Respiration Of Pyruvate Research Articles

Inducible and reversible SOD2 knockdown in mouse skeletal muscle drives impaired pyruvate oxidation and reduced metabolic flexibility.

Skeletal muscle mitochondrial dysfunction is a key characteristic of aging muscle and contributes to age related diseases such as sarcopenia, frailty, and type 2 diabetes. Mitochondrial oxidative distress has been implicated as a driving factor in these age-related diseases, however whether it is a cause, or a consequence of mitochondrial dysfunction remains to be determined. The development of more flexible genetic models is an important tool to test the mechanistic role of mitochondrial oxidative stress on skeletal muscle metabolic dysfunction. We characterize a new model of inducible and reversible mitochondrial redox stress using a tetracycline controlled skeletal muscle specific short hairpin RNA targeted to superoxide dismutase 2 (iSOD2). iSOD2 KD and control (CON) animals were administered doxycycline for 3- or 12- weeks and followed for up to 24 weeks and mitochondrial respiration and muscle contraction were measured to define the time course of SOD2 KD and muscle functional changes and recovery. Maximum knockdown of SOD2 protein occurred by 6 weeks and recovered by 24 weeks after DOX treatment. Mitochondrial aconitase activity and maximum mitochondrial respiration declined in KD muscle by 12 weeks and recovered by 24 weeks. There were minimal changes in gene expression between KD and CON muscle. Twelve-week KD showed a small, but significant decrease in muscle fatigue resistance. The primary phenotype was reduced metabolic flexibility characterized by impaired pyruvate driven respiration when other substrates are present. The pyruvate dehydrogenase kinase inhibitor dichloroacetate partially restored pyruvate driven respiration, while the thiol reductant DTT did not. We use a model of inducible and reversible skeletal muscle SOD2 knockdown to demonstrate that elevated matrix superoxide reversibly impairs mitochondrial substrate flexibility characterized by impaired pyruvate oxidation. Despite the bioenergetic effect, the limited change in gene expression suggests that the elevated redox stress in this model is confined to the mitochondrial matrix.

Exploring the potential for branched chain keto-acids to inhibit the mitochondrial pyruvate carrier

Metabolism of branched-chain amino acids (BCAAs) is often dysregulated in obesity and diabetes. To be oxidized, BCAAs are first converted to branched chain keto-acids (BCKAs), which are structurally similar to pyruvic acid. Previous studies have suggested that BCKAs inhibit the mitochondrial pyruvate carrier (MPC), particularly in hepatocytes, so we sought to investigate this further. Mitochondria were isolated from mouse liver and coupled pyruvate respiration was assessed before and after addition of either 100μM keto-leucine (alpha-ketoisocaproate), keto-isoleucine, keto-valine, an equimolar mixture of the 3 BCKAs, or NaCl control. Keto-leucine and the BCKA mixture reduced respiration by ~35%, but keto-isoleucine and keto-valine had no effect. This was repeated in mouse heart mitochondria, and again, only keto-leucine or the BCKA mixture reduced pyruvate respiration by ~30%. In comparison, 1μM of the synthetic MPC inhibitor UK-5099 reduced pyruvate respiration by 80%. In hepatocytes, most pyruvate is not oxidized but is carboxylated, which is an important first step for hepatic gluconeogenesis. Isolated hepatocytes were treated with pyruvate +/- BCKAs, and while UK-5099 reduced glucose production, none of the BCKAs had any effect. This experiment was repeated with 13C-pyruvate, and while UK-5099 reduced 13C enrichment into the hepatocyte TCA cycle, gluconeogenic intermediates, and glucose in the media, again none of the BCKAs had any effect. This lack of effect on gluconeogenesis may be due to the relatively mild inhibition and the ability of hepatocytes to perform pyruvate-alanine cycling to maintain some pyruvate carbon entry into the TCA cycle. Respiration was then assessed in MPC-deficient cardiac mitochondria, and keto-leucine and the BCKA mixture were no longer able to reduce pyruvate oxidation suggesting the effect is dependent on the MPC. Keto-leucine also had no effect on pyruvate dehydrogenase (PDH) activity of cardiac mitochondrial lysates, suggesting that the reduction in pyruvate oxidation is due to MPC inhibition and not reduced PDH activity. In a bioluminescent resonance energy transfer (BRET) assay which indicates inhibitor binding and MPC conformational change, while UK-5099 increased BRET, keto-leucine had no effect. Lastly, we hypothesized that rapid oxidation of the keto-leucine could explain the relatively mild inhibition of the MPC that we observe. Therefore, we repeated mitochondrial respiration studies in mitochondria isolated from PPM1K−/− mice which is the phosphatase that regulates the branched chain keto acid dehydrogenase (BCKDH) and BCKA oxidation. As expected, PPM1K−/− mitochondria displayed enhanced BCKDH phosphorylation, suggestive of inhibited BCKA oxidation. To our surprise, while pyruvate oxidation was inhibited by keto-leucine in WT mitochondria, this inhibition was completely lost in liver or heart mitochondria from PPM1K−/− mice. Altogether, these results suggest that a downstream metabolite of keto-leucine inhibits the MPC, via a mechanism that differs from synthetic MPC inhibitors. Ongoing studies are being performed to search for this downstream metabolite responsible for MPC inhibition. Saint Louis University School of Medicine internal sponsorship. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Lysine acetylation regulates mitochondrial pyruvate carrier activity and cardiac pyruvate oxidation

Background/Objective: The normal heart can acutely switch fuel sources based on delivery. While this is beneficial in physiological scenarios, this flexibility is often lost in cardiac pathologies. During fasting, blood glucose levels drop and increased free fatty acid delivery causes the heart to shut off glucose/pyruvate oxidation and increase fat oxidation. This balance, known as the Randle cycle, has mainly been attributed to regulation of pyruvate dehydrogenase (PDH) activity via PDH phosphorylation. Hypothesis: Decreased pyruvate transport into the mitochondria via posttranslational modifications of the mitochondrial pyruvate carrier (MPC) plays a role in regulating cardiac pyruvate oxidation. Methods: Wildtype mice were allowed to consume normal chow ad libitum or were fasted for 24 hours. After euthanasia, plasma and hearts were collected and left-ventricular muscle fibers were saponin permeabilized and mitochondrial respiration measured in an Oroboros Oxygraph O2k. Cardiac protein lysates were immunoprecipitated with anti-acetyl-lysine beads, and western blotted using standard procedures. A bioluminescent resonance transfer (BRET) assay to detect conformational changes caused by pyruvate/inhibitor binding the MPC was used, and lysine mutant MPC2 constructs were generated by site-directed mutagenesis. Lastly, MPC2-/- H9C2 cardiomyocytes were generated by CRISPR, and respiration measured by Seahorse bioanalyzer in these cells after transfection with WT or lysine mutant MPC2 constructs. Results: 24h fasting reduced blood glucose and insulin concentrations, and increased circulating free fatty acids. Cardiac mitochondrial oxidation of pyruvate/malate was decreased, while oxidation of fatty acids (palmitoyl-CoA+carnitine) was increased in fasted hearts. Western blotting of cardiac lysates showed the expected increase in phosphorylation of PDH-E1α in fasted hearts, known to decrease PDH activity. Immunoprecipitating cardiac lysates with anti-acetyl-lysine beads and blotting for MPC2 suggested increased acetylation of MPC2 in fasted hearts. Using a model structure of the MPC, we have recently proposed and validated lysine 49 (K49) of MPC2 as a critical pyruvate binding site within the MPC. To assess the possible importance of K49 acetylation we mutated K49 to glutamine to mimic acetylation and observed that this K49Q-MPC2 mutant was no longer able to bind pyruvate or competitive inhibitors in a MPC BRET assay. We next created MPC2 knockout H9C2 cardiomyocytes by CRISPR-Cas9 and observed complete loss of MPC2 and MPC1 expression in these cells which decreased pyruvate respiration compared to wildtype H9C2s. Overexpression of wildtype MPC1 and MPC2 constructs increased pyruvate respiration in these MPC-/- cells, while overexpression of the MPC2-K49Q mutant was unable to improve pyruvate respiration. Conclusion: Fasting results in decreased cardiac oxidation of pyruvate in part by acetylation of the MPC and decreased mitochondrial pyruvate transport. National Institutes of Health and SLU institutional funding. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Myeloperoxidase-derived hypochlorous acid targets human airway epithelial plasmalogens liberating protein modifying electrophilic 2-chlorofatty aldehydes

Neutrophil and airway epithelial cell interactions are critical in the inflammatory response to viral infections including respiratory syncytial virus, Sendai virus, and SARS-CoV-2. Airway epithelial cell dysfunction during viral infections is likely mediated by the interaction of virus and recruited neutrophils at the airway epithelial barrier. Neutrophils are key early responders to viral infection. Neutrophil myeloperoxidase catalyzes the conversion of hydrogen peroxide to hypochlorous acid (HOCl). Previous studies have shown HOCl targets host neutrophil and endothelial cell plasmalogen lipids, resulting in the production of the chlorinated lipid, 2-chlorofatty aldehyde (2-ClFALD). We have previously shown that the oxidation product of 2-ClFALD, 2-chlorofatty acid (2-ClFA) is present in bronchoalveolar lavage fluid of Sendai virus-infected mice, which likely results from the attack of the epithelial plasmalogen by neutrophil-derived HOCl. Herein, we demonstrate small airway epithelial cells contain plasmalogens enriched with oleic acid at the sn-2 position unlike endothelial cells which contain arachidonic acid enrichment at the sn-2 position of plasmalogen. We also show neutrophil-derived HOCl targets epithelial cell plasmalogens to produce 2-ClFALD. Further, proteomics and over-representation analysis using the ω-alkyne analog of the 2-ClFALD molecular species, 2-chlorohexadecanal (2-ClHDyA) showed cell adhesion molecule binding and cell-cell junction enriched categories similar to that observed previously in endothelial cells. However, in contrast to endothelial cells, proteins in distinct metabolic pathways were enriched with 2-ClFALD modification, particularly pyruvate metabolism was enriched in epithelial cells and mitochondrial pyruvate respiration was reduced. Collectively, these studies demonstrate, for the first time, a novel plasmalogen molecular species distribution in airway epithelial cells that are targeted by myeloperoxidase-derived hypochlorous acid resulting in electrophilic 2-ClFALD, which potentially modifies epithelial physiology by modifying proteins.

Maternal obesity causes fetal cardiac hypertrophy and alters adult offspring myocardial metabolism in mice.

Obesity in pregnant women causes fetal cardiac dysfunction and increases offspring cardiovascular disease risk, but its effect on myocardial metabolism is unknown. We hypothesized that maternal obesity alters fetal cardiac expression of metabolism-related genes and shifts offspring myocardial substrate preference from glucose towards lipids. Female mice were fed control or obesogenic diets before and during pregnancy. Fetal hearts were studied in late gestation (embryonic day (E) 18.5; term≈E21), and offspring were studied at 3, 6, 9 or 24months postnatally. Maternal obesity increased heart weight and peroxisome proliferator activated receptor gamma (Pparg) expression in female and male fetuses and caused left ventricular diastolic dysfunction in the adult offspring. Cardiac dysfunction worsened progressively with age in female, but not male, offspring of obese dams, in comparison to age-matched control animals. In 6-month-old offspring, exposure to maternal obesity increased cardiac palmitoyl carnitine-supported mitochondrial respiration in males and reduced myocardial 18 F-fluorodeoxyglucose uptake in females. Cardiac Pparg expression remained higher in adult offspring of obese dams than control dams and was correlated with contractile and metabolic function. Maternal obesity did not affect cardiac palmitoyl carnitine respiration in females or 18 F-fluorodeoxyglucose uptake in males and did not alter cardiac 3 H-oleic acid uptake, pyruvate respiration, lipid content or fatty acid/glucose transporter abundance in offspring of either sex. The results support our hypothesis and show that maternal obesity affects offspring cardiac metabolism in a sex-dependent manner. Persistent upregulation of Pparg expression in response to overnutrition in utero might underpin programmed cardiac impairments mechanistically and contribute to cardiovascular disease risk in children of women with obesity. KEY POINTS: Obesity in pregnant women causes cardiac dysfunction in the fetus and increases lifelong cardiovascular disease risk in the offspring. In this study, we showed that maternal obesity in mice induces hypertrophy of the fetal heart in association with altered expression of genes related to nutrient metabolism. Maternal obesity also alters cardiac metabolism of carbohydrates and lipids in the adult offspring. The results suggest that overnutrition in utero might contribute to increased cardiovascular disease risk in children of women with obesity.

Insulin and IGF-1 receptors regulate complex I-dependent mitochondrial bioenergetics and supercomplexes via FoxOs in muscle.

Decreased skeletal muscle strength and mitochondrial dysfunction are characteristic of diabetes. The actions of insulin and IGF-1 through the insulin receptor (IR) and IGF-1 receptor (IGF1R) maintain muscle mass via suppression of forkhead box O (FoxO) transcription factors, but whether FoxO activation coordinates atrophy in concert with mitochondrial dysfunction is unknown. We show that mitochondrial respiration and complex I activity were decreased in streptozotocin (STZ) diabetic muscle, but these defects were reversed in muscle-specific FoxO1, -3, and -4 triple-KO (M-FoxO TKO) mice rendered diabetic with STZ. In the absence of systemic glucose or lipid abnormalities, muscle-specific IR KO (M-IR-/-) or combined IR/IGF1R KO (MIGIRKO) impaired mitochondrial respiration, decreased ATP production, and increased ROS. These mitochondrial abnormalities were not present in muscle-specific IR, IGF1R, and FoxO1, -3, and -4 quintuple-KO mice (M-QKO). Acute tamoxifen-inducible deletion of IR and IGF1R also decreased muscle pyruvate respiration, complex I activity, and supercomplex assembly. Although autophagy was increased when IR and IGF1R were deleted in muscle, mitophagy was not increased. Mechanistically, RNA-Seq revealed that complex I core subunits were decreased in STZ-diabetic and MIGIRKO muscle, and these changes were not present with FoxO KO in STZ-FoxO TKO and M-QKO mice. Thus, insulin-deficient diabetes or loss of insulin/IGF-1 action in muscle decreases complex I-driven mitochondrial respiration and supercomplex assembly in part by FoxO-mediated repression of complex I subunit expression.

A Single Bout of Premeal Resistance Exercise Improves Postprandial Glucose Metabolism in Obese Men with Prediabetes.

Prediabetes is a major risk factor for type 2 diabetes and cardiovascular diseases. Although resistance exercise (RE) is recommended for individuals with prediabetes, the effects of RE on postprandial glucose metabolism in this population are poorly understood. Therefore, the purpose of this study was to elucidate how RE affects postprandial glucose kinetics, insulin sensitivity, beta cell function, and glucose oxidation during the subsequent meal in sedentary men with obesity and prediabetes. We studied 10 sedentary men with obesity (body mass index, 33 ± 3 kg·m-2) and prediabetes by using a randomized, cross-over study design. After an overnight fast, participants completed either a single bout of whole-body RE (seven exercises, 3 sets of 10-12 repetitions at 80% one-repetition maximum each) or an equivalent period of rest. Participants subsequently completed a mixed meal test in conjunction with an intravenous [6,6-2H2]glucose infusion to determine basal and postprandial glucose rate of appearance (Ra) and disappearance (Rd) from plasma, insulin sensitivity, and the insulinogenic index (a measure of beta cell function). Skeletal muscle biopsies were obtained 90 min postmeal to evaluate pyruvate-supported and maximal mitochondrial respiration. Whole-body carbohydrate oxidation was assessed using indirect calorimetry. RE significantly reduced the postprandial rise in glucose Ra and plasma glucose concentration. Postprandial insulin sensitivity was significantly greater after RE, whereas postprandial plasma insulin concentration was significantly reduced. RE had no effect on the insulinogenic index, postprandial pyruvate respiration, or carbohydrate oxidation. A single bout of RE has beneficial effects on postprandial glucose metabolism in men with obesity and prediabetes by increasing postprandial insulin sensitivity, reducing the postprandial rise in glucose Ra, and reducing postprandial plasma insulin concentration.

299-OR: Loss of Insulin and IGF1 Receptors in Muscle Impairs Complex-I Dependent Mitochondrial Bioenergetics and Supercomplex Formation via Foxo Transcription Factors

Diabetes is associated with decreased muscle strength and mitochondrial metabolism which can lead to disability. Recently, we reported that insulin receptor (IR) and IGF-1 receptor (IGF1R) prevent muscle atrophy via suppression of FoxOs, but how FoxOs interact with mitochondrial function remains unclear. To test whether IR/IGF1R suppression of FoxOs coordinates muscle strength with mitochondrial function, we measured mitochondrial bioenergetics and RNA-Seq in mice lacking IR, IGF1R, and/or FoxO1/3/4 in muscle. Perinatal muscle-specific deletion of IR (MIRKO) showed a mild 20% decrease in pyruvate-driven respiratory capacity in soleus permeabilized fibers, whereas succinate/Rotenone respiration was not different. Deletion of both IR/IGF1R (MIGIRKO) showed a marked 60% decrease in pyruvate respiration and ATP production in soleus fibers. These mitochondrial abnormalities were reversed in muscle-specific IR/IGF1R and FoxO1/3/4 quintuple knockout (QKO), suggesting FoxO activation mediates the mitochondrial decline. FoxOs are known inducers of autophagy, but mitophagy quantification using MitoTIMER or mitoKeima biosensors was not altered in Day 21 Tamoxifen-inducible IR/IGF1R KO in adult muscle (IND-IGIRKO) or by acute in-vitro deletion of IR/IGF1R primary myotubes. Complex-I activity was decreased 50-70% in MIGIRKO and Day 21 IND-IGIRKO muscle, but other complex activities were unchanged. Furthermore, 14 complex-I subunits were decreased in MIGIRKO by RNA-Seq, and 13 of these were reversed in QKO. IND-IGIRKO also showed a decrease in pyruvate respiration, ATP production, complex-I activity, and complex-I/supercomplex content by blue-native gels 21 days after tamoxifen. Thus, loss of IR or both IR/IGF1R in muscle decreases complex-I driven mitochondrial respiration and supercomplex assemblies, at least in part by FoxO-mediated downregulation of mitochondrial genes. Disclosure G. Bhardwaj: None. C.M. Penniman: None. P.A. Suarez Beltran: None. J. Jena: None. C.M. Foster: None. K. Poro: None. T. Junck: None. A. Hinton: None. R. Souvenir: None. J. Fuqua: None. P.E. Morales: None. R. Bravo-Sagua: None. V.A. Lira: None. E.D. Abel: None. B.T. ONeill: None.

Training at Lactate Threshold: Science Based Concept or Irrational Myth?

might detect exercise intensity at or close to the highest prolonged constant power with a constant BLC above resting BLC. Time invariance of BLC indicates a match between rates of glycolytic lactate generation and pyruvate respiration. The highest match between rates of glycolytic lactate generation and pyruvate respiration defines the Maximal Lactate Steady State (MLSS) detected via a series of prolonged constant power tests [5,6]. The between subject variability of both, the MLSS (1.9 to 7.5 mmol l -1

The transcriptional coactivator PGC-1α is essential for maximal and efficient cardiac mitochondrial fatty acid oxidation and lipid homeostasis

High-capacity mitochondrial ATP production is essential for normal function of the adult heart, and evidence is emerging that mitochondrial derangements occur in common myocardial diseases. Previous overexpression studies have shown that the inducible transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha is capable of activating postnatal cardiac myocyte mitochondrial biogenesis. Recently, we generated mice deficient in PGC-1alpha (PGC-1alpha(-/-) mice), which survive with modestly blunted postnatal cardiac growth. To determine if PGC-1alpha is essential for normal cardiac energy metabolic capacity, mitochondrial function experiments were performed on saponin-permeabilized myocardial fibers from PGC-1alpha(-/-) mice. These experiments demonstrated reduced maximal (state 3) palmitoyl-l-carnitine respiration and increased maximal (state 3) pyruvate respiration in PGC-1alpha(-/-) mice compared with PGC-1alpha(+/+) controls. ATP synthesis rates obtained during maximal (state 3) respiration in permeabilized myocardial fibers were reduced for PGC-1alpha(-/-) mice, whereas ATP produced per oxygen consumed (ATP/O), a measure of metabolic efficiency, was decreased by 58% for PGC-1alpha(-/-) fibers. Ex vivo isolated working heart experiments demonstrated that PGC-1alpha(-/-) mice exhibited lower cardiac power, reduced palmitate oxidation, and increased reliance on glucose oxidation, with the latter likely a compensatory response. (13)C NMR revealed that hearts from PGC-1alpha(-/-) mice exhibited a limited capacity to recruit triglyceride as a source for lipid oxidation during beta-adrenergic challenge. Consistent with reduced mitochondrial fatty acid oxidative enzyme gene expression, the total triglyceride content was greater in hearts of PGC-1alpha(-/-) mice relative to PGC-1alpha(+/+) following a fast. Overall, these results demonstrate that PGC-1alpha is essential for the maintenance of maximal, efficient cardiac mitochondrial fatty acid oxidation, ATP synthesis, and myocardial lipid homeostasis.

Dramatic Accumulation of Triglycerides and Precipitation of Cardiac Hemodynamic Dysfunction during Brief Caloric Restriction in Transgenic Myocardium Expressing Human Calcium-independent Phospholipase A2γ

Previously, we identified calcium-independent phospholipase A2gamma (iPLA2gamma) with multiple translation initiation sites and dual mitochondrial and peroxisomal localization motifs. To determine the role of iPLA2gamma in integrating lipid and energy metabolism, we generated transgenic mice containing the alpha-myosin heavy chain promoter (alphaMHC) placed proximally to the human iPLA2gamma coding sequence that resulted in cardiac myocyte-restricted expression of iPLA2gamma (TGiPLA2gamma). TGiPLA2gamma mice possessed multiple phenotypes including: 1) a dramatic approximately 35% reduction in myocardial phospholipid mass in both the fed and mildly fasted states; 2) a marked accumulation of triglycerides during brief caloric restriction that represented 50% of total myocardial lipid mass; and 3) acute fasting-induced hemodynamic dysfunction. Biochemical characterization of the TGiPLA2gamma protein expressed in cardiac myocytes demonstrated over 25 distinct isoforms by two-dimensional SDS-PAGE Western analysis. Immunohistochemistry identified iPLA2gamma in the peroxisomal and mitochondrial compartments in both wild type and transgenic myocardium. Electron microscopy revealed the presence of loosely packed and disorganized mitochondrial cristae in TGiPLA2gamma mice that were accompanied by defects in mitochondrial function. Moreover, markedly elevated levels of 1-hydroxyl-2-arachidonoyl-sn-glycero-3-phosphocholine and 1-hydroxyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine were prominent in the TGiPLA2gamma myocardium identifying the production of signaling metabolites by this enzyme in vivo. Collectively, these results identified the participation of iPLA2gamma in the remarkable lipid plasticity of myocardium, its role in generating signaling metabolites, and its prominent effects in modulating energy storage and utilization in myocardium in different metabolic contexts.

Mitochondrial OXPHOS Functions in R1H Rhabdomyosarcoma and Skeletal Muscles of the Rat

The aim of the study was to determinate mitochondrial oxidative phosphorylation (OXPHOS) functions in rat rhabdomyosarcoma R1H (R1H) and rat skeletal muscles. For that purpose skinned fiber technique and multiple substrate inhibitor titration were adapted to tumor samples. In our animal tumor model (R1H) functional abnormalities of OXPHOS were found compared to skeletal muscles. In R1H the state 3 respiration of pyruvate + malate was decreased: 0.56 +/- 0.28 nmol O(2)/mg/min versus 2.32 +/- 1.19 nmol O(2)/mg/min, P < 0.001, whereas the state 3 respiration of succinate + rotenone was increased: 36 +/- 14% versus 19 +/- 11%, P < 0.001. In R1H the rotenone-insensitive respiration reached higher levels than the antimycin A-insensitive respiration, whereas in normal muscles the converse was observed. Additionally, the obvious difference between the CAT- and the antimycin A-independent respiration indicates an increased part of leak respiration in R1H. By now, the high feasibility of these techniques is appreciated for the investigation of muscles and prospectively for tumors, too.

Creatine protects against 3-nitropropionic acid-induced cell death in murine corticostriatal slice cultures

In murine corticostriatal slice cultures, we studied the protective effects of the bioenergetic compound creatine on neuronal cell death induced by the mitochondrial toxin 3-nitropropionic acid (3-NP). 3-NP caused a dose-dependent neuronal degeneration accompanied by an increased lactate dehydrogenase (LDH) activity in the cell culture medium. An increased ratio of lactate to pyruvate concentration in the medium suggested that metabolic activity shifted to anaerobic energy metabolism. These effects were predominantly observed in the 24-h recovery period after 3-NP exposure. Creatine protected against 3-NP neurotoxicity: LDH activity was reduced and aerobic respiration of pyruvate was stimulated, which resulted in lower lactate levels and less cell death. In both striatum and cortex, apoptosis in 3-NP-exposed slices was demonstrated by increased activation of the pro-apoptotic protein caspase-3 and by numerous cells exhibiting DNA fragmentation detected by the terminal transferase-mediated biotinylated-UTP nick end-labeling (TUNEL) technique. Creatine administration to the 3-NP-exposed corticostriatal slices resulted in a reduced number of TUNEL-positive cells in the recovery period. However, in the striatum, an unexpected increase of both TUNEL-positive cells and caspase-3-immunostained cells was observed in the exposure phase in the presence of creatine. In the recovery phase, caspase-3-immunostaining decreased to basal levels in both striatum and cortex. These findings suggest that 3-NP-induced neuronal degeneration in corticostriatal slices results from apoptosis that in the cortex can be prevented by creatine, while in the more vulnerable striatal cells it may lead to an accelerated and increased execution of apoptotic cell death, preventing further necrosis-related damage in this region.

Native UCP1 Displays Simple Competitive Kinetics between the Regulators Purine Nucleotides and Fatty Acids

Elucidation of the regulation of uncoupling protein 1 (UCP1) activity in its native environment, i.e. the inner membrane of brown-fat mitochondria, has been hampered by the presence of UCP1-independent, quantitatively unresolved effects of investigated regulators on the brown-fat mitochondria themselves. Here we have utilized the availability of UCP1-ablated mice to dissect UCP1-dependent and UCP1-independent effects of regulators. Using a complex-I-linked substrate (pyruvate), we found that UCP1 can mediate a 4-fold increase in thermogenesis when stimulated with the classical positive regulator fatty acids (oleate). After demonstrating that the fatty acids act in their free form, we found that UCP1 increased fatty acid sensitivity approximately 30-fold (as compared with the 1.5-fold increase reported earlier based on nominal fatty acid values). By identifying the UCP1-mediated fraction of the response, we could conclude that the interaction between purine nucleotides (GDP) and fatty acids (oleate) unexpectedly displayed simple competitive kinetics. In GDP-inhibited mitochondria, oleate apparently acted as an activator. However, only a model in which UCP1 is inherently active (i.e."activating" fatty acids cannot be included in the model), where GDP functions as an inhibitor with a K(m) of 0.05 mm, and where oleate functions as a competitive antagonist for the GDP effect (with a K(i) of 5 nm) can fit all of the experimental data. We conclude that, when examined in its native environment, UCP1 functions as a proton (equivalent) carrier in the absence of exogenous or endogenous fatty acids.

The role of polyglucose in oxygen-dependent respiration by a new strain of Desulfovibrio salexigens

Desulfovibrio salexigens strain Mastl was isolated from the oxic/anoxic interface of a marine sediment. Growth under sulfate-reducing conditions was accompanied by polyglucose accumulation in the cell with every substrate tested. Highest polyglucose storage was found with glucose (0.8–1.0 g polyglucose (g protein)−1), but the growth rate with this substrate was very low (0.015 h−1). Anaerobically grown cells of strain Mastl exhibited immediate oxygen-dependent respiration. The endogenous oxygen reduction rate was proportional to the polyglucose content. The rate of aerobic respiration of pyruvate was also directly related to the polyglucose content indicating that this organism was only able to respire with oxygen as long as polyglucose was present. Maximum oxygen reduction rates were found at air saturating concentrations and were relatively low (3–50 nmol O2 min−1 (mg protein)−1). Catalase was constitutively present in anaerobically grown cells. When batch cultures were exposed to oxygen, growth ceased immediately and polyglucose was oxidized to acetate within 40–50 h. Like the oxygen reduction activity, the nitro blue tetrazolium (NBT)-reduction activity in these cells was proportional to the polyglucose content. Under anaerobic starvation conditions there was no correlation between the NBT-reduction activity and polyglucose concentration and polyglucose was degraded slowly within 240 h. The ecological significance of aerobic polyglucose consumption is discussed.

The role of polyglucose in oxygen-dependent respiration by a new strain of Desulfovibrio salexigens

Desulfovibrio salexigens strain Mastl was isolated from the oxic/anoxic interface of a marine sediment. Growth under sulfate-reducing conditions was accompanied by polyglucose accumulation in the cell with every substrate tested. Highest polyglucose storage was found with glucose (0.8–1.0 g polyglucose (g protein) −1), but the growth rate with this substrate was very low (0.015 h −1). Anaerobically grown cells of strain Mastl exhibited immediate oxygen-dependent respiration. The endogenous oxygen reduction rate was proportional to the polyglucose content. The rate of aerobic respiration of pyruvate was also directly related to the polyglucose content indicating that this organism was only able to respire with oxygen as long as polyglucose was present. Maximum oxygen reduction rates were found at air saturating concentrations and were relatively low (3–50 nmol O 2 min −1 (mg protein) −1). Catalase was constitutively present in anaerobically grown cells. When batch cultures were exposed to oxygen, growth ceased immediately and polyglucose was oxidized to acetate within 40–50 h. Like the oxygen reduction activity, the nitro blue tetrazolium (NBT)-reduction activity in these cells was proportional to the polyglucose content. Under anaerobic starvation conditions there was no correlation between the NBT-reduction activity and polyglucose concentration and polyglucose was degraded slowly within 240 h. The ecological significance of aerobic polyglucose consumption is discussed.

Immobilized respiratory chain activities from Escherichia coli utilized to measure D- and L-lactate, succinate, L-malate, 3-glycerophosphate, pyruvate, or NAD(P)H.

The respiratory chain (membranous, multienzymatic system) from Escherichia coli, was coimmobilized with gelatin and insolubilized in film form by tanning with glutaraldehyde. The film was fixed onto an oxygen sensor. The enzyme electrode can be used for measuring NAD(P)H, D- and L-lactate, succinate, L-malate, 3-glycerophosphate, or pyruvate. The range of metabolites concentrations was from 1 to 50 mM. It was possible to discriminate between the different metabolites (if mixed): By inducing during bacterial growth the specific flavoproteins necessary for L-lactate, succinate, L-malate, and 3-glycerophosphate respirations. The constitutive activities are unaltered on glucose or glycerol, namely D-lactate, NAD(P)H, and pyruvate respiration. When intact bacteria were immobilized (with or without induction), D- and L-lactate, succinate, 3-glycerophosphate, and L-malate respiration were measured, no activities of pyruvate and NAD(P)H respiration were obtained. For these last activities, French press breakage (see section on Membrane Preparations) of bacteria prior to immobilization was necessary. Products of reactions can be used as enzyme inhibitors: Pyruvate inhibits D- and L-lactate; fumarate inhibits succinate, and oxaloacetate inhibits L-malate respirations. Heat denaturation of the bacteria at 55 degrees C for 1 h maintains full activity of succinate and pyruvate respiration. On the other hand, no activity of D- and L-lactate, L-malate, or NAD(P)H respiration was measurable. These enzyme electrodes have many applications in basic and applied research.

Oxidation of Branched Chain Amino Acids by Isolated Hearts and Diaphragms of the Rat: THE EFFECT OF FATTY ACIDS, GLUCOSE, AND PYRUVATE RESPIRATION

Abstract Amino acid oxidation was studied in perfused hearts and incubated hemidiaphragms of rats fed ad libitum. Hearts were perfused with Krebs-Henseleit bicarbonate buffer containing glucose and 0.1 mm amino acids. The addition of 1 mm octanoate to the medium markedly stimulated 14CO2 production from [1-14C]leucine, isoleucine, and valine; inhibited 14CO2 production from [1-14C]alanine, pyruvate, and α-ketoglutarate, and did not affect 14CO2 production from [14C]histidine, threonine, glutamate, ornithine, and phenylalanine. Hemidiaphragms incubated with 1 mm hexanoate or 0.1 to 1.0 mm octanoate increased the oxidation of the branched chain amino acids by 80 to 200% and decreased 14CO2 production from and pyruvate. Octanoate stimulated the oxidation of isoleucine by hemidiaphragms at isoleucine concentrations between 0.01 to 1.0 mm; when isoleucine exceeded 2 mm, octanoate became inhibitory. Octanoate stimulated the oxidation of the branched chain amino acids in the presence or absence of glucose and in the presence or absence of insulin from the medium, in diaphragms and hearts. Bovine serum albumin did not affect branched chain amino acid oxidation in the absence of added fatty acids; it inhibited the stimulatory effect of the latter. The inhibition was proportional to the albumin concentration. Palmitate and oleate (1 mm) added to albumin (15 mg per ml) stimulated branched chain amino acid oxidation by hemidiaphragms; the effect was smaller than that observed with equimolar octanoate and albumin. In hearts, no effect of 1 mm palmitate was observed. Butyrate (2 mm) did not affect branched chain amino acid oxidation by hemidiaphragms; 4 mm dl-β-OH butyrate and 4 mm acetate were inhibitory. In hearts and diaphragms, the omission of glucose from the incubation medium stimulated the oxidation of the branched chain amino acids. Pyruvate added to media containing glucose caused further inhibition of branched chain amino acid oxidation. Insulin stimulated 14CO2 production from [14C]leucine in hearts obtained from rats fasted for 48 hours and perfused without glucose. In hearts perfused with [14C]leucine, the addition of octanoate to the perfusate did not affect the tissue concentration of free leucine nor the labeling of the leucine pool. Octanoate stimulated 14CO2 production from leucine by hemidiaphragms which were preloaded with [14C]leucine, indicating stimulation of leucine oxidation beyond the transport of leucine into muscle cells. The apparent inverse relationship between the oxidation of pyruvate and that of the branched chain amino acids suggests that regulation of the latter may occur at the oxidative decarboxylation of the branched chain α-keto acids in muscles. It is suggested that regulation of branched chain amino acid catabolism in muscles may compliment the alanine cycle, and may play a role in homeostasis under conditions of limited availability of glucose (e.g. fasting) or increased utilization (e.g. exercise).

The mode of action of dinitrophenols on the different phosphorylating steps

1. 1. Determinations of the concentrations (caq)opt of a dinitrophenol that induced maximal respiration in a medium deficient in phosphate and phosphate acceptor was carried out with different alkylnitrophenols and substrates (succinate, pyruvate (+ malate), glutamate, β-hydroxybutyrate) at various pH's. 2. 2. The pH optimum of maximally uncoupled succinate oxidation was 7.4, of maximally uncoupled pyruvate oxidation 6.8. 3. 3. The p(c1)opt, where c1 is the calculated concentration of uncoupling phenol in the lipid phase, is a linear function of pH, the slope of the line relating these quantities being o with succinate and — 0.5 for NAD-linked subtrates; an exceptional case is formed when the respiration of pyruvate (+ malate) is uncoupled by alkyldinitrophenols, in which case the slope is o. 4. 4. From a consideration of the reaction kinetics it appears that for systems in which dp(c1)opt/dpH is zero, uncoupling and inhibition by phenols (″) is best explained by the reactions. A∼I+o→A+I−o I−o⇋I+o where A is an oxidized member of the respiratory chain, and A∼I an intermediate of oxidative phosphorylation. When the slope is −0.5 the sequence A∼I+o→A+I−o I−o+H+⇋I+o appears to be the most likely. 5. 5. On basis of the difference in the relation between p(caq)opt and pH for different NAD-linked substrates it is concluded that mitochondrial NAD+ associated with the different substrates is present in different compartments, and that the whole phosphorylation system connected with a particular substrate must be localized in the same mitochondrial compartment. The compartments differ in their lipophilic character.

Osservazioni prellminari sul meccanismo di azione della cocliobolina

Abstract MECHANISM OF COCHLIOBOLIN ACTIVITY: A PREVENTIVE NOTE. — Coch-liobolin caused leakage of phosphate and organic compounds from corn rootlets and potato discs, at room temperature. The leakage does not occur at 1–5°C, but when potato discs are incubated at this temperature and then thoroughly washed and brought at 25°C, a sharp increase of phosphate may be noticed in the incubation solutions. Cochliobolin inhibited partially the aerobic respiration of glucose and endogenous carbon in Micrococcus pyogenes var. aureus resting cells. Aerobic respiration of pyruvate, succinate, fumarate and malate was completely inhibited, while the inhibition of lactate and Lketoglutarate respiration required a short lag period. The hydrogen-ion concentration of the media seemed to be an important factor controlling the rapidity of action of the inhibitor, because at pH 4 and 5 at least 120 minutes were required prior any effect could be observed, while only 30 minutes were required at pH 6. The effects of cochliobolin on Micrococcus resting cells were irreversible In contrast, respiratory activities of acetonic powders were refractory to the substance under aerobic conditions, and oxidation of pyruvate, malate, fumarate, succinate and α-ketoglutarate were not affected by saturated solutions of cochliobolin. It is suggested that the first site of attack is the cell wall-cell membrane unit, altering cell permeability, so that inorganic ions and other cofactors essential to respiration are lost in consequence of the leakage through the cell membrane.