Rates Of Mitochondrial Oxidation Research Articles

Glucagon promotes increased hepatic mitochondrial oxidation and pyruvate carboxylase flux in humans with fatty liver disease

We assessed invivo rates of hepatic mitochondrial oxidation, gluconeogenesis, and β-hydroxybutyrate (β-OHB) turnover by positional isotopomer NMR tracer analysis (PINTA) in individuals with metabolic-dysfunction-associated steatotic liver (MASL) (fatty liver) and MASL disease (MASLD) (steatohepatitis) compared with BMI-matched control participants with no hepatic steatosis. Hepatic fat content was quantified by localized 1H magnetic resonance spectroscopy (MRS). We found that invivo rates of hepatic mitochondrial oxidation were unaltered in the MASL and MASLD groups compared with the control group. A physiological increase in plasma glucagon concentrations increased invivo rates of hepatic mitochondrial oxidation by 50%-75% in individuals with and without MASL and increased rates of glucose production by ∼50% in the MASL group, which could be attributed in part to an ∼30% increase in rates of mitochondrial pyruvate carboxylase flux. These results demonstrate that (1) rates of hepatic mitochondrial oxidation are not substantially altered in individuals with MASL and MASLD and (2) glucagon increases rates of hepatic mitochondrial oxidation.

Combined effects of salinity and intermittent hypoxia on mitochondrial capacity and reactive oxygen species efflux in the Pacific oyster, Crassostrea gigas.

Coastal environments commonly experience fluctuations in salinity and hypoxia-reoxygenation (H/R) stress that can negatively affect mitochondrial functions of marine organisms. Although intertidal bivalves are adapted to these conditions, the mechanisms that sustain mitochondrial integrity and function are not well understood. We determined the rates of respiration and reactive oxygen species (ROS) efflux in the mitochondria of oysters, Crassostrea gigas, acclimated to high (33 psu) or low (15 psu) salinity, and exposed to either normoxic conditions (control; 21% O2) or short-term hypoxia (24 h at <0.01% O2) and subsequent reoxygenation (1.5 h at 21% O2). Further, we exposed isolated mitochondria to anoxia in vitro to assess their ability to recover from acute (∼10 min) oxygen deficiency (<0.01% O2). Our results showed that mitochondria of oysters acclimated to high or low salinity did not show severe damage and dysfunction during H/R stress, consistent with the hypoxia tolerance of C. gigas. However, acclimation to low salinity led to improved mitochondrial performance and plasticity, indicating that 15psu might be closer to the metabolic optimum of C. gigas than 33psu. Thus, acclimation to low salinity increased mitochondrial oxidative phosphorylation rate and coupling efficiency and stimulated mitochondrial respiration after acute H/R stress. However, elevated ROS efflux in the mitochondria of low-salinity-acclimated oysters after acute H/R stress indicates a possible trade-off of higher respiration. The high plasticity and stress tolerance of C. gigas mitochondria may contribute to the success of this invasive species and facilitate its further expansion into brackish regions such as the Baltic Sea.

1558-P: The Mitochondrial Calcium Uniporter Regulates Hepatic Mitochondrial Oxidation and Intracellular Redox In Vivo

The mitochondrial calcium uniporter (MCU) facilitates mitochondrial calcium influx, and several enzymes that catalyze key steps of the TCA cycle require calcium for their activation. As such, it is generally accepted that rates of mitochondrial oxidation correlate with mitochondrial calcium activity. Decreased rates of hepatic mitochondrial oxidation are associated with metabolic dysfunction and ectopic lipid accumulation as seen with type 2 diabetes and nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis. Despite this, a role for MCU and mitochondrial calcium in regulating hepatic mitochondrial oxidation is still unclear. To address this question, we generated a liver-specific MCU knockout (MCU KO) mouse model which displayed reduced mitochondrial calcium ([Ca2+]mt; p&amp;lt;0.05) and increased cytosolic calcium ([Ca2+]cyt; p&amp;lt;0.01). Despite decreased [Ca2+]mt, we found that deletion of hepatic MCU increased rates of isocitrate dehydrogenase flux (VIDH; p&amp;lt;0.01), α-ketoglutarate dehydrogenase flux (VOGDH; p&amp;lt;0.01), and succinate dehydrogenase flux (VSDH; p&amp;lt;0.05) in vivo, using a novel [13C5]glutamine tracer infusion technique (Q-Flux). Further, we show that rates of [14C16]palmitate oxidation (p&amp;lt;0.01) and intrahepatic lipolysis (p&amp;lt;0.05) were significantly increased in liver slices collected from MCU KO mice compared to WT mice, and were associated with reductions in hepatic triacylglycerol content in vivo (p&amp;lt;0.01). Hepatocellular redox state was also altered with MCU KO, as indicated by a decreased cytosolic redox ratio ([lactate]:[pyruvate]; p&amp;lt;0.05) and increased mitochondrial redox ratio ([β-hydroxybutyrate]:[acetoacetate]; p&amp;lt;0.01). Conclusion: Here we show that increases in [Ca2+]cyt potently increase hepatic mitochondrial fat oxidation and alters hepatic redox balance to reduce hepatic lipid content independently of [Ca2+]mt in vivo and targeting [Ca2+]cyt may represent a novel therapeutic target to treat NAFLD. Disclosure T.E.Lamoia: None. B.T.Hubbard: None. M.Guerra: None. R.Goodman: None. M.H.Nathanson: None. G.I.Shulman: Consultant; Novo Nordisk, DiCerna Pharmaceuticals, Inc., Bayer Consumer Care AG, Kriya Therapeutics, Arrowhead Pharmaceuticals, Inc., ESPERION Therapeutics, Inc., Other Relationship; AstraZeneca, Merck &amp; Co., Inc., Janssen Research &amp; Development, LLC, iMetabolic Biopharma Corporation, Maze Therapeutics, 89bio, Inc., Equator Therapeutics, Inc., Generian Pharmaceuticals, Fortress Biotech, Inc., OrsoBio, Aro Biotherapeutics Company, Stock/Shareholder; Levels Health, Inc. Funding National Institutes of Health (F31DK126362, T32GM007324)

Evidence for inefficient contraction and abnormal mitochondrial activity in sarcopenia using magnetic resonance spectroscopy.

Mitochondrial dysfunction has been implicated in sarcopenia. 31 P magnetic resonance spectroscopy (MRS) enables non-invasive measurement of adenosine triphosphate (ATP) synthesis rates to probe mitochondrial function. Here, we assessed muscle energetics in older sarcopenic and non-sarcopenic men and compared with muscle biopsy-derived markers of mitochondrial function. Twenty Chinese men with sarcopenia (SARC, age=73.1±4.1years) and 19 healthy aged and sex-matched controls (CON, age=70.3±4.2years) underwent assessment of strength, physical performance, and magnetic resonance imaging. Concentrations of phosphocreatine (PCr), ATP and inorganic phosphate (Pi) as well as muscle pH were measured at rest and during an interleaved rest-exercise protocol to probe muscle mitochondrial function. Results were compared to biopsy-derived mitochondrial complex activity and expression to understand underlying metabolic perturbations. Despite matched muscle contractile power (strength/cross-sectional area), the ATP contractile cost was higher in SARC compared with CON (low-intensity exercise: 1.06±0.59 vs. 0.57±0.22, moderate: 0.93±0.43 vs. 0.58±0.68, high: 0.70±0.57 vs. 0.43±0.51mmolL-1 min-1 bar-1 cm-2 , P=0.003, <0.0001 and <0.0001, respectively). Post-exercise mitochondrial oxidative synthesis rates (a marker of mitochondrial function) tended to be longer in SARC but did not reach significance (17.3±6.4 vs. 14.6±6.5mmolL-1 min-1 , P=0.2). However, relative increases in end-exercise ADP in SARC (31.8±9.9 vs. 24.0±7.3mmolL-1 , P=0.008) may have been a compensatory mechanism. Mitochondrial complex activity was found to be associated with exercise-induced drops in PCr [citrate synthetase activity (CS), Spearman correlation rho=-0.42, P=0.03] and end-exercise ADP (complex III, rho=-0.52, P=0.01; CS rho=-0.45, P=0.02; SDH rho=-0.45, P=0.03), with CS also being strongly associated with the PCr recovery rate following low intensity exercise (rho=-0.47, P=0.02), and the cost of contraction at high intensity (rho=-0.54, P=0.02). Interestingly, at high intensity, the fractional contribution of oxidative phosphorylation to exercise was correlated with activity in complex II (rho=0.5, P=0.03), CS (rho=0.47, P=0.02) and SDH (rho=0.46, P=0.03), linking increased mitochondrial complex activity with increased ability to generate energy through oxidative pathways. This study used 31 P MRS to assess ATP utilization and resynthesis in sarcopenic muscle and demonstrated abnormal increases in the energy cost during exercise and perturbed mitochondrial energetics in recovery. Associations between mitochondrial complex activity and the fractional contribution to energy requirement during exercise indicate increased ability to generate energy oxidatively in those with better mitochondrial complex activity.

Antagonistic Interactions in Mitochondria ROS Signaling Responses to Manganese.

Antagonistic interaction refers to opposing beneficial and adverse signaling by a single agent. Understanding opposing signaling is important because pathologic outcomes can result from adverse causative agents or the failure of beneficial mechanisms. To test for opposing responses at a systems level, we used a transcriptome-metabolome-wide association study (TMWAS) with the rationale that metabolite changes provide a phenotypic readout of gene expression, and gene expression provides a phenotypic readout of signaling metabolites. We incorporated measures of mitochondrial oxidative stress (mtOx) and oxygen consumption rate (mtOCR) with TMWAS of cells with varied manganese (Mn) concentration and found that adverse neuroinflammatory signaling and fatty acid metabolism were connected to mtOx, while beneficial ion transport and neurotransmitter metabolism were connected to mtOCR. Each community contained opposing transcriptome-metabolome interactions, which were linked to biologic functions. The results show that antagonistic interaction is a generalized cell systems response to mitochondrial ROS signaling.

Inhibition of pathologic immunoglobulin E in food allergy by EBF-2 and active compound berberine associated with immunometabolism regulation.

Food allergy is a significant public health problem with limited treatment options. As Food Allergy Herbal Formula 2 (FAHF-2) showed potential as a food allergy treatment, we further developed a purified version named EBF-2 and identified active compounds. We investigated the mechanisms of EBF-2 on IgE-mediated peanut (PN) allergy and its active compound, berberine, on IgE production. IgE plasma cell line U266 cells were cultured with EBF-2 and FAHF-2, and their effects on IgE production were compared. EBF-2 was evaluated in a murine PN allergy model for its effect on PN-specific IgE production, number of IgE+ plasma cells, and PN anaphylaxis. Effects of berberine on IgE production, the expression of transcription factors, and mitochondrial glucose metabolism in U266 cells were evaluated. EBF-2 dose-dependently suppressed IgE production and was over 16 times more potent than FAHF-2 in IgE suppression in U266 cells. EBF-2 significantly suppressed PN-specific IgE production (70%, p<0.001) and the number of IgE-producing plasma cells in PN allergic mice, accompanied by 100% inhibition of PN-induced anaphylaxis and plasma histamine release (p<0.001) without affecting IgG1 or IgG2a production. Berberine markedly suppressed IgE production, which was associated with suppression of XBP1, BLIMP1, and STAT6 transcription factors and a reduced rate of mitochondrial oxidation in an IgE-producing plasma cell line. EBF-2 and its active compound berberine are potent IgE suppressors, associated with cellular regulation of immunometabolism on IgE plasma cells, and may be a potential therapy for IgE-mediated food allergy and other allergic disorders.

Abstract 9485: Role of Fatty Acid Oxidation in Heart Function

Introduction: More than 70% of the energy used by myocardial cells is from fatty acid oxidation (FAO), but excessive FAO may also cause cardiac dysfunction due to increased cardiac oxygen consumption. Inhibition of cardiac FAO has been tested in animal models and human subjects with chronic heart failure, but its absolute benefit is still unclear. Here, we generated myocyte-specific ACC1 and 2 knockout mice (ACC dHKO) to investigate the role of FAO in cardiac function. Methods: ACC dHKO were generated by crossing ACC1 and ACC2 floxed mice with Myh6-CRE transgenic mice. Cardiac FAO was measured by Langendorff perfusion. Etomoxir was administered to inhibit FA oxidation. FA composition and cardiolipins were measured by LC/MS and mitochondrial electron transport chain (mETC) was assessed using Seahorse. Results: ACC dHKO mice developed a dilated cardiomyopathy at 7-8 weeks, which progressed with age (A, B). Perfusion studies demonstrated that hearts from ACC mice exhibit increased FA oxidation. Cardiac fatty acid composition analysis showed a severe deficiency of cardiolipin due to a significant decrease in linoleic acid, presumably due to excessive FA oxidation (C). Cardiac mETC in ACC dHKO was significantly compromised compared to control mouse (D). Etomoxir, which blocks FAO, normalized levels of cardiac linoleic acid and cardiolipins, and prevented the development of the dilated cardiomyopathy in ACC dHKO (E). Conclusions: Deletion of ACCs in heart led to unrestrained FAO, mitochondrial dysfunction, and a dilated cardiomyopathy., which could be prevented by administration of drugs that suppress FA oxidation. This study provides evidence that elevated rates of mitochondrial FA oxidation can be detrimental and suggests that efforts to further increase FA oxidation in a failing heart may not enhance function.

Obesity elicits a unique metabolomic signature in human airway smooth muscle cells.

Obesity can aggravate asthma by enhancing airway hyperresponsiveness (AHR) and attenuating response to treatment. However, the precise mechanisms linking obesity and asthma remain unknown. Human airway smooth muscle (HASM) cells exhibit amplified excitation-contraction (EC) coupling and force generation in obesity. Therefore, we posit that airway smooth muscle (ASM) cells obtained from obese donors manifest a metabolomic phenotype distinct from that of nonobese donor cells and that a differential metabolic phenotype, at least in part, drives enhanced ASM cell EC coupling. HASM cells derived from age-, sex-, and race-matched nonobese [body mass index (BMI) ≤ 24.9 kg·m-2] and obese (BMI ≥ 29.9 kg·m-2) lung donors were subjected to unbiased metabolomic screening. The unbiased metabolomic screening identified differentially altered metabolites linked to glycolysis and citric acid cycle in obese donor-derived cells compared with nonobese donor cells. The Seahorse assay measured the bioenergetic profile based on glycolysis, mitochondrial respiration, palmitate oxidation, and glutamine oxidation rates in HASM cells. Glycolytic rate and capacity were elevated in obese donor-derived HASM cells, whereas mitochondrial respiration, palmitate oxidation, and glutamine oxidation rates were comparable between obese and nonobese groups. PFKFB3 mRNA and protein expression levels were also elevated in obese donor-derived HASM cells. Furthermore, pharmacological inhibition of PFKFB3 attenuated agonist-induced myosin light chain (MLC) phosphorylation in HASM cells derived from obese and nonobese donors. Our findings identify elevated glycolysis as a signature metabolic phenotype of obesity and inhibition of glycolysis attenuates MLC phosphorylation in HASM cells. These findings identify novel therapeutic targets to mitigate AHR in obesity-associated asthma.

SAT104 - The effect of glucagon on rates of hepatic mitochondrial oxidation and pyruvate carboxylase flux in man assessed by positional isotopomer tracer analysis (PINTA)

SAT104 - The effect of glucagon on rates of hepatic mitochondrial oxidation and pyruvate carboxylase flux in man assessed by positional isotopomer tracer analysis (PINTA)

219-LB: The Mitochondrial Calcium Uniporter Regulates Hepatic Lipid Accumulation and Mitochondrial Oxidation

The mitochondrial calcium uniporter (MCU) facilitates mitochondrial calcium influx, and several enzymes that catalyze key steps of the tricarboxylic acid (TCA) cycle require calcium for their activation. As such, it is generally accepted that rates of mitochondrial oxidation correlate with mitochondrial calcium activity. Decreased rates of hepatic mitochondrial oxidation are associated with metabolic dysfunction and ectopic lipid accumulation as seen with type 2 diabetes and nonalcoholic fatty liver disease. Despite this, a role for MCU and mitochondrial calcium in regulating hepatic mitochondrial oxidation is still unclear. To address this question, we generated a liver-specific MCU knockout (MCU KO) mouse model which displayed significantly reduced mitochondrial calcium ([Ca2+]mt, p&amp;lt;0.05) and increased cytosolic calcium ([Ca2+]cyt, p&amp;lt;0.01) in isolated hepatocytes. Despite decreased [Ca2+]mt, we found that deletion of hepatic MCU resulted in increased rates of isocitrate dehydrogenase flux (VIDH, p&amp;lt;0.01) , α-ketoglutarate dehydrogenase flux (VOGDH, p&amp;lt;0.01) , and succinate dehydrogenase flux (VSDH, p&amp;lt;0.05) , using a novel in vivo [13C5]glutamine tracer technique. Further, we report that rates of [14C16]palmitate oxidation (p&amp;lt;0.01) and intrahepatic lipolysis (p&amp;lt;0.05) were significantly increased in precision-cut liver slices collected from MCU KO mice compared to WT, consistent with [Ca2+]cyt activation of CAMKII (p&amp;lt;0.01) and ATGL (p&amp;lt;0.001) in MCU KO mice, and were associated with reductions in hepatic triacylglycerol content in vivo (p&amp;lt;0.01) . Taken together, these data demonstrate that hepatic mitochondrial activity can be dissociated from mitochondrial calcium concentrations and reveal a key role for cytosolic calcium and mitochondrial anaplerosis in the regulation of hepatic mitochondrial fat oxidation. Disclosure T. E. Lamoia: n/a. B. T. Hubbard: None. M. Guerra: None. R. Goodman: None. M. H. Nathanson: None. V. Mootha: Advisory Panel; 5AM Ventures, LLC, Janssen Research &amp; Development, LLC. G. I. Shulman: Advisory Panel; 89bio, Inc., AstraZeneca, Equator Therapeutics, Inc., Janssen Research &amp; Development, LLC, Merck &amp; Co., Inc., Consultant; DiCerna Pharmaceuticals, Inc. , Novo Nordisk, Other Relationship; Generian Pharmaceuticals, iMetabolic Biopharma Corporation, Maze Therapeutics, The Liver Company, Stock/Shareholder; Levels Health, Inc. . Funding F31 DK126362/DK/NIDDK NIH HHST32 GM007324/GM/NIGMS NIH HHS

Rates of warming impact oxidative stress in zebrafish (Danio rerio).

Potentially negative effects of thermal variation on physiological functions may be modulated by compensatory responses, but their efficacy depends on the time scale of phenotypic adjustment relative to the rate of temperature change. Increasing temperatures in particular can affect mitochondrial bioenergetics and rates of reactive oxygen species (ROS) production. Our aim was to test whether different rates of temperature increase affect mitochondrial bioenergetics and modulate oxidative stress. We exposed zebrafish (Danio rerio) to warming from 20°C to 28°C over 3, 6, 24 or 48 h, and compared these with a control group that was kept at constant 20°C. Fish exposed to the fastest (3 h) and slowest (48 h) rates of warming had significantly higher rates of H2O2 production relative to the control treatment, and the proportion of O2 converted to H2O2 (H2O2/O2 ratio) was significantly greater in these groups. However, ROS production was not paralleled by differences in mitochondrial substrate oxidation rates, leak respiration rates or coupling (respiratory control ratios). Increased rates of ROS production did not lead to damage of proteins or membranes, which may be explained by a moderate increase in catalase activity at the fastest, but not the slowest, rate of warming. The increase in ROS production at the slowest rate of warming indicates that even seemingly benign environments may be stressful. Understanding how animals respond to different rates of temperature change is important, because the rate determines the time period for phenotypic adjustments and it also alters the environmental thermal signal that triggers compensatory pathways.

282-OR: The Effect of Glucagon on Rates of Hepatic Mitochondrial Oxidation and Pyruvate Carboxylase Flux in Man Assessed by Positional Isotopomer NMR Tracer Analysis (PINTA)

Dual GLP-1/glucagon agonists are now in clinical trials for the treatment of T2D and NASH. In addition to promoting further reduction in food intake on top of GLP-1, the glucagon component of these dual agonists may also promote increased rates of hepatic mitochondrial fat oxidation. In order to investigate this possibility we assessed the glucagon-specific effects of a dual agonist on rates of hepatic mitochondrial oxidation (VCS) and pyruvate carboxylase flux (VPC) in humans by infusing glucagon to achieve a physiological increase in plasma glucagon concentrations in combination with a novel 13C PINTA method to assess rates of hepatic VCS and VPC in 15 healthy volunteers using a paired study design. Following an overnight fast a 3-h IV infusion of [3-13C]lactate (4.3 μmol/Kg-min) and [1,2,3,4,5,6,6-2H7]glucose (0.22 μmol/Kg-min) was begun and during the last 2 hours glucagon (GLG) was co-infused [6 ng/(Kg-min)] or no GLG (CON). The GLG infusion resulted in a ~2.4 fold increase in plasma GLG conc. (from 75±11 to 183±20 pg/mL; P&amp;lt;0.0001), which was associated with an ~20% increase in both plasma C-peptide (from 1.89±0.17 to 2.30±0.26 ng/mL; P=0.02) and plasma insulin conc. (from 9.9±2.0 to 12.0±2.4 μU/mL; P=0.06). Plasma glucose conc. increased from 4.90±0.16 to 5.64±0.19 mmol/L (P=0.0001) which was associated with a tendency for increased rates of glucose production (CON:1042±123 vs. GLG:1148±122 μmol/min; p=0.086) despite no change in rates of VPC flux (CON: 403±69 vs. GLG: 373±55 μmol/min; p=NS). In contrast, this physiological increase in plasma GLG concentrations caused an 85% increase in rates of hepatic VCS (P&amp;lt;0.05). Conclusion: Taken together these studies demonstrate that a physiological increase in plasma glucagon promotes increased rates of hepatic mitochondrial oxidation in humans, which may provide additional benefits to the anorexic effects of dual GLP-1/glucagon agonists to reduce hepatic steatosis. Disclosure K. Petersen: Advisory Panel; Spouse/Partner; AstraZeneca, iMetabolic Biopharma Corporation, Janssen Research &amp; Development, LLC, Merck &amp; Co., Inc., Consultant; Spouse/Partner; Aegerion Pharmaceuticals Inc., Novo Nordisk, Research Support; Self; Gilead Sciences, Inc., Merck &amp; Co., Inc. G. I. Shulman: Consultant; Self; 89bio, Inc., BridgeBio, Ionis Pharmaceuticals, Maze Therapeutics, Novo Nordisk, Other Relationship; Self; AstraZeneca, Esperion Therapeutics, Inc, Generian Pharmaceuticals, Inc., Gilead Sciences, Inc., iMetabolic Biopharma Corporation, Janssen Research &amp; Development, LLC, Merck &amp; Co., Inc., The Liver Company. Funding Merck &amp; Co., Inc. (MISP583402); National Institutes of Health (R01DK113984)

Mitochondrial Fission Governed by Drp1 Regulates Exogenous Fatty Acid Usage and Storage in Hela Cells.

In the presence of high abundance of exogenous fatty acids, cells either store fatty acids in lipid droplets or oxidize them in mitochondria. In this study, we aimed to explore a novel and direct role of mitochondrial fission in lipid homeostasis in HeLa cells. We observed the association between mitochondrial morphology and lipid droplet accumulation in response to high exogenous fatty acids. We inhibited mitochondrial fission by silencing dynamin-related protein 1(DRP1) and observed the shift in fatty acid storage-usage balance. Inhibition of mitochondrial fission resulted in an increase in fatty acid content of lipid droplets and a decrease in mitochondrial fatty acid oxidation. Next, we overexpressed carnitine palmitoyltransferase-1 (CPT1), a key mitochondrial protein in fatty acid oxidation, to further examine the relationship between mitochondrial fatty acid usage and mitochondrial morphology. Mitochondrial fission plays a role in distributing exogenous fatty acids. CPT1A controlled the respiratory rate of mitochondrial fatty acid oxidation but did not cause a shift in the distribution of fatty acids between mitochondria and lipid droplets. Our data reveals a novel function for mitochondrial fission in balancing exogenous fatty acids between usage and storage, assigning a role for mitochondrial dynamics in control of intracellular fuel utilization and partitioning.

Progesterone receptor membrane component 1 reduces cardiac steatosis and lipotoxicity via activation of fatty acid oxidation and mitochondrial respiration

Obesity is implicated in cardiovascular disease and heart failure. When fatty acids are transported to and not adequately oxidized in cardiac cells, they accumulate, causing lipotoxicity in the heart. Since hepatic progesterone receptor membrane component 1 (Pgrmc1) suppressed de novo lipogenesis in a previous study, it was questioned whether cardiac Pgrmc1 protects against lipotoxicity. Hence, we focused on the role of cardiac Pgrmc1 in basal (Resting), glucose-dominant (Refed) and lipid-dominant high-fat diet (HFD) conditions. Pgrmc1 KO mice showed high FFA levels and low glucose levels compared to wild-type (WT) mice. Pgrmc1 KO mice presented low number of mitochondrial DNA copies in heart, and it was concomitantly observed with low expression of TCA cycle genes and oxidative phosphorylation genes. Pgrmc1 absence in heart presented low fatty acid oxidation activity in all conditions, but the production of acetyl-CoA and ATP was in pronounced suppression only in HFD condition. Furthermore, HFD Pgrmc1 KO mice resulted in high cardiac fatty acyl-CoA levels and TG level. Accordingly, HFD Pgrmc1 KO mice were prone to cardiac lipotoxicity, featuring high levels in markers of inflammation, endoplasmic reticulum stress, oxidative stress, fibrosis, and heart failure. In vitro study, it was also confirmed that Pgrmc1 enhances rates of mitochondrial respiration and fatty acid oxidation. This study is clinically important because mitochondrial defects in Pgrmc1 KO mice hearts represent the late phase of cardiac failure.

Inhibition of autophagy aggravates molybdenum-induced mitochondrial dysfunction by aggravating oxidative stress in duck renal tubular epithelial cells

Excessive molybdenum (Mo) has adverse effects on animals. To elucidate the effects of autophagy on Mo-induced nephrotoxicity, the duck renal tubular epithelial cells were cultured in medium in absence and presence of (NH4)6Mo7O24.4H2O (0, 480, 720, 960 μM Mo), 3-Methyladenine (3-MA) (2.5 μM), and the combination of Mo and 3-MA for 12 h. After 12 h exposure, the MDC staining, morphologic observation, LC3 puncta, cell viability, autophagy-related genes mRNA and proteins levels, lactate dehydrogenase (LDH) release, reactive oxygen species (ROS) level, antioxidant indices, mitochondrial membrane potential (MMP), mitochondrial mass, mitochondrial respiratory control ratio (RCR) and oxidative phosphorylation rate (OPR) were determined. The results showed that excessive Mo exposure significantly elevated the number of autophagosome and LC3 puncta, upregulated Beclin-1, Atg5, LC3A and LC3B mRNA levels, and LC3II/LC3I and Beclin-1 protein levels, decreased mTOR, p62 and Dynein mRNA levels and p62 protein level. Besides, co-treatment with Mo and 3-MA dramatically increased LDH release, ROS level, hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents as well as cell dam age, reduced cell viability, the activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT), MMP, mitochondrial mass, mitochondrial RCR and OPR compared to treatment with Mo alone. Taken together, these results suggest that excessive Mo exposure can induce autophagy in duck renal tubular epithelial cells, inhibition of autophagy aggravates Mo-induced mitochondrial dysfunction by regulating oxidative stress.

224-LB: Role of the Mitochondrial Calcium Uniporter in the Regulation of Hepatic Mitochondrial Metabolism and Gluconeogenesis

The mitochondrial calcium uniporter (MCU) is a key regulator of mitochondrial calcium, however, the role of MCU in mediating hepatic mitochondrial metabolism and gluconeogenesis has received limited attention. In order to address these questions, we examined rates of mitochondrial oxidation in both liver slices and in awake liver-specific MCU knockout (MCU KO) mice. We found that deletion of MCU significantly increases glucagon-induced intrahepatic lipolysis (p&amp;lt;0.04) and glucose production (p&amp;lt;0.04) in ex vivo liver slices. We also observed significantly reduced liver triglyceride accumulation (p&amp;lt;0.05) and increased hepatic long chain acyl-CoA (p&amp;lt;0.02) and acetyl-CoA (p=0.05) content in MCU KO mice, consistent with increased intrahepatic lipolysis and fat oxidation. This effect was not due to changes in white adipose tissue lipolysis. Deletion of MCU also impaired glucose tolerance in chow-fed mice, indicating dysregulated hepatic glucose production in these mice. These metabolic alterations could be attributed to reduced mitochondrial buffering of cytoplasmic calcium, leading to a 77% increase in cytoplasmic calcium (p&amp;lt;0.0001). This led to activation of CAMKII (p&amp;lt;0.04) and subsequent increase in ATGL phosphorylation (p&amp;lt;0.04) in hepatocytes lacking MCU. In conclusion, these results demonstrate a critical role for MCU in mediating hepatic mitochondrial oxidation and hepatic gluconeogenesis. Disclosure T.E. LaMoia: None. J. Lee: None. M. Guerra: None. R. Goodman: None. G.I. Shulman: Advisory Panel; Self; AstraZeneca, Janssen Research &amp; Development, LLC, Merck &amp; Co., Inc. Advisory Panel; Spouse/Partner; Merck &amp; Co., Inc. Consultant; Self; Novo Nordisk A/S. Consultant; Spouse/Partner; Novo Nordisk A/S. Other Relationship; Self; Gilead Sciences, Inc., iMetabolic Biopharma Corporation. Other Relationship; Spouse/Partner; iMetabolic Biopharma Corporation. Other Relationship; Self; Maze Therapeutics. Funding National Institutes of Health (T32GM0007324)

Antagonistic Pleiotropy in Mitochondria ROS Signaling Responses to Manganese

SUMMARY Antagonistic pleiotropy refers to opposing beneficial and adverse signaling by a single agent. Understanding opposing signaling is important because pathologic outcome can result from adverse causative agents or failure of beneficial mechanisms. To test for opposing responses at a systems level, we used a transcriptome-metabolome-wide association study (TMWAS) with the rationale that metabolite changes provide a phenotypic readout of gene expression, and gene expression provides a phenotypic readout of signaling metabolites. We incorporated measures of mitochondrial oxidative stress (mtOx) and oxygen consumption rate (mtOCR) with TMWAS of cells with varied manganese (Mn) concentration and found that adverse neuroinflammatory signaling and fatty acid metabolism were connected to mtOx while beneficial ion transport and neurotransmitter metabolism were connected to mtOCR. Each community contained opposing transcriptome-metabolome interactions, which were linked to biologic functions. The results show that antagonistic pleiotropy is a generalized cell systems response to mitochondrial ROS signaling.

Mismatched light and temperature cues disrupt locomotion and energetics via thyroid-dependent mechanisms.

Animals integrate information from different environmental cues to maintain performance across environmental gradients. Increasing average temperature and variability induced by climate change can lead to mismatches between seasonal cues. We used mosquitofish (Gambusia holbrooki) to test the hypotheses that mismatches between seasonal temperature and light regimes (short days and warm temperature and vice versa) decrease swimming performance, metabolic rates and mitochondrial efficiency and that the responses to light and temperature are mediated by thyroid hormone. We show that day length influenced thermal acclimation of swimming performance through thyroid-dependent mechanisms. Oxygen consumption rates were influenced by acclimation temperature and thyroid hormone. Mitochondrial substrate oxidation rates (state three rates) were modified by the interaction between temperature and day length, and mitochondrial efficiency (P/O ratios) increased with warm acclimation. Using P/O ratios to calibrate metabolic (oxygen consumption) scope showed that oxygen consumption did not predict adenosine triphosphate (ATP) production. Unlike oxygen consumption, ATP production was influenced by day length in a thyroid-dependent manner. Our data indicate that oxygen consumption alone should not be used as a predictor of ATP production. Overall, the effects of thyroid hormone on locomotion and energetics were reversed by mismatches such as warm temperatures on short days. We predict that mid to high latitudes in North America and Asia will be particularly affected by mismatches as a result of high seasonality and predicted warming over the next 50 years.

Hypertonic external medium represses cellular respiration and promotes Warburg/Crabtree effect.

Hyperosmotic conditions are associated to several pathological states. In this article, we evaluate the consequence of hyperosmotic medium on cellular energy metabolism. We demonstrate that exposure of cells to hyperosmotic conditions immediately reduces the mitochondrial oxidative phosphorylation rate. This causes an increase in glycolysis, which represses further respiration. This is known as the Warburg or Crabtree effect. In addition to aerobic glycolysis, we observed two other cellular responses that would help to preserve cellular ATP level and viability: A reduction in the cellular ATP turnover rate and a partial mitochondrial uncoupling which is expected to enhance ATP production by Krebs cycle. The latter is likely to constitute another metabolic adaptation to compensate for deficient oxidative phosphorylation that, importantly, is not dependent on glucose.

Controlled-release mitochondrial protonophore (CRMP) reverses dyslipidemia and hepatic steatosis in dysmetabolic nonhuman primates.

Nonalcoholic fatty liver disease (NAFLD) is estimated to affect up to one-third of the general population, and new therapies are urgently required. Our laboratory previously developed a controlled-release mitochondrial protonophore (CRMP) that is functionally liver-targeted and promotes oxidation of hepatic triglycerides. Although we previously demonstrated that CRMP safely reverses hypertriglyceridemia, fatty liver, hepatic inflammation, and fibrosis in diet-induced rodent models of obesity, there remains a critical need to assess its safety and efficacy in a model highly relevant to humans. Here, we evaluated the impact of longer-term CRMP treatment on hepatic mitochondrial oxidation and on the reversal of hypertriglyceridemia, NAFLD, and insulin resistance in high-fat, fructose-fed cynomolgus macaques (n = 6) and spontaneously obese dysmetabolic rhesus macaques (n = 12). Using positional isotopomer nuclear magnetic resonance tracer analysis (PINTA), we demonstrated that acute CRMP treatment (single dose, 5 mg/kg) increased rates of hepatic mitochondrial fat oxidation by 40%. Six weeks of CRMP treatment reduced hepatic triglycerides in both nonhuman primate models independently of changes in body weight, food intake, body temperature, or adverse reactions. CRMP treatment was also associated with a 20 to 30% reduction in fasting plasma triglycerides and low-density lipoprotein (LDL)-cholesterol in dysmetabolic nonhuman primates. Oral administration of CRMP reduced endogenous glucose production by 18%, attributable to a 20% reduction in hepatic acetyl-coenzyme A (CoA) content [as assessed by whole-body β-hydroxybutyrate (β-OHB) turnover] and pyruvate carboxylase flux. Collectively, these studies provide proof-of-concept data to support the development of liver-targeted mitochondrial uncouplers for the treatment of metabolic syndrome in humans.