41Exercise capacity is a strong predictor of all-cause mortality. Skeletal muscle mitochondrial 42 respiratory capacity, its biggest contributor, adapts robustly to changes in energy demands 43 induced by contractile activity. While transcriptional regulation of mitochondrial enzymes has 44 been extensively studied, there is limited information on how mitochondrial membrane lipids are 45 regulated. Herein, we show that exercise training or muscle disuse alters mitochondrial 46 membrane phospholipids including phosphatidylethanolamine (PE). Addition of PE promoted, 47 whereas removal of PE diminished, mitochondrial respiratory capacity. Surprisingly, skeletal 48 muscle-specific inhibition of mitochondrial-autonomous synthesis of PE caused a respiratory 49 failure due to metabolic insults in the diaphragm muscle. While mitochondrial PE deficiency 50 coincided with increased oxidative stress, neutralization of the latter did not rescue lethality. 51 These findings highlight the previously underappreciated role of mitochondrial membrane 52 phospholipids in dynamically controlling skeletal muscle energetics and function. 53 54 55 157 3B&C), without changes in abundance of ETS enzymes (Figure 3D). PE molecules are bound to 158 ETS complexes I, II, III, and IV, likely facilitating conformational changes and acting as an 159
Drosophila simulans identification. We thank undergraduate students Damani Fitzgerald, Aaron Johnson, Imani Lowery, and Angela Sehres for technical assistance. This study was supported in parts by NIH R01 DK096907 (PDN), NIH-NIGMS (KMB), and R15ES029673 (AKM).
Compensatory changes in energy expenditure occur in response to positive and negative energy balance, but the underlying mechanism remains unclear. Under low energy demand, the mitochondrial electron transport system (ETS) is particularly sensitive to added energy supply (i.e., reductive stress) which exponentially increases the rate of H2O2 (JH2O2) production. H2O2 is reduced to H2O by electrons supplied by NADPH. NADP+ is reduced back to NADPH by activation of mitochondrial membrane potential-dependent nicotinamide nucleotide transhydrogenase (NNT). The coupling of reductive stress-induced JH2O2 production to NNT-linked redox buffering circuits provides a potential means of integrating energy balance with energy expenditure. To test this hypothesis, energy supply was manipulated by varying flux rate through β-oxidation in muscle mitochondria minus/plus pharmacological or genetic inhibition of redox buffering circuits. Here we show during both non-ADP and low-ADP stimulated respiration that accelerating flux through β-oxidation generates a corresponding increase in mitochondrial JH2O2 production, that the majority (∼70-80%) of H2O2 produced is reduced to H2O by electrons drawn from redox buffering circuits supplied by NADPH, and that the rate of electron flux through redox buffering circuits is directly linked to changes in oxygen consumption mediated by NNT. These findings provide evidence that redox reactions within β-oxidation and the ETS serve as a barometer of substrate flux relative to demand, continuously adjusting JH2O2 production and, in turn, the rate at which energy is expended via NNT-mediated proton conductance. This variable flux through redox circuits provides a potential compensatory mechanism for fine-tuning energy expenditure to energy balance in real-time.
Hepatocellular carcinoma (HCC) is the most prevalent form of liver malignancy and carries poor prognoses due to late presentation of symptoms. Treatment of late-stage HCC relies heavily on chemotherapeutics, many of which target cellular energy metabolism. A key platform for testing candidate chemotherapeutic compounds is the intrahepatic orthotopic xenograft (IOX) model in rodents. Translational efficacy from the IOX model to clinical use is limited (in part) by variation in the metabolic phenotypes of the tumor-derived cells that can be induced by selective adaptation to subculture conditions.
In this study, a detailed multilevel systems approach combining microscopy, respirometry, potentiometry, and extracellular flux analysis (EFA) was utilized to examine metabolic adaptations that occur under aglycemic growth media conditions in HCC-derived (HEPG2) cells. We hypothesized that aglycemic growth would result in adaptive “aerobic poise” characterized by enhanced capacity for oxidative phosphorylation over a range of physiological energetic demand states.
Aglycemic growth did not invoke adaptive changes in mitochondrial content, network complexity, or intrinsic functional capacity/efficiency. In intact cells, aglycemic growth markedly enhanced fermentative glycolytic substrate-level phosphorylation during glucose refeeding and enhanced responsiveness of both fermentation and oxidative phosphorylation to stimulated energy demand. Additionally, aglycemic growth induced sensitivity of HEPG2 cells to the provitamin menadione at a 25-fold lower dose compared to control cells.
These findings indicate that growth media conditions have substantial effects on the energy metabolism of subcultured tumor-derived cells, which may have significant implications for chemotherapeutic sensitivity during incorporation in IOX testing panels. Additionally, the metabolic phenotyping approach used in this study provides a practical workflow that can be incorporated with IOX screening practices to aid in deciphering the metabolic underpinnings of chemotherapeutic drug sensitivity.
Alterations to branched-chain keto acid (BCKA) oxidation have been implicated in a wide variety of human diseases, ranging from diabetes to cancer. Although global shifts in BCKA metabolism—evident by gene transcription, metabolite profiling, and in vivo flux analyses have been documented across various pathological conditions, the underlying biochemical mechanism(s) within the mitochondrion remain largely unknown. In vitro experiments using isolated mitochondria represent a powerful biochemical tool for elucidating the role of the mitochondrion in driving disease. Such analyses have routinely been utilized across disciplines to shed valuable insight into mitochondrial-linked pathologies. That said, few studies have attempted to model in vitro BCKA oxidation in isolated organelles. The impetus for the present study stemmed from the knowledge that complete oxidation of each of the three BCKAs involves a reaction dependent upon bicarbonate and ATP, both of which are not typically included in respiration experiments. Based on this, it was hypothesized that the inclusion of exogenous bicarbonate and stimulation of respiration using physiological shifts in ATP-free energy, rather than excess ADP, would allow for maximal BCKA-supported respiratory flux in isolated mitochondria. This hypothesis was confirmed in mitochondria from several mouse tissues, including heart, liver and skeletal muscle. What follows is a thorough characterization and validation of a novel biochemical tool for investigating BCKA metabolism in isolated mitochondria.
Cholinergic and sympathetic counter-regulatory networks control numerous physiologic functions including learning/memory/cognition, stress responsiveness, blood pressure, heart rate and energy balance. As neurons primarily utilize glucose as their primary metabolic energy source, we generated mice with increased glycolysis in cholinergic neurons by specific deletion of the fructose-2,6-phosphatase protein TIGAR. Steady-state and stable isotope flux analyses demonstrated increased rates of glycolysis, acetyl-CoA production, acetylcholine levels and density of neuromuscular synaptic junction clusters with enhanced acetylcholine release. The increase in cholinergic signaling reduced blood pressure and heart rate with a remarkable resistance to cold-induced hypothermia. These data directly demonstrate that increased cholinergic signaling through the modulation of glycolysis has several metabolic benefits particularly to increase energy expenditure and heat production upon cold exposure.
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