Search

Mitochondrial membrane potential directly powers many critical functions of mitochondria, including ATP production, mitochondrial protein import, and metabolite transport. Its loss is a cardinal feature of aging and mitochondrial diseases, and cells closely monitor membrane potential as an indicator of mitochondrial health. Given its central importance, it is logical that cells would modulate mitochondrial membrane potential in response to demand and environmental cues, but there has been little exploration of this question. We report that loss of the Sit4 protein phosphatase in yeast increases mitochondrial membrane potential, both by inducing the electron transport chain and the phosphate starvation response. Indeed, a similarly elevated mitochondrial membrane potential is also elicited simply by phosphate starvation or by abrogation of the Pho85-dependent phosphate sensing pathway. This enhanced membrane potential is primarily driven by an unexpected activity of the ADP/ATP carrier. We also demonstrate that this connection between phosphate limitation and enhancement of mitochondrial membrane potential is observed in primary and immortalized mammalian cells as well as in Drosophila. These data suggest that mitochondrial membrane potential is subject to environmental stimuli and intracellular signaling regulation and raise the possibility for therapeutic enhancement of mitochondrial function even in defective mitochondria.

Thermogenesis by uncoupling protein 1 (UCP1) is one of the primary mechanisms by which brown adipose tissue (BAT) increases energy expenditure. UCP1 resides in the inner mitochondrial membrane (IMM), where it dissipates membrane potential independent of adenosine triphosphate (ATP) synthase. Here, we provide evidence that phosphatidylethanolamine (PE) modulates UCP1-dependent proton conductance across the IMM to modulate thermogenesis. Mitochondrial lipidomic analyses revealed PE as a signature molecule whose abundance bidirectionally responds to changes in thermogenic burden. Reduction in mitochondrial PE by deletion of phosphatidylserine decarboxylase (PSD) made mice cold intolerant and insensitive to β3 adrenergic receptor agonist-induced increase in whole-body oxygen consumption. High-resolution respirometry and fluorometry of BAT mitochondria showed that loss of mitochondrial PE specifically lowers UCP1-dependent respiration without compromising electron transfer efficiency or ATP synthesis. These findings were confirmed by a reduction in UCP1 proton current in PE-deficient mitoplasts. Thus, PE performs a previously unknown role as a temperature-responsive rheostat that regulates UCP1-dependent thermogenesis.

Cyanide-a fast-acting poison-is easy to obtain given its widespread use in manufacturing industries. It is a high-threat chemical agent that poses a risk of occupational exposure in addition to being a terrorist agent. FDA-approved cyanide antidotes must be given intravenously, which is not practical in a mass casualty setting due to the time and skill required to obtain intravenous access. Glyoxylate is an endogenous metabolite that binds cyanide and reverses cyanide-induced redox imbalances independent of chelation. Efficacy and biochemical mechanistic studies in an FDA-approved preclinical animal model have not been reported. Therefore, in a swine model of cyanide poisoning, we evaluated the efficacy of intramuscular glyoxylate on clinical, metabolic, and biochemical endpoints. Animals were instrumented for continuous hemodynamic monitoring and infused with potassium cyanide. Following cyanide-induced apnea, saline control or glyoxylate was administered intramuscularly. Throughout the study, serial blood samples were collected for pharmacokinetic, metabolite, and biochemical studies, in addition, vital signs, hemodynamic parameters, and laboratory values were measured. Survival in glyoxylate-treated animals was 83% compared with 12% in saline-treated control animals (p

Carbohydrates and lipids provide the majority of substrates to fuel mitochondrial oxidative phosphorylation. Metabolic inflexibility, defined as an impaired ability to switch between these fuels, is implicated in a number of metabolic diseases. Here, we explore the mechanism by which physical inactivity promotes metabolic inflexibility in skeletal muscle. We developed a mouse model of sedentariness, small mouse cage (SMC), that, unlike other classic models of disuse in mice, faithfully recapitulated metabolic responses that occur in humans. Bioenergetic phenotyping of skeletal muscle mitochondria displayed metabolic inflexibility induced by physical inactivity, demonstrated by a reduction in pyruvate- stimulated respiration ([JO.sub.2]) in the absence of a change in palmitate-stimulated [JO.sub.2]. Pyruvate resistance in these mitochondria was likely driven by a decrease in phosphatidylethanolamine (PE) abundance in the mitochondrial membrane. Reduction in mitochondrial PE by heterozygous deletion of phosphatidylserine decarboxylase (PSD) was sufficient to induce metabolic inflexibility measured at the whole-body level, as well as at the level of skeletal muscle mitochondria. Low mitochondrial PE in C2C12 myotubes was sufficient to increase glucose flux toward lactate. We further implicate that resistance to pyruvate metabolism is due to attenuated mitochondrial entry via mitochondrial pyruvate carrier (MPC). These findings suggest a mechanism by which mitochondrial PE directly regulates MPC activity to modulate metabolic flexibility in mice., Introduction Chronic physical inactivity increases all-cause mortality by 30%, accounting for one death every 44 seconds (1-4). Sedentary behavior exacerbates the risk for many chronic diseases, such as type 2 [...]

Although cyanide's biological effects are pleiotropic, its most obvious effects are as a metabolic poison. Cyanide potently inhibits cytochrome c oxidase and potentially other metabolic enzymes, thereby unleashing a cascade of metabolic perturbations that are believed to cause lethality. From systematic screens of human metabolites using a zebrafish model of cyanide toxicity, we have identified the TCA-derived small molecule glyoxylate as a potential cyanide countermeasure. Following cyanide exposure, treatment with glyoxylate in both mammalian and non-mammalian animal models confers resistance to cyanide toxicity with greater efficacy and faster kinetics than known cyanide scavengers. Glyoxylate-mediated cyanide resistance is accompanied by rapid pyruvate consumption without an accompanying increase in lactate concentration. Lactate dehydrogenase is required for this effect which distinguishes the mechanism of glyoxylate rescue as distinct from countermeasures based solely on chemical cyanide scavenging. Our metabolic data together support the hypothesis that glyoxylate confers survival at least in part by reversing the cyanide-induced redox imbalances in the cytosol and mitochondria. The data presented herein represent the identification of a potential cyanide countermeasure operating through a novel mechanism of metabolic modulation.

Aging-related chronic inflammation is a risk factor for many human disorders through incompletely understood mechanisms. Aged mice deficient in microRNA (miRNA/miR)-146a succumb to life-shortening chronic inflammation. In this study, we report that miR-155 in T cells contributes to shortened lifespan of miR-146a-/- mice. Using single-cell RNA sequencing and flow cytometry, we found that miR-155 promotes the activation of effector T cell populations, including T follicular helper cells, and increases germinal center B cells and autoantibodies in mice aged over 15 months. Mechanistically, aerobic glycolysis genes are elevated in T cells during aging, and upon deletion of miR-146a, in a T cell miR-155-dependent manner. Finally, skewing T cell metabolism toward aerobic glycolysis by deleting mitochondrial pyruvate carrier recapitulates age-dependent T cell phenotypes observed in miR-146a-/- mice, revealing the sufficiency of metabolic reprogramming to influence immune cell functions during aging. Altogether, these data indicate that T cell-specific miRNAs play pivotal roles in regulating lifespan through their influences on inflammaging.

Metabolic pathways regulate T cell development and function, but many remain understudied. Recently, the mitochondrial pyruvate carrier (MPC) was identified as the transporter that mediates pyruvate entry into mitochondria, promoting pyruvate oxidation. Here we find that deleting Mpc1, an obligate MPC subunit, in the hematopoietic system results in a specific reduction in peripheral αβ T cell numbers. MPC1-deficient T cells have defective thymic development at the β-selection, intermediate single positive (ISP)-to-double-positive (DP), and positive selection steps. We find that early thymocytes deficient in MPC1 display alterations to multiple pathways involved in T cell development. This results in preferred escape of more activated T cells. Finally, mice with hematopoietic deletion of Mpc1 are more susceptible to experimental autoimmune encephalomyelitis. Altogether, our study demonstrates that pyruvate oxidation by T cell precursors is necessary for optimal αβ T cell development and that its deficiency results in reduced but activated peripheral T cell populations.

Brown adipose tissue (BAT) is composed of thermogenic cells that convert chemical energy into heat to maintain a constant body temperature and counteract metabolic disease. The metabolic adaptations required for thermogenesis are not fully understood. Here, we explore how steady state levels of metabolic intermediates are altered in brown adipose tissue in response to cold exposure. Transcriptome and metabolome analysis revealed changes in pathways involved in amino acid, glucose, and TCA cycle metabolism. Using isotopic labeling experiments, we found that activated brown adipocytes increased labeling of pyruvate and TCA cycle intermediates from U13C-glucose. Although glucose oxidation has been implicated as being essential for thermogenesis, its requirement for efficient thermogenesis has not been directly tested. We show that mitochondrial pyruvate uptake is essential for optimal thermogenesis, as conditional deletion of Mpc1 in brown adipocytes leads to impaired cold adaptation. Isotopic labeling experiments using U13C-glucose showed that loss of MPC1 led to impaired labeling of TCA cycle intermediates. Loss of MPC1 in BAT increased 3-hydroxybutyrate levels in blood and BAT in response to the cold, suggesting that ketogenesis provides an alternative fuel source to compensate. Collectively, these studies highlight that complete glucose oxidation is essential for optimal brown fat thermogenesis.

Eukaryotic cells employ the ribosome-associated quality control complex (RQC) to maintain homeostasis despite defects that cause ribosomes to stall. The RQC comprises the E3 ubiquitin ligase Ltn1p, the ATPase Cdc48p, Rqc1p, and Rqc2p. Upon ribosome stalling and splitting, the RQC assembles on the 60S species containing unreleased peptidyl-tRNA (60S:peptidyl-tRNA). Ltn1p and Rqc1p facilitate ubiquitination of the incomplete nascent chain, marking it for degradation. Rqc2p stabilizes Ltn1p on the 60S and recruits charged tRNAs to the 60S to catalyze elongation of the nascent protein with carboxy-terminal alanine and threonine extensions (CAT tails). By mobilizing the nascent chain, CAT tailing can expose lysine residues that are hidden in the exit tunnel, thereby supporting efficient ubiquitination. If the ubiquitin-proteasome system is overwhelmed or unavailable, CAT-tailed nascent chains can aggregate in the cytosol or within organelles like mitochondria. Here we identify Vms1p as a tRNA hydrolase that releases stalled polypeptides engaged by the RQC.

Cyanide is a potent toxic agent, and the few available antidotes are not amenable to rapid deployment in mass exposures. As a result, there are ongoing efforts to exploit different animal models to identify novel countermeasures. We have created a pipeline that combines high-throughput screening in zebrafish with subsequent validation in two mammalian small animal models as well as a porcine large animal model. We found that zebrafish embryos in the first 3 days post fertilization (dpf) are highly resistant to cyanide, becoming progressively more sensitive thereafter. Unbiased analysis of gene expression in response to several hours of ultimately lethal doses of cyanide in both 1 and 7 dpf zebrafish revealed modest changes in iron-related proteins associated with the age-dependent cyanide resistance. Metabolomics measurements demonstrated significant age-dependent differences in energy metabolism during cyanide exposure which prompted us to test modulators of the tricarboxylic acid cycle and related metabolic processes as potential antidotes. In cyanide-sensitive 7 dpf larvae, we identified several such compounds that offer significant protection against cyanide toxicity. Modulators of the pyruvate dehydrogenase complex, as well as the small molecule sodium glyoxylate, consistently protected against cyanide toxicity in 7 dpf zebrafish larvae. Together, our results indicate that the resistance of zebrafish embryos to cyanide toxicity during early development is related to an altered regulation of cellular metabolism, which we propose may be exploited as a potential target for the development of novel antidotes against cyanide poisoning.

Most differentiated cells convert glucose to pyruvate in the cytosol through glycolysis, followed by pyruvate oxidation in the mitochondria. These processes are linked by the mitochondrial pyruvate carrier (MPC), which is required for efficient mitochondrial pyruvate uptake. In contrast, proliferative cells, including many cancer and stem cells, perform glycolysis robustly but limit fractional mitochondrial pyruvate oxidation. We sought to understand the role this transition from glycolysis to pyruvate oxidation plays in stem cell maintenance and differentiation. Loss of the MPC in Lgr5-EGFP-positive stem cells, or treatment of intestinal organoids with an MPC inhibitor, increases proliferation and expands the stem cell compartment. Similarly, genetic deletion of the MPC in Drosophila intestinal stem cells also increases proliferation, whereas MPC overexpression suppresses stem cell proliferation. These data demonstrate that limiting mitochondrial pyruvate metabolism is necessary and sufficient to maintain the proliferation of intestinal stem cells.

Although normally dormant, hair follicle stem cells (HFSCs) quickly become activated to divide during a new hair cycle. The quiescence of HFSCs is known to be regulated by a number of intrinsic and extrinsic mechanisms. Here we provide several lines of evidence to demonstrate that HFSCs utilize glycolytic metabolism and produce significantly more lactate than other cells in the epidermis. Furthermore, lactate generation appears to be critical for the activation of HFSCs as deletion of lactate dehydrogenase (Ldha) prevented their activation. Conversely, genetically promoting lactate production in HFSCs through mitochondrial pyruvate carrier 1 (Mpc1) deletion accelerated their activation and the hair cycle. Finally, we identify small molecules that increase lactate production by stimulating Myc levels or inhibiting Mpc1 carrier activity and can topically induce the hair cycle. These data suggest that HFSCs maintain a metabolic state that allows them to remain dormant and yet quickly respond to appropriate proliferative stimuli.