Nature Metabolism

Adipocyte lipid metabolism is coordinated by circadian rhythms, diet, and environmental temperature. Yet how these diverse signals are molecularly integrated remains unknown. Here we show that clock, diet, and temperature cues converge on the orphan mitochondrial transporter, SLC25A34, to orchestrate thermogenic cycling of lipid synthesis and oxidation. During sleep, the clock suppresses Slc25a34 transcription through REV-ERB. Waking, lipid-rich diets, or cold exposure abolish this repression, allowing lipolytic signals to stimulate Slc25a34 expression via PPAR. SLC25A34 then imports oxaloacetate into mitochondria to accelerate the export of substrates used for acetyl-CoA production in the cytosol. This feeds into cytosolic lipid synthesis and transcriptional induction of mitochondrial biogenesis, which collectively promote mitochondrial lipid oxidation. Thus, SLC25A34 confers circadian, dietary, and environmental control of thermogenic metabolism through interorganellar lipid cycling.

The gut microbiota produces metabolites that circulate to host tissues and are increasingly linked to metabolic health, yet the mechanisms by which individual microbial products regulate liver glucose metabolism remain poorly defined. Here, we identify the tryptophan-derived microbial metabolite indole-3-propionic acid (IPA) as a direct modulator of hepatic glucose production. In primary hepatocytes, a focused screen of indole metabolites revealed that several indole-containing compounds suppress glucagon-stimulated glucose output, with IPA emerging as a physiologically relevant candidate. IPA selectively reduced glucose production from mitochondrial-dependent gluconeogenic substrates while largely preserving glycerol-supported glucose production, suggesting that it does not simply shut down gluconeogenesis but instead alters how hepatocytes use metabolic fuels. Mechanistic analyses showed that IPA redirects lactate-derived carbon away from glucose production and reshapes mitochondrial metabolism, including redox balance, ATP availability, and urea cycle-linked metabolic activity. These effects occurred without detectable disruption of proximal insulin or glucagon signaling, supporting a model in which IPA acts primarily through intracellular metabolic remodeling. In mice, endogenous IPA levels varied with nutritional state, and short-term IPA administration improved fasting glycemia and glucose handling in Western diet-fed animals. Finally, microbiome-depleted mice colonized with IPA-producing Clostridium sporogenes displayed increased circulating IPA and improved glucose tolerance compared with mice colonized with an IPA-deficient mutant C. Sporogenes strain. Together, these findings identify IPA as a microbial metabolite that directly connects gut tryptophan metabolism to hepatic mitochondrial function and systemic glucose regulation, highlighting a mechanistic gut-liver pathway with potential therapeutic relevance to metabolic disease.

The liver- cell axis is a finely tuned biological rheostat that regulates whole body amino acid availability. Pancreatic cells secrete glucagon that regulates amino acid catabolism through gluconeogenesis and ureagenesis, yet the mechanisms linking amino acid levels to cell growth and function are not fully understood. Here, we identify glutaminase, the enzyme that catalyzes glutamine catabolism, as a critical cell regulator. Glutaminase is highly enriched in cells across species. cell expression of glutaminase is required for nutrient-dependent mTORC1 activation, suppression of AMPK signaling, and sustained expression of the glutamine transporter SLC38A5. This establishes a feed-forward loop linking glutamine metabolism to amino acid sensing and growth. Reduced glutaminase activity impairs dynamic glucagon secretion in response to low glucose and amino acids. Together, these findings highlight the importance of glutamine metabolism in cell growth and hormone secretion and suggest it may play a role in cell adaptation to hyperaminoacidemia.

Coordination between innate immune signaling and glucose metabolism is fundamental to organismal homeostasis, yet despite decades of study linking immunity and metabolism, the mechanisms by which metabolic cells restrain antiviral innate signaling while preserving glycolytic competence during overnutrition remain poorly defined. Here we identify Tetherin (BST2) as a unique cell-intrinsic immunometabolic checkpoint that couples restraint of type I interferon (IFN-I) signaling to preservation of glycolytic capacity in adipocytes. Tetherin localizes to endoplasmic reticulum and organizes an interactome enriched for antiviral sensing regulators and glycolytic control nodes in adipocytes. Mechanistically, Tetherin directly engages the ubiquitin-dependent degradation machinery NDFIP1 and RNF128 to terminate IRF3 activation, thereby limiting pro-inflammatory, anti-glycolytic signaling and protecting adipocytes from metabolic dysfunction. In parallel, multiomics integration reveals that Tetherin also acts as a scaffold that binds and spatially organizes and activates PFKFB3 to increase glycolytic capacity and restrain MAVS-IRF3 innate immune signalling. In vivo, adipocyte-specific loss of Tetherin amplifies high sucrose diet and high-fat-diet-induced glucose intolerance and liver steatosis, whereas overexpression of human Tetherin in adipocyte suppresses obesity-driven interferon signaling, restores glycolytic pathway, and improves metabolic homeostasis. Orthogonal perturbations in cancer and insulinoma cells further confirm an immunometabolic role for Tetherin. Together, these findings define Tetherin as a dual node immunometabolic checkpoint that couples restraint of antiviral innate inflammatory signaling to maintenance of glycolytic competence, thereby safeguarding adipocyte metabolic homeostasis.

Mitochondria and lipid droplets (LDs) are functionally coupled to coordinate fatty acid utilization and storage. However, a comprehensive understanding of mitochondria-LD alliances remains elusive. We have identified a previously unrecognized role for optical atrophy 1 (OPA1), a mitochondrial fusion factor, in the regulation of fatty acid release from LDs. We demonstrated that OPA1s exon 4 adapts an amphipathic helix to target OPA1 to LDs. OPA1 localized to LDs promote fatty acid release by facilitating the recruitment of lipases to LDs. In addition, OPA1s residence on LDs competes with its mitochondrial entry, influencing mitochondria fusion and connectivity. Furthermore, the S158N polymorphism within OPA1s exon 4 exhibiting attenuated fatty acid release from LDs is associated with changes in metabolic traits in pediatric cancer survivors. Altogether, our findings reveal that OPA1 actively mediates fatty acid release from LDs and provide a mechanistic link between OPA1 and human metabolism.

Alzheimers disease and related tauopathies are escalating public health threats, particularly in the context of obesity and metabolic dysfunction, which accelerate cerebral glucose hypometabolism, tau pathology, neurodegeneration, and cognitive decline. Ketogenic therapies reconfigure systemic fuel metabolism, with emerging evidence for neuroprotection. (R,S)-1,3-butanediol (BD) raises circulating D- and L-{beta}-hydroxybutyrate ({beta}OHB) concentrations. To evaluate whether BD improves cognitive function across dietary contexts, male and female tau-transgenic mice and littermate controls received 10% BD in drinking water for 20 or 30 weeks starting at 6 weeks of age. BD rapidly induced ketosis (1.5-3.0 mM {beta}OHB) in chow-fed mice, with L-{beta}OHB contributing to [~]75% of the circulating {beta}OHB pool. Despite minimal effects of BD on body weight and glucose homeostasis, and no effect on histopathological tau signal, 20-week BD treatment improved memory to control levels in chow-fed female tauopathy mice. Isotope-tracing untargeted metabolomics revealed that BD-treatment differentially affected glucose-derived 13C-enrichment of metabolites in brains of male and female mice. BD-induced cognitive benefits in tau-transgenic mice were abrogated when mice were maintained on BD for 30 weeks on standard chow or when mice were administered BD over 20 weeks while maintained on a high-fat, Western diet, Notably, BD-induced ketosis was blunted in mice consuming Western diet. Moreover, intermittent ketogenic diet-induced ketosis failed to improve cognition in Western diet-fed tauopathy mice. These results suggest BD-induced ketosis extends cognitive benefits in a manner dependent on biological sex and nutritional metabolic status. Taken together, these data contextualize the roles of {beta}OHB as modulators of cognitive resilience in tauopathy.

Mitochondrial protein homeostasis intersects with metabolic control, but the in vivo roles of specific mitochondrial co-chaperones remain unclear. The chaperone mtHSP70 plays a key role in import and folding of nuclear-encoded proteins targeted to mitochondrial matrix. Its protein folding cycle is regulated by the GrpE-like nucleotide exchange factor GRPEL1. Vertebrates also have a GRPEL2 paralog, postulated as the stress-sensitive counterpart, but its physiological relevance is not known. We show here that GRPEL2 is not essential for viability in mice, and its absence does not induce proteotoxic stress responses in stark contrast to GRPEL1. However, we find that GRPEL2 has a role in regulating body weight homeostasis. GRPEL2 knockout mice are protected from age- and diet-induced weight gain and maintain a better metabolic health and insulin sensitivity. Transcriptional profiling revealed minimal changes in liver and skeletal muscle, whereas white adipose tissue from Grpel2-deficient mice lacked the obesity-associated remodeling seen in controls. We propose that GRPEL2 fine-tunes metabolic setpoints without broadly perturbing mitochondrial protein import, thereby maintaining adipose tissue health during nutritional excess. These findings show that subtle alterations in mitochondrial chaperone systems reshape systemic metabolism and could suggest strategies to mitigate obesity and insulin resistance through targeted modulation of mitochondrial proteostasis.

Systemic glucose regulation depends on coordinated signaling among metabolically specialized tissues, yet most human in vitro models capture only limited portions of this network. Here, we developed and benchmarked a perfused human six-tissue MPS by combining AnthroHive, a recirculating perfusion platform, with MOTIVE-6, a six-compartment Multiorgan Tissue Interaction Vessel, to culture human gut epithelium, pancreatic islets, liver organoids, adipocytes, skeletal muscle, and midbrain-patterned brain organoids in a microphysiological system. Shared perfusion redirected engineered tissue states toward tissue-aligned metabolic, endocrine, absorptive, contractile, and neural-associated programs while reducing selected isolation-associated stress and remodeling signatures. Under High nutrient conditions, however, multi-tissue interaction shifted liver and islet responses toward inflammatory and nutrient-stress-associated gene expression, indicating context-dependent effects of cross-compartment signaling. Graded nutrient exposure resolved a staged circuit trajectory: Low nutrient conditions supported maintenance-associated programs, Mid nutrient exposure induced compensatory endocrine and anabolic remodeling with declining net glucose depletion, and High nutrient exposure shifted the system toward stress-associated metabolic dysfunction. Under High conditions, metformin and semaglutide produced distinct response modes. Metformin preserved circuit-level glucose handling without increasing insulin or C-peptide accumulation, while semaglutide remodeled gut, brain organoid, islet, and liver organoid transcriptional programs linked to nutrient sensing, epithelial maintenance, endocrine signaling, and neurometabolic state. Together, this study establishes a benchmarked human six-tissue MPS resource, paired with tissue-resolved transcriptomic, shared-media metabolomic, functional, endocrine, and inflammatory datasets, for investigating how tissue interaction, nutrient availability, and metabolic therapies reshape glucose-regulatory networks. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/726943v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@181cdfforg.highwire.dtl.DTLVardef@fb211eorg.highwire.dtl.DTLVardef@13b7284org.highwire.dtl.DTLVardef@1db7543_HPS_FORMAT_FIGEXP M_FIG C_FIG Created in BioRender. Trapecar, M. (2026) https://BioRender.com/a4tl7nv

Brown adipose tissue (BAT) regulates systemic metabolism beyond thermogenesis, yet the circulating mediators through which BAT communicates with other organs remain less defined. Here, we performed comprehensive serum metabolomics and lipidomics in BAT-ablated mice and human cohorts with varying BAT activity to delineate how BAT activity shapes the circulating metabolome. By integrating datasets across serum, tissues, extracellular fluids, and conditioned media, we assembled BAT-linked circulating molecular signatures. The analyses support a role for BAT in the clearance of circulating branched-chain amino acids and triglycerides, and also identify a cold-inducible metabolite, 3-hydroxystearic acid (3-OHSA), produced by BAT and released into circulation. 3-OHSA serves as a circulating readout of cold-activated BAT and acts on the liver to reduce mitochondrial membrane potential and reactive oxygen species (ROS) production, thereby limiting oxidative stress. This work provides a framework for identifying BAT-derived mediators and uncovers a BAT-liver axis that coordinates adaptation to metabolic stress. HIGHLIGHTSO_LIComprehensive analyses of BAT-linked circulating metabolome and lipidome in mice and humans. C_LIO_LIMulti-level metabolomics supports the role of BAT in circulating BCAA and triglyceride clearance. C_LIO_LICold-inducible 3-OHSA is secreted by BAT and signals to the liver. C_LIO_LI3-OHSA decreases hepatic oxidative stress by decreasing mitochondrial membrane potential. C_LI

Dietary fructose is a major risk factor driving the progression of metabolic dysfunction-associated steatohepatitis (MASH) and hepatocellular carcinoma (HCC). However, the underlying fructose-induced nutrient-sensing pathway remains unclear. Here, we report that fructose facilitates iron absorption through the (Ketohexokinase) KHK/PKM2/HIF-2 axis, driving MASH and HCC development. Fructose aberrantly stabilizes intestinal HIF-; this effect is abrogated by a KHK inhibitor and genetic Khk deletion. Mechanistically, fructose-induced metabolic reprogramming drives glutamine-dependent oxidative phosphorylation, leading to HIF- stabilization, which is mediated by pyruvate kinase M2 (PKM2). A selective PKM2 inhibitor rescues reduced intestinal HIF- stability in Khk-deficient mice. Furthermore, dietary fructose increases plasma iron levels. Conversely, Khk-deficient mice exhibit spontaneous systemic iron deficiency, characterized by hypochromic anemia. Moreover, Khk deficiency inhibits iron absorption in a HIF-2-dependent manner. Finally, fructose promotes MASH and HCC progression in an iron-dependent manner. This study reveals a unique, therapeutically targetable nutrient-sensing pathway utilized by dietary fructose. In briefMitchell et al. demonstrate that fructose consumption increases plasma iron levels, while KHK deficiency inhibits iron absorption in a HIF-2-dependent manner. Mechanistically, dietary fructose-induced metabolic reprogramming aberrantly stabilizes intestinal HIF-, which is mediated by PKM2. Fructose promotes MASH and HCC progression in an iron-dependent manner. HighlightsO_LIFructose aberrantly stabilizes intestinal HIF- C_LIO_LIKHK is required for intestinal HIF- stability C_LIO_LIKHK deficiency inhibits iron absorption in a HIF-2-dependent manner C_LIO_LIDietary fructose promotes MASH and HCC progression in an iron-dependent manner C_LI

Metabolic plasticity of cancer cells plays a major role in metastasis. Use of alternative carbon fuels (e.g., lactate) boosts metabolic plasticity, but the underlying mechanisms remain obscure. We show that lactate dehydrogenase B (LDHB) cooperates with the hydride ion transfer complex (HTC) comprised of pyruvate carboxylase (PC), malic enzyme (ME1) and malate dehydrogenase (MDH1) to form a metabolon that confers metabolic flexibility through lactate assimilation under physiological conditions. HTC/LDHB metabolon assembles in the aggressive breast cancer subtypes and reprograms nicotinamide adenine dinucleotide (NAD) metabolism to promote migration, invasion and escape from anoikis - thereby driving metastasis. Altogether, this work identifies a lactate-fueled metabolon that propels metastatic dissemination of breast cancer.

Mammalian energy homeostasis depends on coordinated metabolism across tissues, with the liver acting as a central hub for systemic energy balance and biosynthetic precursor supply. Although hepatic mitochondrial dysfunction is implicated in diverse pathologies, mitochondrial regulation across liver microanatomical space and time remains incompletely defined. Here, we mapped how mitochondrial- and nuclear-encoded genes supporting mitochondrial function vary along spatial gradients within the lobule and across the feeding-fasting cycle in mice. Integrating these transcriptomic features with quantitative measurements of mitochondrial morphology in periportal and pericentral hepatocytes, we showed that functional hepatocyte subtypes are distinguished by pronounced mitochondrial divergence, including cells with exceptionally low mitochondrial gene expression and reduced secretory protein production. We described that higher periportal oxidative phosphorylation relies on an exceptionally high periportal mitochondrial transcript fraction, while nuclear mitochondrial-function genes do not follow this pattern. The increased periportal mitochondrial transcript abundance coincided with substantially increased periportal cytoplasmic mitochondrial density. In humans, we recapitulated the higher periportal mitochondrial transcript abundance and showed that mitochondrial-function genes exhibited rhythmic expression patterns, more so in women. Together, these data establish a spatially and temporally resolved reference dataset of hepatic mitochondrial regulation that provides a reference for interpreting liver single-cell datasets and mechanistic pathophysiological studies.

Mitochondrial communication coordinates adaptive responses across organelles to sustain cellular homeostasis, a network that declines with age and contributes to loss of proteostasis. Here, we identify MOTS-c, an exercise-induced mitochondrial-derived peptide (MDP) encoded within the 12S rRNA locus, as an inter-organellar arm of the mitochondrial stress response (MSR) that links mitochondrial signaling to endoplasmic reticulum (ER) proteostasis and enables adaptation to chronic stress. Using progressive stress media (PSM), a model of gradual and multifactorial metabolic stress, we show that MOTS-c enables adaptation through a biphasic program: acutely, a reversible, ATF4-independent suppression of protein synthesis; and chronically, an ATF6-biased ER unfolded protein response (UPRER) with tempered ATF4 engagement and coordinated metabolic remodeling. Whereas mitochondrial unfolded protein response (UPRmt) pathways have been extensively characterized in acute, genetic, and sustained models of mitochondrial perturbation, this work reveals how mitochondrial communication actively engages ER proteostasis during progressive and persistent metabolic stress. By expanding proteostatic capacity while tempering terminal stress signaling, MOTS-c enables cells to withstand chronic stress. Together, these findings define a MOTS-c-dependent arm of the MSR that integrates mitochondrial communication with ER proteostasis to promote chronic metabolic stress adaptation.

Malonate is often described as an endogenous inhibitor of complex II of the electron transport chain. However, the cellular source of malonate is unclear, and current knowledge concerning its metabolism is limited to the action of a single enzyme, Acyl-CoA Synthetase Family Member 3 (ACSF3), which converts malonate to malonyl-CoA in the mitochondrial matrix. One potential route of malonate metabolism downstream of ACSF3 is its consumption by the mitochondrial fatty acid synthesis (mtFAS) pathway. However, studies examining the link between ACSF3 and mtFAS have yielded conflicting results. We developed a novel mass spectrometry approach to perform stable isotope tracing into products of mtFAS, and found that while malonate is in fact a carbon source for mtFAS, ACSF3 is not required for malonate incorporation into mtFAS products. Using this method to trace other nutrients into mtFAS, we also found evidence of acetyl-CoA carboxylase 1 (ACC1)-dependent malonate synthesis from glucose. We further show that ACC1 is required for optimal mtFAS activity, with downstream effects on oxidative phosphorylation. Together these findings establish the malonate as a regulated endogenous intermediate that supports mtFAS activity and mitochondrial oxidative function.

Excess fatty acids can disrupt membrane and organelle function. Cells buffer fatty acid toxicity by synthesizing and storing triglycerides (TGs) in lipid droplets, but their capacity for TG storage is limited. Here, using hepatocytes with impaired TG synthesis, we identified adaptive pathways that restore homeostasis during lipid overload. One arm of the response is transcriptional activation of peroxisome proliferator-activated receptors to promote fatty acid oxidation. The other suppresses sterol regulatory element-binding protein 1 (SREBP1)-mediated lipogenesis, reducing fatty acid synthesis and desaturation. Mechanistically, SREBP1 cleavage-activation occurs with changes in membrane fluidity: impaired TG synthesis increased membrane fluidity and suppressed SREBP1 activation, whereas saturated fatty acids exerted opposite effects. These findings reveal feedback regulation that maintains fatty acid homeostasis by coordinating their synthesis and oxidation. They also support a model in which ER membrane fluidity regulates SREBP1 activity to maintain membrane lipid homeostasis, a finding with broad implications for physiology and disease. HighlightsO_LIImpaired triglyceride synthesis induces feedback regulation of fatty acid metabolism to restore fatty acid homeostasis. C_LIO_LIHomeostasis is restored via peroxisome proliferator-activated receptor transcriptional activity to enhance fatty acid oxidation. C_LIO_LIReduced lipogenesis occurs by suppression of sterol regulatory element-binding protein 1 (SREBP1)-mediated fatty acid synthesis and desaturation. C_LIO_LIChanges in ER membrane fluidity regulate SREBP1 activity to maintain membrane lipid homeostasis. C_LI

BackgroundThe systemic long-term health effects and underlying biological mechanisms of early-life sugar restriction remain poorly defined. MethodsWe exploited a natural experiment created by the abrupt end of UK postwar sugar rationing (September 1953), including 60,768 UK Biobank participants born between October 1951 and March 1956. Exposure to early-life sugar restriction was defined by birth date relative to the policy end. We assessed 27 incident disease outcomes and all-cause mortality. In subsets, we performed plasma proteomic and metabolomic profiling, evaluated 81 adult phenotypes, and applied formal mediation analysis. FindingsOf 60,768 participants (mean age 54.6 years; 56.2% female), 38,453 (63.3%) were exposed to early-life sugar restriction. Longer exposure was associated with dose-dependent risk reductions for infections, cancer, mental and behavioural disorders, nervous system, digestive, musculoskeletal, genitourinary, and skin disorders (adjusted HRs 0.83-0.93), and with lower all-cause mortality (adjusted HR 0.79; 95% CI 0.73-0.87). The exposure was associated with a distinct molecular signature and an adult phenotype marked by higher fat-free mass and lower basal metabolic rate, with no difference in BMI. Mediation analyses identified a modest "molecular memory" pathway (3-9% of effect) and a dominant "physiological programming" pathway (4-13% of effect). InterpretationSugar restriction in the first 1,000 days programs multisystem resilience that substantially reduces risk of chronic diseases and later-life mortality. This protection operates through a hierarchical biological architecture dominated by a metabolically efficient physiological phenotype, providing mechanistic support for stricter regulation of added sugars in infant foods. FundingsNational Natural Science Foundation of China and other funding sources. Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSWe searched PubMed, Web of Science, and medRxiv up to January 31, 2026, using "sugar rationing", "natural experiment", and "UK Biobank". Studies using the 1953 end of UK sugar rationing as a natural experiment have reported reduced risks of type 2 diabetes, hypertension, cardiovascular disease, metabolic dysfunction-associated steatotic liver disease, respiratory conditions, heart failure, and anxiety. All previous studies examined single disease outcomes in isolation; none tested for coordinated protection across organ systems or examined underlying biological mechanisms with multi-omics profiling. Added value of this studyThis study demonstrates that early-life sugar restriction confers dose-dependent protection across 27 incident outcomes spanning multiple organ systems--including infections, cancer, mental and behavioural disorders, nervous system, digestive, musculoskeletal, and genitourinary diseases--and reduces all-cause mortality, establishing a pattern of multisystem resilience. Through proteomic and metabolomic profiling, it identifies a molecular signature of early-life sugar restriction and a corresponding adult physiological phenotype characterized by higher fat-free mass and lower basal metabolic rate, with no difference in BMI. Formal mediation analysis reveals a hierarchical dual-pathway mechanism: a modest molecular memory (3-9% of total effect) and a dominant physiological programming pathway mediated by fat-free mass and basal metabolic rate (4-13%). Conventional risk traits, including visceral adiposity and dysglycemia, showed no significant mediation. Implications of all the available evidenceEarly-life nutrition programs lifelong multisystem resilience through a hierarchical biological architecture dominated by physiological reprogramming. These findings provide a mechanistic mandate for stricter regulation of added sugars in infant and toddler foods as primary prevention of non-communicable diseases, and highlight fat-free mass and basal metabolic rate as physiological pathways for future risk assessment and intervention.

Translation fidelity is generally viewed as a constitutive process that deteriorates under stress and aging. Here we show that the fidelity of amino acid incorporation is instead dynamically regulated by the circadian clock. In Neurospora crassa, methionine (Met) misincorporation into proteins exhibits robust daily rhythms, peaking at night coincident with elevated reactive oxygen species (ROS). Rhythmic Met misincorporation requires the circadian clock, the ERK-family MAPK MAK1, and MAK1-dependent phosphorylation of methionyl-tRNA synthetase (MetRS), linking circadian signaling to regulated mistranslation associated with oxidative stress resistance. Preventing MetRS phosphorylation abolishes rhythmic Met misincorporation, impairs growth, and increases sensitivity to oxidative stress, whereas a phosphomimetic MetRS mutant enhances oxidative stress survival. Proteome-wide analyses identified thousands of Met misincorporation events, including a rhythmic subset that oscillates independently of corresponding protein abundance, suggesting that mistranslation dynamically remodels proteome composition across the day. Together, these findings establish translation fidelity as a regulated circadian output and support a model in which the circadian clock temporally regulates mistranslation to enhance oxidative stress resilience. Significance StatementBiological clocks regulate translation termination fidelity, but whether they also control the accuracy of amino acid incorporation during protein synthesis was unknown. We show that the circadian clock drives rhythmic methionine misincorporation into proteins through ERK-family MAPK signaling and phosphorylation of methionyl-tRNA synthetase. Methionine misincorporation peaks during periods of elevated oxidative stress, and disrupting this regulation compromises oxidative stress survival, whereas constitutive activation enhances resistance. Together with previous work on translation termination fidelity, these findings reveal that biological clocks regulate multiple layers of translation fidelity and identify adaptive mistranslation as a mechanism that promotes cellular resilience.

Stress involves the activation of cellular, physiological, and emotional processes that cost energy--nothing is free in biology. In mammals, the stress response involves hormone release, including norepinephrine (NE), which increases energy expenditure. To quantify the energetic cost of NE signaling in a simple cellular system, we interrogated the dose (0-10 M NE) and time-dependent (up to 10 hours) effects of adrenergic signaling in primary human fibroblasts. Oxygen consumption rates (OCR, reflecting ATP generated by mitochondria) and extracellular acidification rate (ECAR, reflecting ATP generated by glycolysis) were measured continuously using extracellular flux analysis, allowing us to estimate the ATP turnover rates, and thus cellular energy expenditure. Within the first 18 minutes (early phase), glycolysis increases up to 47% whereas respiration decreased 2-5%. Both parameters normalized within 1-2 hours for low NE concentrations. This was followed by an increase in oxidative phosphorylation (OxPhos), peaking around 9-12% by 2-6 hours (mid or late-phase). These minutes-to-hours data reveal the temporal dynamics whereby NE increases cellular energy expenditure in fibroblasts. Blocking OxPhos with oligomycin or piericidin A abolished OxPhos changes post-NE addition while conserving the glycolytic response. Withdrawal of glucose from the media significantly dampened the absolute rise in ECAR in response to NE, and instead increased OxPhos, revealing the metabolic flexibility in fibroblasts. Finally, cells with genetic defects impairing OxPhos exhibited a 50% blunted NE-driven metabolic response, consistent with the existence of an energy constraint in mitochondrial diseases. In summary, we have resolved the dynamics and flexible bioenergetic recalibrations associated with NE-driven hypermetabolism in primary human fibroblasts. Mapping the nature and magnitude of these recalibrations in humans would advance our understanding of the potential energetic forces underlying the damage to health by chronic stress.

Astrocytes are glycolytic cells that convert a substantial fraction of glucose-derived pyruvate into lactate, a metabolite implicated in supporting neuronal energy demand and modulating excitability, plasticity and memory. This view has placed astrocytic lactate production and export at the centre of astrocyte-neuron metabolic coupling, but whether mitochondrial pyruvate utilization in astrocytes is dispensable in vivo or fulfils an essential function in the intact brain remains unknown. Here we show that adult astrocyte-specific deletion of Mpc2, encoding an obligatory mitochondrial pyruvate carrier subunit, causes motor deficits, neuronal hyperexcitability and seizure-associated lethality. Metabolic profiling revealed pyruvate diversion toward alanine as an unsuccessful compensatory bypass, together with impaired tricarboxylic acid-cycle metabolism and an imbalance in neurotransmitter-related pools, including glutamate, glutamine and {gamma}-aminobutyric acid. Thus, astrocytic mitochondrial pyruvate import is not primarily required for bioenergetic purposes but acts as a non-redundant anaplerotic gate that maintains neurotransmitter homeostasis, excitation-inhibition balance and seizure resistance in vivo.

Nicotinamide adenine dinucleotide (NAD+) and its precursor nicotinamide mononucleotide (NMN) are strictly compartmentalized, yet how individual organelles maintain local metabolic homeostasis remains unresolved. Here, we report FrNADS and FrNMNS1.0, a FRET-based biosensor toolkit that maps NAD+ and NMN dynamics in living cells with subcellular resolution, including the oxidizing lumen of the endoplasmic reticulum. We find that NAD+ recovery in the nucleus following PARP1 activation depends on NAMPT mediated salvage synthesis, while peroxisomes buffer NAD+ via NUDT12 and SLC25A17. In mitochondria, NMNAT3 acts as a NAD+ hydrolase that counterbalances import through SLC25A51; HINT2 functionally enhances this activity. Furthermore, SLC25A48 functions as a critical regulatory node that modulates the compartmental redistribution of the generated NMN. These findings establish a mitochondrial NAD+/NMN regulatory circuit and reveal how organelles independently resolve metabolic stress.