Sestrin2 is a member of a family of stress responsive proteins, which controls cell viability via antioxidant activity and regulation of the mammalian target of rapamycin protein kinase (mTOR). Sestrin2 is induced by different stress insults, which diminish ATP production and induce energetic stress in the cells. Glucose is a critical substrate for ATP production utilized via glycolysis and mitochondrial respiration as well as for glycosylation of newly synthesized proteins in the endoplasmic reticulum (ER) and Golgi. Thus, glucose starvation causes both energy deficiency and activation of ER stress followed by the unfolding protein response (UPR). Here, we show that UPR induces Sestrin2 via ATF4 and NRF2 transcription factors and demonstrate that Sestrin2 protects cells from glucose starvation-induced cell death. Sestrin2 inactivation sensitizes cells to necroptotic cell death that is associated with a decline in ATP levels and can be suppressed by Necrostatin 7. We propose that Sestrin2 protects cells from glucose starvation-induced cell death via regulation of mitochondrial homeostasis.

Huntington disease (HD) is a fatal neurodegenerative disorder caused by a CAG expansion in the huntingtin (HTT) gene, leading to selective and progressive neuronal death predominantly in the striatum. Mutant HTT expression causes dysfunctional cortico-striatal (CS) transmission, loss of CS synapses, and striatal medium spiny neuron (MSN) dendritic spine instability prior to neuronal death. Co-culturing cortical and striatal neurons in vitro promotes the formation of functional CS synapses and is a widely used approach to elucidate pathogenic mechanisms of HD and to validate potential synapto-protective therapies. A number of relevant in vivo synaptic phenotypes from the YAC128 HD mouse model, which expresses full-length transgenic human mutant HTT, are recapitulated in CS co-culture by 21 days in vitro (DIV). However, striatal spine loss, which occurs in HD patients and in vivo animal models, has been observed in YAC128 CS co-culture in some studies but not in others, leading to difficulties in reproducing and interpreting results. Here, we investigated whether differences in the relative proportion of cortical and striatal neurons alter YAC128 synaptic phenotypes in this model.

Dysregulation of oxidative phosphorylation is associated with increased mitochondrial reactive oxygen species production and some of the most prevalent human diseases including obesity, cancer, diabetes, neurodegeneration, and heart disease. Chemical 'mitochondrial uncouplers' are lipophilic weak acids that transport protons into the mitochondrial matrix via a pathway that is independent of ATP synthase, thereby uncoupling nutrient oxidation from ATP production. Mitochondrial uncouplers also lessen the proton motive force across the mitochondrial inner membrane and thereby increase the rate of mitochondrial respiration while decreasing production of reactive oxygen species. Thus, mitochondrial uncouplers are valuable chemical tools that enable the measurement of maximal mitochondrial respiration and they have been used therapeutically to decrease mitochondrial reactive oxygen species production. However, the most widely used protonophore uncouplers such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and 2,4-dinitrophenol have off-target activity at other membranes that lead to a range of undesired effects including plasma membrane depolarization, mitochondrial inhibition, and cytotoxicity. These unwanted properties interfere with the measurement of mitochondrial function and result in a narrow therapeutic index that limits their usefulness in the clinic. To identify new mitochondrial uncouplers that lack off-target activity at the plasma membrane we screened a small molecule chemical library. Herein we report the identification and validation of a novel mitochondrial protonophore uncoupler (2-fluorophenyl){6-[(2-fluorophenyl)amino](1,2,5-oxadiazolo[3,4-e]pyrazin-5-yl)}amine, named BAM15, that does not depolarize the plasma membrane. Compared to FCCP, an uncoupler of equal potency, BAM15 treatment of cultured cells stimulates a higher maximum rate of mitochondrial respiration and is less cytotoxic. Furthermore, BAM15 is bioactive in vivo and dose-dependently protects mice from acute renal ischemic-reperfusion injury. From a technical standpoint, BAM15 represents an effective new tool that allows the study of mitochondrial function in the absence of off-target effects that can confound data interpretation. From a therapeutic perspective, BAM15-mediated protection from ischemia-reperfusion injury and its reduced toxicity will hopefully reignite interest in pharmacological uncoupling for the treatment of the myriad of diseases that are associated with altered mitochondrial function.

Targeting the mitochondria during ischemia/reperfusion (IR) can confer cardioprotection leading to improved clinical outcomes. The cardioprotective potential of (-)-epicatechin (EPI) during IR via modulation of mitochondrial function was evaluated.

Glutamate is the dominant excitatory neurotransmitter in the brain, but under conditions of metabolic stress it can accumulate to excitotoxic levels. Although pharmacologic modulation of excitatory amino acid receptors is well studied, minimal consideration has been given to targeting mitochondrial glutamate metabolism to control neurotransmitter levels. Here we demonstrate that chemical inhibition of the mitochondrial pyruvate carrier (MPC) protects primary cortical neurons from excitotoxic death. Reductions in mitochondrial pyruvate uptake do not compromise cellular energy metabolism, suggesting neuronal metabolic flexibility. Rather, MPC inhibition rewires mitochondrial substrate metabolism to preferentially increase reliance on glutamate to fuel energetics and anaplerosis. Mobilizing the neuronal glutamate pool for oxidation decreases the quantity of glutamate released upon depolarization and, in turn, limits the positive-feedback cascade of excitotoxic neuronal injury. The finding links mitochondrial pyruvate metabolism to glutamatergic neurotransmission and establishes the MPC as a therapeutic target to treat neurodegenerative diseases characterized by excitotoxicity.

The etiology of nonalcoholic fatty liver disease (NAFLD) is poorly understood, with males and certain populations exhibiting markedly increased susceptibility. Using a systems genetics approach involving multi-omic analysis of ∼100 diverse inbred strains of mice, we recently identified several candidate genes driving NAFLD. We investigated the role of one of these, liver pyruvate kinase (L-PK or Pklr), in NAFLD by using patient samples and mouse models.

Cerebral ischemia/reperfusion (IR) drives oxidative stress and injurious metabolic processes that lead to redox imbalance, inflammation, and tissue damage. However, the key mediators of reperfusion injury remain unclear, and therefore, there is considerable interest in therapeutically targeting metabolism and the cellular response to oxidative stress.

Huntington disease (HD) is a fatal neurodegenerative disease caused by a pathogenic expansion of a CAG repeat in the huntingtin (HTT) gene. There are no disease-modifying therapies for HD. Artificial microRNAs targeting HTT transcripts for degradation have shown preclinical promise and will soon enter human clinical trials. Here, we examine the tolerability and efficacy of non-selective HTT lowering with an AAV5 encoded miRNA targeting human HTT (AAV5-miHTT) in the humanized Hu128/21 mouse model of HD. We show that intrastriatal administration of AAV5-miHTT results in potent and sustained HTT suppression for at least 7 months post-injection. Importantly, non-selective suppression of huntingtin was generally tolerated, however high dose AAV5-miHTT did induce astrogliosis. We observed an improvement of select behavioural and modest neuropathological HD-like phenotypes in Hu128/21 mice, suggesting a potential therapeutic benefit of miRNA-mediated non-selective HTT lowering. Finally, we also observed that potent reduction of wild type HTT (wtHTT) in Hu21 control mice was tolerated up to 7 months post-injection but may induce impairment of motor coordination and striatal atrophy. Taken together, our data suggests that in the context of HD, the therapeutic benefits of mHTT reduction may outweigh the potentially detrimental effects of wtHTT loss following non-selective HTT lowering.

Elevated levels of branched chain amino acids (BCAAs) and branched-chain α-ketoacids (BCKAs) are associated with cardiovascular and metabolic disease, but the molecular mechanisms underlying a putative causal relationship remain unclear. The branched-chain ketoacid dehydrogenase kinase (BCKDK) inhibitor BT2 is often used in preclinical models to increase BCAA oxidation and restore steady-state BCAA and BCKA levels. BT2 administration is protective in various rodent models of heart failure and metabolic disease, but confoundingly, targeted ablation of Bckdk in specific tissues does not reproduce the beneficial effects conferred by pharmacologic inhibition. Here we demonstrate that BT2, a lipophilic weak acid, can act as a mitochondrial uncoupler. Measurements of oxygen consumption, mitochondrial membrane potential, and patch-clamp electrophysiology show BT2 increases proton conductance across the mitochondrial inner membrane independently of its inhibitory effect on BCKDK. BT2 is roughly five-fold less potent than the prototypical uncoupler 2,4-dinitrophenol (DNP), and phenocopies DNP in lowering de novo lipogenesis and mitochondrial superoxide production. The data suggest the therapeutic efficacy of BT2 may be attributable to the well-documented effects of mitochondrial uncoupling in alleviating cardiovascular and metabolic disease.

Changes in mitochondrial morphology are associated with nutrient utilization, but the precise causalities and the underlying mechanisms remain unknown. Here, using cellular models representing a wide variety of mitochondrial shapes, we show a strong linear correlation between mitochondrial fragmentation and increased fatty acid oxidation (FAO) rates. Forced mitochondrial elongation following MFN2 over-expression or DRP1 depletion diminishes FAO, while forced fragmentation upon knockdown or knockout of MFN2 augments FAO as evident from respirometry and metabolic tracing. Remarkably, the genetic induction of fragmentation phenocopies distinct cell type-specific biological functions of enhanced FAO. These include stimulation of gluconeogenesis in hepatocytes, induction of insulin secretion in islet β-cells exposed to fatty acids, and survival of FAO-dependent lymphoma subtypes. We find that fragmentation increases long-chain but not short-chain FAO, identifying carnitine O-palmitoyltransferase 1 (CPT1) as the downstream effector of mitochondrial morphology in regulation of FAO. Mechanistically, we determined that fragmentation reduces malonyl-CoA inhibition of CPT1, while elongation increases CPT1 sensitivity to malonyl-CoA inhibition. Overall, these findings underscore a physiologic role for fragmentation as a mechanism whereby cellular fuel preference and FAO capacity are determined.

Miner1 is a redox-active 2Fe2S cluster protein. Mutations in Miner1 result in Wolfram Syndrome, a metabolic disease associated with diabetes, blindness, deafness, and a shortened lifespan. Embryonic fibroblasts from Miner1(-/-) mice displayed ER stress and showed hallmarks of the unfolded protein response. In addition, loss of Miner1 caused a depletion of ER Ca(2+) stores, a dramatic increase in mitochondrial Ca(2+) load, increased reactive oxygen and nitrogen species, an increase in the GSSG/GSH and NAD(+)/NADH ratios, and an increase in the ADP/ATP ratio consistent with enhanced ATP utilization. Furthermore, mitochondria in fibroblasts lacking Miner1 displayed ultrastructural alterations, such as increased cristae density and punctate morphology, and an increase in O2 consumption. Treatment with the sulphydryl anti-oxidant N-acetylcysteine reversed the abnormalities in the Miner1 deficient cells, suggesting that sulphydryl reducing agents should be explored as a treatment for this rare genetic disease.

Recent studies have suggested that human pluripotent stem cells (hPSCs) depend primarily on glycolysis and only increase oxidative metabolism during differentiation. Here, we demonstrate that both glycolytic and oxidative metabolism can support hPSC growth and that the metabolic phenotype of hPSCs is largely driven by nutrient availability. We comprehensively characterized hPSC metabolism by using (13)C/(2)H stable isotope tracing and flux analysis to define the metabolic pathways supporting hPSC bioenergetics and biosynthesis. Although glycolytic flux consistently supported hPSC growth, chemically defined media strongly influenced the state of mitochondrial respiration and fatty acid metabolism. Lipid deficiency dramatically reprogramed pathways associated with fatty acid biosynthesis and NADPH regeneration, altering the mitochondrial function of cells and driving flux through the oxidative pentose phosphate pathway. Lipid supplementation mitigates this metabolic reprogramming and increases oxidative metabolism. These results demonstrate that self-renewing hPSCs can present distinct metabolic states and highlight the importance of medium nutrients on mitochondrial function and development.

Carnitine palmitoyltransferase-1 (CPT-1) is a rate-controlling enzyme for long-chain fatty acid oxidation. This manuscript provides protocols for measuring CPT-1-mediated respiration in permeabilized, adherent cell monolayers and mitochondria freshly isolated from tissue, along with examples to assess the potency and specificity of interventions targeting CPT-1. Strengths of the approach include ease, speed, and breadth of analysis, whereas drawbacks include loss of physiological regulation in reductionist systems and indirect assessment of CPT-1 enzymatic activity. For complete details on the use and execution of this protocol, please refer to Divakaruni et al. (2018).

Advanced prostate cancers are treated with therapies targeting the androgen receptor (AR) signaling pathway. While many tumors initially respond to AR inhibition, nearly all develop resistance. It is critical to understand how prostate tumor cells respond to AR inhibition in order to exploit therapy-induced phenotypes prior to the outgrowth of treatment-resistant disease. Here, we comprehensively characterize the effects of AR blockade on prostate cancer metabolism using transcriptomics, metabolomics, and bioenergetics approaches. The metabolic response to AR inhibition is defined by reduced glycolysis, robust elongation of mitochondria, and increased reliance on mitochondrial oxidative metabolism. We establish DRP1 activity and MYC signaling as mediators of AR-blockade-induced metabolic phenotypes. Rescuing DRP1 phosphorylation after AR inhibition restores mitochondrial fission, while rescuing MYC restores glycolytic activity and prevents sensitivity to complex I inhibition. Our study provides insight into the regulation of treatment-induced metabolic phenotypes and vulnerabilities in prostate cancer.

Pancreatic β cell physiology changes substantially throughout life, yet the mechanisms that drive these changes are poorly understood. Here, we performed comprehensive in vivo quantitative proteomic profiling of pancreatic islets from juvenile and 1-year-old mice. The analysis revealed striking differences in abundance of enzymes controlling glucose metabolism. We show that these changes in protein abundance are associated with higher activities of glucose metabolic enzymes involved in coupling factor generation as well as increased activity of the coupling factor-dependent amplifying pathway of insulin secretion. Nutrient tracing and targeted metabolomics demonstrated accelerated accumulation of glucose-derived metabolites and coupling factors in islets from 1-year-old mice, indicating that age-related changes in glucose metabolism contribute to improved glucose-stimulated insulin secretion with age. Together, our study provides an in-depth characterization of age-related changes in the islet proteome and establishes metabolic rewiring as an important mechanism for age-associated changes in β cell function.

Pyruvate lies at a central biochemical node connecting carbohydrate, amino acid, and fatty acid metabolism, and the regulation of pyruvate flux into mitochondria represents a critical step in intermediary metabolism impacting numerous diseases. To characterize changes in mitochondrial substrate utilization in the context of compromised mitochondrial pyruvate transport, we applied (13)C metabolic flux analysis (MFA) to cells after transcriptional or pharmacological inhibition of the mitochondrial pyruvate carrier (MPC). Despite profound suppression of both glucose and pyruvate oxidation, cell growth, oxygen consumption, and tricarboxylic acid (TCA) metabolism were surprisingly maintained. Oxidative TCA flux was achieved through enhanced reliance on glutaminolysis through malic enzyme and pyruvate dehydrogenase (PDH) as well as fatty acid and branched-chain amino acid oxidation. Thus, in contrast to inhibition of complex I or PDH, suppression of pyruvate transport induces a form of metabolic flexibility associated with the use of lipids and amino acids as catabolic and anabolic fuels.

The assumption underlying current diabetes treatment is that lowering the level of time-averaged glucose concentrations, measured as HbA1c, prevents microvascular complications. However, 89% of variation in risk of retinopathy, microalbuminuria, or albuminuria is due to elements of glycemia not captured by mean HbA1c values. We show that transient exposure to high glucose activates a multicomponent feedback loop that causes a stable left shift of the glucose concentration-reactive oxygen species (ROS) dose-response curve. Feedback loop disruption by the GLP-1 cleavage product GLP-1(9-36)(amide) reverses the persistent left shift, thereby normalizing persistent overproduction of ROS and its pathophysiologic consequences. These data suggest that hyperglycemic spikes high enough to activate persistent ROS production during subsequent periods of normal glycemia but too brief to affect the HbA1c value are a major determinant of the 89% of diabetes complications risk not captured by HbA1c. The phenomenon and mechanism described in this study provide a basis for the development of both new biomarkers to complement HbA1c and novel therapeutic agents, including GLP-1(9-36)(amide), for the prevention and treatment of diabetes complications.

Long-chain fatty acid (LCFA) oxidation has been shown to play an important role in interleukin-4 (IL-4)-mediated macrophage polarization (M(IL-4)). However, many of these conclusions are based on the inhibition of carnitine palmitoyltransferase-1 with high concentrations of etomoxir that far exceed what is required to inhibit enzyme activity (EC90 < 3 μM). We employ genetic and pharmacologic models to demonstrate that LCFA oxidation is largely dispensable for IL-4-driven polarization. Unexpectedly, high concentrations of etomoxir retained the ability to disrupt M(IL-4) polarization in the absence of Cpt1a or Cpt2 expression. Although excess etomoxir inhibits the adenine nucleotide translocase, oxidative phosphorylation is surprisingly dispensable for M(IL-4). Instead, the block in polarization was traced to depletion of intracellular free coenzyme A (CoA), likely resulting from conversion of the pro-drug etomoxir into active etomoxiryl CoA. These studies help explain the effect(s) of excess etomoxir on immune cells and reveal an unappreciated role for CoA metabolism in macrophage polarization.

Mitochondrial depolarization can initiate reversal activity of ATP synthase, depleting ATP by its hydrolysis. We have recently shown that increased ATP hydrolysis contributes to ATP depletion leading to a maladaptation in mitochondrial disorders, where maximal hydrolytic capacity per CV content is increasing. However, despite its importance, ATP hydrolysis is not a commonly studied parameter because of the limitations of the currently available methods. Methods that measure CV hydrolytic activity indirectly require the isolation of mitochondria and involve the introduction of detergents, preventing their utilization in clinical studies or any high-throughput analyses. Here, we describe a novel approach to assess maximal ATP hydrolytic capacity and maximal respiratory capacity in a single assay in cell lysates, PBMCs, and tissue homogenates that were previously frozen. The methodology described here has the potential to be used in clinical samples to determine adaptive and maladaptive adjustments of CV function in diseases, with the added benefit of being able to use frozen samples in a high-throughput manner and to explore ATP hydrolysis as a drug target for disease treatment.

Coenzyme A (CoA) is an essential cofactor for dozens of reactions in intermediary metabolism. Dysregulation of CoA synthesis or acyl CoA metabolism can result in metabolic or neurodegenerative disease. Although several methods use liquid chromatography coupled with mass spectrometry/mass spectrometry (LC-MS/MS) to quantify acyl CoA levels in biological samples, few allow for simultaneous measurement of intermediates in the CoA biosynthetic pathway. Here we describe a simple sample preparation and LC-MS/MS method that can measure both short-chain acyl CoAs and biosynthetic precursors of CoA. The method does not require use of a solid phase extraction column during sample preparation and exhibits high sensitivity, precision, and accuracy. It reproduces expected changes from known effectors of cellular CoA homeostasis and helps clarify the mechanism by which excess concentrations of etomoxir reduce intracellular CoA levels.