metabolic diseases. However, recent in vitro data indicate that MPC inhibition diverts glutamine/glutamate away from glutathione synthesis and toward glutaminolysis to compensate for loss of pyruvate oxidation, possibly sensitizing cells to oxidative insult. Here, we explored this using the clinically relevant acetaminophen (APAP) overdose model of acute liver injury, which is driven by oxidative stress. We report that MPC inhibition does indeed sensitize the liver to APAP-induced injury in vivo, but only with concomitant loss of alanine aminotransferase 2 (ALT2). Pharmacologic and genetic manipulation of neither MPC2 nor ALT2 alone affected APAP toxicity, but liver-specific double knockout (DKO) of these proteins significantly worsened the liver damage. Further investigation confirmed that DKO impaired glutathione synthesis and increased urea cycle flux, consistent with increased glutaminolysis. Furthermore, APAP toxicity was exacerbated by inhibition of both the MPC and ALT in vitro. Thus, increased glutaminolysis and susceptibility to oxidative stress requires loss of both the MPC and ALT2 in vivo and exacerbates them in vitro. Finally, induction of ALT2 reduced APAP-induced injury.

About 20-30% of cancer-associated deaths are due to complications from cachexia which is characterized by skeletal muscle atrophy. Metabolic reprogramming in cancer cells causes body-wide metabolic and proteomic remodeling, which remain poorly understood. Here, we present evidence that the oncometabolite D-2-hydroxylgutarate (D2-HG) impairs NAD+ redox homeostasis in skeletal myotubes, causing atrophy via deacetylation of LC3-II by the nuclear deacetylase Sirt1. Overexpression of p300 or silencing of Sirt1 abrogate its interaction with LC3, and subsequently reduced levels of LC3 lipidation. Using RNA-sequencing and mass spectrometry-based metabolomics and proteomics, we demonstrate that prolonged treatment with the oncometabolite D2-HG in mice promotes cachexia in vivo and increases the abundance of proteins and metabolites, which are involved in energy substrate metabolism, chromatin acetylation and autophagy regulation. We further show that D2-HG promotes a sex-dependent adaptation in skeletal muscle using network modeling and machine learning algorithms. Our multi-omics approach exposes new metabolic vulnerabilities in response to D2-HG in skeletal muscle and provides a conceptual framework for identifying therapeutic targets in cachexia.

Cancer cachexia is an involuntary weight loss condition characterized by systemic metabolic disorder. A comprehensive flux characterization of this condition however is lacking. Here, we systematically isotope traced eight major circulating nutrients in mice bearing cachectic C26 tumors (cxC26) and food intake-matched mice bearing non-cachectic C26 tumors (ncxC26). We found no difference in whole-body lipolysis and proteolysis, ketogenesis, or fatty acid and ketone oxidation by tissues between the two groups. In contrast, compared to ncxC26 mice ad libitum, glucose turnover flux decreased in food intake-controlled ncxC26 mice but not in cxC26 mice. Similarly, sustained glucose turnover flux was observed in two autochthonous cancer cachexia models despite reduced food intake. We identified glutamine and alanine as responsible for sustained glucose production and tissues with altered use of glucose and lactate in cxC26 mice. We provide a comprehensive view of metabolic alterations in cancer cachexia revealing those distinct from decreased nutrient intake. HighlightsO_LIQuantitative fluxomics of cancer cachexia under matched food intake and body weight C_LIO_LIIntact lipolysis, proteolysis, ketogenesis, and lipid oxidation in cachectic mice C_LIO_LISustained glucose consumption in cachectic mice despite reduced food intake C_LIO_LIIncreased glucose production from glutamine and alanine in cachectic mice C_LI

Individual tissues perform highly specialized metabolic functions to maintain whole-body metabolic homeostasis. Although Drosophila serves as a powerful model for studying human metabolic diseases, modeling tissue-specific metabolism has been limited in this organism. To address this gap, we reconstruct 32 tissue-specific genome-scale metabolic models (GEMs) by integrating a curated Drosophila metabolic network with pseudo-bulk single-nuclei transcriptomics data, revealing distinct metabolic network structures and subsystem coverage across tissues. We validate enriched pathways identified through tissue-specific GEMs, particularly in muscle and fat body, using metabolomics and pathway analysis. Moreover, to demonstrate the utility in disease modeling, we apply muscle-GEM to investigate high sugar diet (HSD)-induced metabolic dysregulation. Constraint-based semi-quantitative flux and sensitivity analyses identify altered NAD(H)-dependent reactions and distributed control of glycolytic flux, including GAPDH. This prediction is further validated through in vivo 13C-glucose isotope tracing study. Notably, decreased glycolytic flux, including GAPDH, is linked to increased redox modifications. Finally, our pathway-level flux analyses identify dysregulation in fructose metabolism. Together, this work establishes a quantitative framework for tissue-specific metabolic modeling in Drosophila, demonstrating its utility for identifying dysregulated reactions and pathways in muscle in response to HSD.

Skeletal muscle contains a resident population of somatic stem cells capable of both self-renewal and differentiation. The signals that regulate this important decision have yet to be fully elucidated. Here we use metabolomics and mass spectrometry imaging (MSI) to identity a state of localized hyperglycaemia following skeletal muscle injury. We show that committed muscle progenitor cells exhibit an enrichment of glycolytic and TCA cycle genes and that extracellular monosaccharide availability regulates intracellular citrate levels and global histone acetylation. Muscle stem cells exposed to a reduced (or altered) monosaccharide environment demonstrate reduced global histone acetylation and transcription of myogenic determination factors (including myod1). Importantly, reduced monosaccharide availability was linked directly to increased rates of asymmetric division and muscle stem cell self-renewal in regenerating skeletal muscle. Our results reveal an important role for the extracellular metabolic environment in the decision to undergo self-renewal or myogenic commitment during skeletal muscle regeneration.

Type 1 diabetes (T1D) results from autoimmune destruction of the insulin producing pancreatic {beta} cells. The bodys lipid metabolism is strongly regulated during this process but there is a need to understand how this regulation contributes to the {beta}-cell death. Here, we show that fatty acids are released from plasma lipoproteins in children during islet autoimmunity, prior to T1D onset. These fatty acids (FFAs) enhanced cytokine-mediated apoptosis in cultured insulin-producing cells by downregulating the production of nicotinamide adenosine dinucleotide (NAD) via its salvage pathway, as well as deregulated central carbon metabolism and impaired levels of ATP. Downregulation of the NAD salvage pathway and central carbon metabolism enzymes were further observed during T1D development, supporting that the pathways for NAD and energy production are compromised in vivo. Our findings show that fatty acids are released during islet autoimmunity, accelerating disease development through impaired NAD metabolism.

The molecular basis of caloric restriction (CR) has been defined primarily at a metabolic steady state, leaving the initiating events that drive the transition from ad libitum feeding to an adapted CR state largely unresolved. Here, we combine continuous indirect calorimetry with longitudinal bulk RNA-seq of liver and inguinal white adipose tissue (iWAT) sampled at six circadian timepoints across four stages of adaptation to 30% CR in male C57BL/6J mice. We show that whole-body metabolic adaptation proceeds through two discrete adaptive phases separated by a threshold at approximately 14 days; during this initial transition, consolidated feeding attenuates ketogenesis, establishing a distinct whole-body metabolic phenotype prior to long-term adaptation. To elucidate the molecular mechanisms underlying these physiological shifts, weighted gene co-expression network analysis (WGCNA) was performed, revealing that hepatic transcriptional remodeling is organized proportionally to fasting duration, whereas iWAT remodeling remains restricted to specific circadian timepoints. Because systemic adaptation requires coordinated inter-tissue communication, we conducted a cartographic analysis to evaluate network topology and inter-modular connectivity. This approach identifies restricted populations of early kinless and connector hub genes, nucleated by Casp3 in the liver and Lpl in iWAT, whose structural integration is established prior to the broader transcriptional remodeling observed at later timepoints. Functional annotation indicates the hepatic hub network is enriched for mitochondrial bioenergetics and FOXO/TP53-mediated transcription, while the iWAT hub network exhibits a bifurcated enrichment spanning ribosomal biosynthesis and immune-regulatory signaling. Although these tissues exhibit distinct transcriptional profiles, projecting both datasets onto a shared phenotypic eigenspace reveals a unified systemic response; as CR is maintained, dynamically regulated transcripts in both liver and iWAT converge on an adiponectin-coupled state. Ultimately, the identification of adiponectin as an integrative signal coordinating chronic adaptation across metabolically distinct tissues delineates the temporal sequence of early CR adaptation; furthermore, it establishes a mechanistic framework defining how early molecular and physiological shifts converge to achieve steady-state metabolic homeostasis.

Caloric restriction (CR) extends organismal lifespan and health span by improving glucose homeostasis mechanisms. How CR affects organellar structure and function of pancreatic beta cells over the lifetime of the animal remains unknown. Here, we used single nucleus transcriptomics to show that CR increases the expression of genes for beta cell identity, protein processing, and organelle homeostasis. Gene regulatory network analysis link this transcriptional phenotype to transcription factors involved in beta cell identity (Mafa) and homeostasis (Atf6). Imaging metabolomics further demonstrates that CR beta cells are more energetically competent. In fact, high-resolution light and electron microscopy indicates that CR reduces beta cell mitophagy and increases mitochondria mass, increasing mitochondrial ATP generation. Finally, we show that long-term CR delays the onset of beta cell aging and senescence to promote longevity by reducing beta cell turnover. Therefore, CR could be a feasible approach to preserve compromised beta cells during aging and diabetes.

Intermuscular adipose tissue (IMAT) expansion is closely associated with cardiometabolic disease, yet its cellular organization and regulatory mechanisms remain poorly defined. Here, we define a human IMAT gene signature using bulk transcriptomics and identify candidate regulators for IMAT function including adipogenic transcription factor early B-cell factor 2 (EBF2). To determine how these programs are organized in situ, we mapped this signature in a mouse model of diet-induced CMD using spatial transcriptomics. We found that IMAT expansion occurs within discrete stromal niches surrounding muscle fibers, characterized by coordinated activation of adipogenic, extracellular matrix, inflammatory, and metabolic pathways. Spatial analyses showed that fibro-adipogenic progenitor (FAP) abundance does not predict adipocyte formation, supporting a model of localized and context-dependent lineage transitions. Cross-species comparison revealed partial conservation of human IMAT gene programs, validating the mouse model and highlighting species-specific features. Functional experiments in human primary myoblasts showed that EBF2 is sufficient to induce adipogenic reprogramming. Our findings establish IMAT as an active, spatially organized remodeling niche and identify lineage plasticity as a central mechanism driving its expansion in metabolic disease

Obesity and metabolic dysfunction are among the strongest risk factors for poor brain and mental health, yet the neural mechanisms linking metabolism, brain, and behaviour remains poorly understood. Here, we provide the first evidence for two distinct large-scale brain network configurations--one associated with metabolic health and another with obesity-- identified using resting-state fMRI data and metabolic phenotypes from a large community cohort (N = 564). While obesity was linked to enhanced coupling between subcortical reward and higher-order cortical networks, metabolic health was characterized by functional integration among default mode, salience, and frontoparietal control regions (metabolic health functional connectivity; MHFC). The MHFC network mediated the relationship between eating restraint and metabolic health, independent on individuals body weight and metabolic status, and it was replicated with data from a different time point. Longitudinal analysis showed that change of MHFC strength predicted metabolic indicators over time, suggesting a role for this network as a potential marker of metabolic resilience. These findings reveal a neurobiological pathway through which executive and interoceptive regulatory systems contribute to metabolic health, offering new insights into the brain mechanisms linking eating behaviour, metabolism, and brain function.

Obesity remains a major global health challenge with limited durable pharmacotherapies. Disulfiram (DSF), an FDA-approved drug reported to inhibit gasdermin D (GSDMD), has been proposed to improve metabolic outcomes through suppression of inflammasome signaling. Here, we demonstrate that GSDMD is dispensable for high-fat diet-induced obesity and insulin resistance, as neither genetic deletion nor antisense-mediated inhibition of GSDMD confers metabolic protection. In contrast, DSF robustly protects against obesity and IR through a GSDMD-independent mechanism. These effects are not attributable to reduced caloric intake but instead reflect a coordinated reprogramming of systemic lipid handling. Under steady-state conditions, DSF suppresses basal lipid oxidation while promoting fecal fatty acid excretion. In striking contrast, during acute lipid challenge, DSF enhances tissue lipid utilization and accelerates systemic clearance. Together, these findings overturn the prevailing inflammasome-centric model and establish context-dependent regulation of lipid partitioning--rather than inflammasome inhibition--as the primary mechanism underlying DSFs anti-obesity effects

There is an increasing interest in the pathway of L-serine synthesis and its. Although L-serine and downstream products can be obtained from the diet, serine deficiency has been documented in neurological disorders, macular degeneration and aging. This evidence calls for strategies to induce serine synthesis. Here I address this problem taking advantage of the wealth of data deposited in the gene expression omnibus database. I uncover that low protein and ketogenic diets increase the expression of serine synthesis genes in the liver and the brain relative to control diets. I discover oestrogen medications, the antifolate methotrexate and serine synthesis inhibitors as classes of compounds inducing the expression of serine synthesis genes in the liver. Future work is required to investigate the use of these interventions for the management of serine deficiency disorders.Competing Interest StatementThe authors have declared no competing interest.View Full Text

Metabolic diseases including type 2 diabetes and obesity pose a significant global health burden. Plant-based diets, including vegan diets, are linked to favorable metabolic outcomes, yet the underlying mechanisms remain unclear. In a randomized trial involving 21 pairs of identical twins, we investigated the effects of vegan and omnivorous diets on the host metabolome, immune system, and gut microbiome. Vegan diets induced significant shifts in serum and stool metabolomes, cytokine profiles, and gut microbial composition. Despite lower dietary glycine intake, vegan diet subjects exhibited elevated serum glycine levels linked to reduced abundance of the gut pathobiont Bilophila wadsworthia. Functional studies demonstrated that B. wadsworthia metabolizes glycine via the glycine reductase pathway and modulates host glycine availability. Removing B. wadsworthia from a complex microbiota in mice elevated glycine levels and improved metabolic markers. These findings reveal a previously underappreciated mechanism by which diet regulates host metabolic status via the gut microbiota.

Management of patients with mitochondrial respiratory chain diseases is challenging, in part because of our incomplete understanding of pathogenesis and a lack of biomarkers. Unknown metabolites account for >90% of detected features in modern metabolomics experiments and hold immense untapped promise for new basic and biomedical research. We recently used mass spectrometry-based metabolomics to identify and validate 19 circulating blood-based biomarkers for patients with the mitochondrial DNA (mtDNA) m.3243A>G pathogenic variant, which is the most frequent cause of the mitochondrial disorder MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes). However, the most significantly changing biomarker corresponded to an "unknown" metabolite. Here, we combine cheminformatics with analytical chemistry and identify that feature as 4,5-dihydroxyhexanoic acid (4,5-DHHA), a metabolite previously associated with inherited defects of gamma-aminobutyric acid (GABA) catabolism, but with no prior links to mitochondrial respiratory chain disorders. We validate this finding in an independent MELAS cohort and further show that 4,5-DHHA levels correlate with disease severity and are elevated in patients with other forms of mitochondrial disease and sepsis. Furthermore, brain 4,5-DHHA levels were elevated in two genetic mouse models of mitochondrial disease. In vitro and tissue culture experiments indicate that 4,5-DHHA is generated when the GABA catabolite succinic semialdehyde reacts with an intermediate of the pyruvate dehydrogenase reaction and is sensitive to mitochondrial complex I function. Our work identifies 4,5-DHHA as a robust plasma and urine marker of mitochondrial dysfunction in humans and reveals new connections between the respiratory chain and GABA metabolism. Significance StatementInborn errors of the mitochondrial respiratory chain cause severe, progressive diseases, yet effective treatments and biomarkers remain limited. Modern metabolomics detects thousands of molecules in biofluids, but the vast majority are unidentified. In this study, we investigate the most significantly altered blood metabolite in patients with the most common mitochondrial disease - MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) - and identify it as an 4,5-dihydroxyhexanoic acid (4,5-DHHA). We show that 4,5-DHHA is reproducibly elevated and correlates with severity. Levels are increased across multiple mitochondrial disorders as well as in sepsis and rise when respiratory chain function is impaired. These findings establish 4,5-DHHA as a promising biomarker of mitochondrial dysfunction and reveal a link to dysregulated GABA metabolism.

The one-carbon metabolic pathway is essential for proliferating cells and has recently been identified as an immunomodulatory target in CD4 T cells. However, its role in other immune cell types has not been fully established. We investigated the function of the one-carbon pathway in CD8 T cells, which are the primary effectors responsible for the destruction of pancreatic beta cells that causes type 1 diabetes. Enzymes involved in the one-carbon pathway, as well as levels of formate--a critical intermediate--were upregulated during CD8 T-cell activation. Pharmacological inhibition of MTHFD2, a mitochondrial enzyme involved in one-carbon metabolism, suppressed CD8 T-cell activation, proliferation, and effector function. Mechanistically, this effect was mediated by reduced signaling through KRAS and the mTORC1 downstream targets HIF1, S6, and STAT3. As previously shown in CD4 T cells, formate supplementation reversed the effects of MTHFD2 inhibition on activation, proliferation, and function of CD8+ T cells, and prevented the reduction of the TCF1high CD8 progenitor cell population, which has been shown to drive anti-beta cell autoimmunity. Formate levels were elevated in the immune cells isolated from pancreatic lymph nodes during the insulitis stage in non-obese diabetic mice. Treatment of euglycemic non-obese diabetic mice with an MTHFD2 inhibitor during the insulitis stage delayed CD8 T-cell infiltration into pancreatic islets and postponed the onset of type 1 diabetes. These findings reveal a new paradigm for preventing and delaying the onset of type 1 diabetes.

Mitochondria warrant cellular energy demands by generating energy equivalents in central carbon metabolism. They are also able to newly synthesize fatty acids via mitochondrial fatty acid synthesis (mtFAS), however, the role of mtFAS for systemic metabolism has been poorly investigated. Here we show that mitochondrial Trans-2-Enoyl-CoA Reductase (MECR), a key enzyme of mtFAS, critically regulates cellular and systemic glucose and lipid homeostasis. In mice, liver or adipose tissue-specific deletion of Mecr reduces the capacity for aerobic glycolytic catabolism and lipogenesis and causes severe mitochondrial as well as fatal parenchymal organ dysfunction. Mechanistically, mtFAS is essential for pyruvate dehydrogenase activity, resulting in low NAD(P)H synthesis and reduced non-mitochondrial lipogenesis. In different human mitochondriopathies we further identify a dysregulation of mtFAS-associated lipid species, thus linking inherited mitochondrial disease to mtFAS. In summary, we introduce mtFAS as an important player in metabolic health via facilitating cellular glycolysis-derived metabolite transformation ultimately linking mtFAS to mitochondrial function and diseases.

Ergothioneine (EGT) is a diet-derived, atypical amino acid that accumulates to high levels in human tissues. Reduced EGT levels have been linked to age-related disorders, including neurodegenerative and cardiovascular diseases, while EGT supplementation is protective in a broad range of disease and aging models in mice. Despite these promising data, the direct and physiologically relevant molecular target of EGT has remained elusive. Here we use a systematic approach to identify how mitochondria remodel their metabolome in response to exercise training. From this data, we find that EGT accumulates in muscle mitochondria upon exercise training. Proteome-wide thermal stability studies identify 3-mercaptopyruvate sulfurtransferase (MPST) as a direct molecular target of EGT; EGT binds to and activates MPST, thereby boosting mitochondrial respiration and exercise training performance in mice. Together, these data identify the first physiologically relevant EGT target and establish the EGT-MPST axis as a molecular mechanism for regulating mitochondrial function and exercise performance.

Adaptive thermogenesis is a key homeostatic mechanism that primarily occurs in brown adipocytes and enables the maintenance of body temperature. Although this process involves coordinated responses in multiple tissues, including the browning of white adipocytes, the precise inter-organ crosstalk underlying adaptive thermogenesis is unclear. Here, we investigate the pivotal role of the GDNF family receptor alpha-like (GFRAL) neuronal axis in modulating compensatory thermogenic responses in brown and white adipose depots under stress conditions, specifically the mitochondrial unfolded protein response resulting from genetic modification and cold exposure. We employed a mouse model with targeted deletion of Crif1 in the mitoribosomes of brown adipocytes, and cold-exposed mice and immortalized adipocytes, to uncover the mechanism by which mitochondrial stress-induced growth differentiation factor 15 (GDF15) expression affects metabolism and facilitates adaptive thermogenesis. We found that Crif1 deletion resulted in browning of inguinal white adipose depots, increased energy expenditure, reduced food intake, and resistance to weight gain. Retrograde neuronal tracing established that GFRAL-positive neurons in the hindbrain and sympathetic preganglionic neurons in the spinal cord mediated the GDF15-associated browning of inguinal white adipose tissue. Intervention studies using antisense oligonucleotides to inhibit Gfral expression blunted the effect of Crif1 deletion on energy expenditure and food intake, further confirming the essential role the GFRAL axis plays in GDF15-driven thermogenic adaptation in white adipose tissue. Our findings suggest that the GFRAL neuronal axis is key in coordinating the adaptive thermogenic response across multiple tissues and adipose depots, thereby ensuring metabolic homeostasis during mitochondrial stress.

Hepatic gluconeogenesis (GNG) is essential for maintaining euglycemia during prolonged fasting. However, GNG becomes pathologically elevated and drives chronic hyperglycemia in type 2 diabetes (T2D). Lactate/pyruvate is a major GNG substrate known to be imported into mitochondria for GNG. Yet, the subsequent mitochondrial carbon export mechanisms required to supply the extra-mitochondrial canonical GNG pathway have not been genetically delineated. Here, we evaluated the role of the mitochondrial dicarboxylate carrier (DiC) in mediating GNG from lactate/pyruvate. We generated liver-specific DiC knockout (DiC LivKO) mice. During lactate/pyruvate tolerance tests, DiC LivKO decreased plasma glucose excursion and 13C-lactate/-pyruvate flux into hepatic and plasma glucose. In a Western diet (WD) feeding model of T2D, acute DiC LivKO after induction of obesity decreased lactate/pyruvate-driven GNG, hyperglycemia, and hyperinsulinemia. Our results show that mitochondrial carbon export through the DiC mediates GNG and that the DiC contributes to impaired glucose homeostasis in a mouse model of T2D.