Deficiency in SLC25A1, encoding the mitochondrial citrate carrier, causes combined D-2- and L-2-hydroxyglutaric aciduria

The Krebs cycle is of fundamental importance for the generation of the energetic and molecular needs of both prokaryotic and eukaryotic cells. Both enantiomers of metabolite 2-hydroxyglutarate are directly linked to this pivotal biochemical pathway and are found elevated not only in several cancers, but also in different variants of the neurometabolic disease 2-hydroxyglutaric aciduria. Recently we showed that cancer-associated IDH2 germline mutations cause one variant of 2-hydroxyglutaric aciduria. Complementary to these findings, we now report recessive mutations in SLC25A1, the mitochondrial citrate carrier, in 12 out of 12 individuals with combined D-2- and L-2-hydroxyglutaric aciduria. Impaired mitochondrial citrate efflux, demonstrated by stable isotope labeling experiments and the absence of SLC25A1 in fibroblasts harboring certain mutations, suggest that SLC25A1 deficiency is pathogenic. Our results identify defects in SLC25A1 as a cause of combined D-2- and L-2-hydroxyglutaric aciduria.

Effect of remifentanil on mitochondrial oxygen consumption of cultured human hepatocytes

During sepsis, liver dysfunction is common, and failure of mitochondria to effectively couple oxygen consumption with energy production has been described. In addition to sepsis, pharmacological agents used to treat septic patients may contribute to mitochondrial dysfunction. This study addressed the hypothesis that remifentanil interacts with hepatic mitochondrial oxygen consumption. The human hepatoma cell line HepG2 and their isolated mitochondria were exposed to remifentanil, with or without further exposure to tumor necrosis factor-α (TNF-α). Mitochondrial oxygen consumption was measured by high-resolution respirometry, Caspase-3 protein levels by Western blotting, and cytokine levels by ELISA. Inhibitory κBα (IκBα) phosphorylation, measurement of the cellular ATP content and mitochondrial membrane potential in intact cells were analysed using commercial ELISA kits. Maximal cellular respiration increased after one hour of incubation with remifentanil, and phosphorylation of IκBα occurred, denoting stimulation of nuclear factor κB (NF-κB). The effect on cellular respiration was not present at 2, 4, 8 or 16 hours of incubation. Remifentanil increased the isolated mitochondrial respiratory control ratio of complex-I-dependent respiration without interfering with maximal respiration. Preincubation with the opioid receptor antagonist naloxone prevented a remifentanil-induced increase in cellular respiration. Remifentanil at 10× higher concentrations than therapeutic reduced mitochondrial membrane potential and ATP content without uncoupling oxygen consumption and basal respiration levels. TNF-α exposure reduced respiration of complex-I, -II and -IV, an effect which was prevented by prior remifentanil incubation. Furthermore, prior remifentanil incubation prevented TNF-α-induced IL-6 release of HepG2 cells, and attenuated fragmentation of pro-caspase-3 into cleaved active caspase 3 (an early marker of apoptosis). Our data suggest that remifentanil increases cellular respiration of human hepatocytes and prevents TNF-α-induced mitochondrial dysfunction. The results were not explained by uncoupling of mitochondrial respiration.

Differential modulation of ROS signals and other mitochondrial parameters by the antioxidants MitoQ, resveratrol and curcumin in human adipocytes

Abstract Mitochondrial reactive oxygen species (ROS) have been demonstrated to play an important role as signaling and regulating molecules in human adipocytes. In order to evaluate the differential modulating roles of antioxidants, we treated human adipocytes differentiated from human bone marrow-derived mesenchymal stem cells with MitoQ, resveratrol and curcumin. The effects on ROS, viability, mitochondrial respiration and intracellular ATP levels were examined. MitoQ lowered both oxidizing and reducing ROS. Resveratrol decreased reducing and curcumin oxidizing radicals only. All three substances slightly decreased state III respiration immediately after addition. After 24 h of treatment, MitoQ inhibited both basal and uncoupled oxygen consumption, whereas curcumin and resveratrol had no effect. Intracellular ATP levels were not altered. This demonstrates that MitoQ, resveratrol and curcumin exert potent modulating effects on ROS signaling in human adipocyte with marginal effects on metabolic parameters.

Hint2, a mitochondrial apoptotic sensitizer down-regulated in hepatocellular carcinoma

BACKGROUND ; AIMS: Hints, histidine triad nucleotide-binding proteins, are adenosine monophosphate-lysine hydrolases of uncertain biological function. Here we report the characterization of human Hint2. METHODS: Tissue distribution was determined by real-time quantitative polymerase chain reaction and immunoblotting, cellular localization by immunocytochemistry, and transfection with green fluorescent protein constructs. Enzymatic activities for protein kinase C and adenosine phosphoramidase in the presence of Hint2 were measured. HepG2 cell lines with Hint2 overexpressed or knocked down were established. Apoptosis was assessed by immunoblotting for caspases and by flow cytometry. Tumor growth was measured in SCID mice. Expression in human tumors was investigated by microarrays. RESULTS: Hint2 was predominantly expressed in liver and pancreas. Hint2 was localized in mitochondria. Hint2 hydrolyzed adenosine monophosphate linked to an amino group (AMP-pNA; k(cat):0.0223 s(-1); Km:128 micromol/L). Exposed to apoptotic stress, fewer HepG2 cells overexpressing Hint2 remained viable (32.2 +/- 0.6% vs 57.7 +/- 4.6%), and more cells displayed changes of the mitochondrial membrane potential (87.8 +/- 2.35 vs 49.7 +/- 1.6%) with more cleaved caspases than control cells. The opposite was observed in HepG2 cells with knockdown expression of Hint2. Subcutaneous injection of HepG2 cells overexpressing Hint2 in SCID mice resulted in smaller tumors (0.32 +/- 0.13 g vs 0.85 +/- 0.35 g). Microarray analyses revealed that HINT2 messenger RNA is downregulated in hepatocellular carcinomas (-0.42 +/- 0.58 log2 vs -0.11 +/- 0.28 log2). Low abundance of HINT2 messenger RNA was associated with poor survival. CONCLUSION: Hint2 defines a novel class of mitochondrial apoptotic sensitizers down-regulated in hepatocellular carcinoma.

Effects of prolonged endotoxemia on liver, skeletal muscle and kidney mitochondrial function

INTRODUCTION: Sepsis may impair mitochondrial utilization of oxygen. Since hepatic dysfunction is a hallmark of sepsis, we hypothesized that the liver is more susceptible to mitochondrial dysfunction than the peripheral tissues, such as the skeletal muscle. We studied the effect of prolonged endotoxin infusion on liver, muscle and kidney mitochondrial respiration and on hepatosplanchnic oxygen transport and microcirculation in pigs. METHODS: 20 anesthetized pigs were randomized to receive endotoxin or saline infusion for 24 hours. Muscle, liver and kidney mitochondrial respiration was assessed. Cardiac output (thermodilution), carotid, superior mesenteric and kidney arterial, portal venous (ultrasound Doppler) and microcirculatory blood flow (laser Doppler) were measured, and systemic and regional oxygen transport and lactate exchange were calculated. RESULTS: Endotoxin infusion induced hyperdynamic shock and impaired the glutamate- and succinate-dependent mitochondrial respiratory control ratio (RCR) in the liver (glutamate: endotoxemia: median [range] 2.8 [2.3-3.8] vs. controls: 5.3 [3.8-7.0]; p<0.001; succinate: endotoxemia: 2.9 [1.9-4.3] vs. controls: 3.9 [2.6-6.3] p=0.003). While the ADP:O ratio was reduced with both substrates, maximal ATP production was impaired only in the succinate-dependent respiration. Hepatic oxygen consumption and extraction, and liver surface laser Doppler blood flow remained unchanged. Glutamate-dependent respiration in the muscle and kidney was unaffected. CONCLUSIONS: Endotoxemia reduces the efficiency of hepatic but neither skeletal muscle nor kidney mitochondrial respiration, independent of regional and microcirculatory blood flow changes.

Lactate, not glucose, up-regulates mitochondrial oxygen consumption both in sham and lateral fluid percussed rat brains

OBJECTIVE: Failure of energy metabolism after traumatic brain injury may be a major factor limiting outcome. Although glucose is the primary metabolic substrate in the healthy brain, the well documented surge in tissue lactate after traumatic brain injury suggests that lactate may provide an energy need that cannot be met by glucose. We hypothesized, therefore, that administration of lactate or the combination of lactate and supraphysiological oxygen may improve mitochondrial oxidative respiration in the brain after rat fluid percussion injury. We measured oxygen consumption (VO2) to determine what effects glucose, lactate, oxygen, and the combination of lactate and oxygen have on mitochondrial respiration in both injured and uninjured rat brain tissue. METHODS: Anesthetized Sprague-Dawley rats were intubated and ventilated with either 0.21 or 1.0 fraction of inspired oxygen (FIO2). Brain tissue from acute sham animals was subjected in vitro to 1.1 mM, 12 mM and 100 mM concentrations of glucose and L-lactate. In another group, injury (fluid percussion injury of 2.5 +/- 0.02 atmospheres) was induced over the left hemisphere. The VO2 of mug amounts of brain tissues were measured in a microrespirometry system (Cartesian diver). RESULTS: The VO2 was found to be independent of glucose concentrations, but dose-dependent for lactate. Moreover, the lactate dependent VO2s were all significantly higher than those generated by glucose. Injured rats on FIO2 0.21 had brain tissue VO2 rates that were significantly lower than those of shams or preinjury levels. In injured rats treated with FIO2 1.0, the reduction in VO2 levels was prevented. Injured rats that received an intravenous infusion of 100 mM lactate had VO2 rates that were significantly higher than those obtained with FIO2 1.0. Combined treatment further boosted the lactate generated VO2 rates by approximately 15%. CONCLUSION: Glucose sustains mitochondrial respiration at a low level "fixed" rate because, despite increasing its concentration nearly 100-fold, it cannot up-regulate VO2 after fluid percussion injury. Lactate produces a dose-dependent VO2 response, possibly enabling mitochondria to meet the increased energy needs of the injured brain.

Acetyl-L-carnitine feeding to unloaded rats triggers in soleus muscle the coordinated expression of genes involved in mitochondrial biogenesis

The expressional profile of mitochondrial transcripts and of genes involved in the mitochondrial biogenesis pathway induced by ALCAR daily supplementation in soleus muscle of control and unloaded 3-month-old rats has been analyzed. It has been found that ALCAR treatment is able to upregulate the expression level of mitochondrial transcripts (COX I, ATP6, ND6, 16 S rRNA) in both control and unloaded animals. Interestingly, ALCAR feeding to unloaded rats resulted in the increase of transcript level for master factors involved in mitochondrial biogenesis (PGC-1alpha, NRF-1, TFAM). It also prevented the unloading-induced downregulation of mRNA levels for kinases able to transduce metabolic (AMPK) and neuronal stimuli (CaMKIIbeta) into mitochondrial biogenesis. No significant effect on the expressional level of such genes was found in control ALCAR-treated rats. In addition, ALCAR feeding was able to prevent the loss of mitochondrial protein content due to unloading condition. Correlation analysis revealed a strong coordination in the expression of genes involved in mitochondrial biogenesis only in ALCAR-treated suspended animals, supporting a differentiated effect of ALCAR treatment in relation to the loading state of the soleus muscle. In conclusions, we demonstrated the ability of ALCAR supplementation to promote only in soleus muscle of hindlimb suspended rats an orchestrated expression of genes involved in mitochondrial biogenesis, which might counteract the unloading-induced metabolic changes, preventing the loss of mitochondrial proteins.

Effect of endotoxin, dobutamine and dopamine on muscle mitochondrial respiration in vitro

INTRODUCTION: Mitochondrial respiration is impaired during endotoxemia. While catecholamines are frequently used in sepsis, their effects on mitochondrial function are controversial. We assessed effects of dobutamine and dopamine endotoxin on isolated muscle mitochondria. MATERIALS AND METHODS: Sternocleidomastoid muscle mitochondria were isolated from six anesthetized pigs. Each sample was divided into six different groups. Three groups were incubated with endotoxin, three with vehicle. After 1 h, dopamine and dobutamine at final concentrations of 100 microM were added to the vehicle and endotoxin groups. After 2 h, state 3 and 4 respiration rates were determined for all mitochondrial complexes. Oxygen consumption was determined with a Clark-type electrode. RESULTS: Endotoxin increased glutamate-dependent state 4 respiration from 9.3 +/- 3.6 to 31.9 +/- 9.1 (P = 0.001) without affecting state 3 respiration. This reduced the efficiency of mitochondrial respiration (RCR; state 3/state 4, 9.9 +/- 1.9 versus 3.6 +/- 0.6; P < 0.001). The other complexes were unaffected. Catecholamine partially restored the endotoxin-induced increase in complex I state 4 respiration rate (31.9 +/- 9.1 versus 17.1 +/- 6.4 and 20.1 +/- 12.2) after dopamine and dobutamine, respectively (P = 0.007), and enhanced the ADP:O ratio (P = 0.033). CONCLUSIONS: Dopamine and dobutamine enhanced the efficiency of mitochondrial respiration after short-term endotoxin exposure.