Changes In Mitochondrial Metabolism Research Articles

Alteration in mitochondrial Ca(2+) uptake disrupts insulin signaling in hypertrophic cardiomyocytes.

BackgroundCardiac hypertrophy is characterized by alterations in both cardiac bioenergetics and insulin sensitivity. Insulin promotes glucose uptake by cardiomyocytes and its use as a substrate for glycolysis and mitochondrial oxidation in order to maintain the high cardiac energy demands. Insulin stimulates Ca2+ release from the endoplasmic reticulum, however, how this translates to changes in mitochondrial metabolism in either healthy or hypertrophic cardiomyocytes is not fully understood.ResultsIn the present study we investigated insulin-dependent mitochondrial Ca2+ signaling in normal and norepinephrine or insulin like growth factor–1-induced hypertrophic cardiomyocytes. Using mitochondrion-selective Ca2+-fluorescent probes we showed that insulin increases mitochondrial Ca2+ levels. This signal was inhibited by the pharmacological blockade of either the inositol 1,4,5-triphosphate receptor or the mitochondrial Ca2+ uniporter, as well as by siRNA-dependent mitochondrial Ca2+ uniporter knockdown. Norepinephrine-stimulated cardiomyocytes showed a significant decrease in endoplasmic reticulum-mitochondrial contacts compared to either control or insulin like growth factor–1-stimulated cells. This resulted in a reduction in mitochondrial Ca2+ uptake, Akt activation, glucose uptake and oxygen consumption in response to insulin. Blocking mitochondrial Ca2+ uptake was sufficient to mimic the effect of norepinephrine-induced cardiomyocyte hypertrophy on insulin signaling.ConclusionsMitochondrial Ca2+ uptake is a key event in insulin signaling and metabolism in cardiomyocytes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-014-0068-4) contains supplementary material, which is available to authorized users.

Impaired in vivo mitochondrial Krebs cycle activity after myocardial infarction assessed using hyperpolarized magnetic resonance spectroscopy.

Myocardial infarction (MI) is one of the leading causes of heart failure. An increasing body of evidence links alterations in cardiac metabolism and mitochondrial function with the progression of heart disease. The aim of this work was to, therefore, follow the in vivo mitochondrial metabolic alterations caused by MI, thereby allowing a greater understanding of the interplay between metabolic and functional abnormalities. Using hyperpolarized carbon-13 ((13)C)-magnetic resonance spectroscopy, in vivo alterations in mitochondrial metabolism were assessed for 22 weeks after surgically induced MI with reperfusion in female Wister rats. One week after MI, there were no detectable alterations in in vivo cardiac mitochondrial metabolism over the range of ejection fractions observed (from 28% to 84%). At 6 weeks after MI, in vivo mitochondrial Krebs cycle activity was impaired, with decreased (13)C-label flux into citrate, glutamate, and acetylcarnitine, which correlated with the degree of cardiac dysfunction. These changes were independent of alterations in pyruvate dehydrogenase flux. By 22 weeks, alterations were also seen in pyruvate dehydrogenase flux, which decreased at lower ejection fractions. These results were confirmed using in vitro analysis of enzyme activities and metabolomic profiles of key intermediates. The in vivo decrease in Krebs cycle activity in the 6-week post-MI heart may represent an early maladaptive phase in the metabolic alterations after MI in which reductions in Krebs cycle activity precede a reduction in pyruvate dehydrogenase flux. Changes in mitochondrial metabolism in heart disease are progressive and proportional to the degree of cardiac impairment.

Changes in mitochondrial metabolism and morphology contribute to sepsis‐associated acute kidney injury (1134.10)

Acute Kidney Injury (AKI) is commonly seen in septic patients, but there are no effective treatments to prevent or treat it. New perspectives on the pathogenesis of AKI in sepsis are needed to improve the current standard of treatment. We examined renal mitochondrial function and expression of proteins with known roles in mitochondrial function and dynamics in the cecal ligation and puncture (CLP) model of sepsis in rats. At 24 hrs post CLP or sham surgery, kidney tissue and mitochondria were isolated for protein expression analyses and mitochondrial functional assays including mitochondrial oxygen consumption (QO2), ATP generation and reactive oxygen species (ROS) generation. Data represents mean ± SEM.In isolated mitochondria, QO2 rates were higher in CLP compared to sham; with the largest difference observed during state 3 (199.9 ± 31.6 pMol/min vs. 163.2 ± 28.81 pMol/min, p<0.05). However, ATP generation was significantly lower in the mitochondria from CLP kidneys. With western blot expression, we found increased expression of mitochondrial fission proteins DRP1 (1.9‐fold higher in CLP, P<.0004) and Fis1 (trend for higher expression) in CLP kidneys. We also observed decreased expression of PGC‐1α, a key transcription factor in mitochondrial biogenesis. Analysis of the expression of the expression of the ETC complexes in the mitochondrial fraction showed a 25% reduction (P 蠄 .001) in Complex Va and Complex III in CLP mitochondria. In conclusion, our data indicate significant increase in renal oxidative metabolism but without the associated ATP generation and reduction in the expression of ETC complexes. Mitochondrial analyses reveal increased mitochondrial fission but with reduced biogenesis, indicating increased mitochondrial stress. Additional morphological and functional analyses are being conducted. We are also evaluating mechanisms underlying the altered mitochondrial function to identify potential therapeutic targets for the treatment of kidney injury during sepsis.Grant Funding Source: NIH

Investigating mitochondrial metabolism in contracting HL-1 cardiomyocytes following hypoxia and pharmacological HIF activation identifies HIF-dependent and independent mechanisms of regulation.

Hypoxia is a consequence of cardiac disease and downregulates mitochondrial metabolism, yet the molecular mechanisms through which this occurs in the heart are incompletely characterized. Therefore, we aimed to use a contracting HL-1 cardiomyocyte model to investigate the effects of hypoxia on mitochondrial metabolism. Cells were exposed to hypoxia (2% O2) for 6, 12, 24, and 48 hours to characterize the metabolic response. Cells were subsequently treated with the hypoxia inducible factor (HIF)-activating compound, dimethyloxalylglycine (DMOG), to determine whether hypoxia-induced mitochondrial changes were HIF dependent or independent, and to assess the suitability of this cultured cardiac cell line for cardiovascular pharmacological studies. Hypoxic cells had increased glycolysis after 24 hours, with glucose transporter 1 and lactate levels increased 5-fold and 15-fold, respectively. After 24 hours of hypoxia, mitochondrial networks were more fragmented but there was no change in citrate synthase activity, indicating that mitochondrial content was unchanged. Cellular oxygen consumption was 30% lower, accompanied by decreases in the enzymatic activities of electron transport chain (ETC) complexes I and IV, and aconitase by 81%, 96%, and 72%, relative to controls. Pharmacological HIF activation with DMOG decreased cellular oxygen consumption by 43%, coincident with decreases in the activities of aconitase and complex I by 26% and 30%, indicating that these adaptations were HIF mediated. In contrast, the hypoxia-mediated decrease in complex IV activity was not replicated by DMOG treatment, suggesting HIF-independent regulation of this complex. In conclusion, 24 hours of hypoxia increased anaerobic glycolysis and decreased mitochondrial respiration, which was associated with changes in ETC and tricarboxylic acid cycle enzyme activities in contracting HL-1 cells. Pharmacological HIF activation in this cardiac cell line allowed both HIF-dependent and independent mitochondrial metabolic changes to be identified.

Endogenous Differences in Cochlear Sensory and Supporting Cell Mitochondrial Metabolism Bias Free Radical Production during Ototoxin Exposure

Aminoglycosides, including gentamicin (GM), are the most frequently used antibiotics in the world despite irreversible cochlear damage and hearing loss associated with their use. Although there are numerous causes of deafness, reactive oxygen species (ROS) are key regulators of multiple pathologies including: ototoxicity, noise-induced and age-related hearing loss. Unfortunately, the source of these cell-damaging ROS remains controversial. Given that ROS are normal byproducts of ATP synthesis, intrinsic differences in cochlear sensory (inner and outer hair cell, I/OHC) and supporting (pillar) cell mitochondrial metabolism may explain why high-frequency OHCs are profoundly sensitive to a host of challenges. Mitochondrial metabolism was compared in low-frequency and high-frequency IHCs, OHCs and pillar cells from acutely cultured cochlear explants. Intensity-based changes in the metabolic intermediate, nicotinamide adenine dinucleotide (NADH), were used to measure endogenous, mitochondrial toxin and GM-induced differences in sensory and supporting cell mitochondrial metabolism. Sensory cell mitochondrial metabolism was significantly enhanced relative to supporting cells. Despite similar amounts of mitochondria in IHCs and OHCs, endogenous levels of NADH were greatest in high-frequency OHCs. Metabolic profiling of NADH metabolism revealed basal turn, high-frequency OHCs to be metabolically responsive to a number of changes in their microenvironment including metabolic toxins and GM, while high-frequency IHCs, low-frequency I/OHCs and, pillar cells are substantially less sensitive. DHR-123 was used to detect sensory and supporting cell ROS production during GM exposure. Within 30 minutes of GM application, ROS are dramatically and specifically increased in high-frequency sensory cells. GM-induced changes in mitochondrial metabolism and cell-damaging free radical production were greatest in high-frequency OHCs. This metabolic predisposition biases basal turn OHC responses to a variety of cochlear insults, particularly those involving energy metabolism and ROS production.

Integrated 3D Simulation of Cardiomyocyte Revealed the Distinct Functional Characteristics between Subsarcolemmal and Interfibrillar Mitochondria

It has been reported that two subpopulations of mitochondria exist in cardiomyocyte: subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM). However, their functional characteristics have yet to be clarified, due to the experimental difficulties in differential isolation and assessment techniques. We have already developed a 3-D computational model of cardiomyocyte with subcellular structure integrating electrophysiology, metabolism, and mechanics. In this study, this model was further improved to include the intracellular gradient of oxygen and myoglobin distribution to test the hypothesis that the difference in location within the cell can introduce significant changes in mitochondrial metabolism. For this purpose, all mitochondria in the model were made to have the same membrane permeability and enzymatic activities. When compared the responses of [Ca2+], TCA activity, [NADH] and mitochondrial inner membrane potential to an abrupt changes in pacing frequency (0.25 Hz to 2 Hz) between SSM and IFM, IFM reached higher plateau levels in all of these parameters. These differences seemed to be related to the intracellular gradient in [Ca2+]. We also examined the effect of reduction in [O2]. Under normal condition intracellular [O2] is much higher than the half saturation concentration for oxidative phosphorylation even for the IFM located in the core region of the cell. However under limited extracellular [O2] environment, [NADH] and inner membrane potential gradually decreased in IFM compared to SSM reflecting the intracellular [O2] gradient from the cell surface to the core. Although further validations are required, the current simulation results suggested that only the difference in intracellular location can cause the different functional characteristics of mitochondrial metabolism in cardiomyocytes.

Assessment of Cellular Responses after Short- and Long-Term Exposure to Silver Nanoparticles in Human Neuroblastoma (SH-SY5Y) and Astrocytoma (D384) Cells

Silver nanoparticle (AgNP, 20 nm) neurotoxicity was evaluated by an integrated in vitro testing protocol employing human cerebral (SH-SY5Y and D384) cell lines. Cellular response after short-term (4–48 h, 1–100 μg/ml) and prolonged exposure (up to 10 days, 0.5–50 μg/ml) to AgNP was assessed by MTT, calcein-AM/PI, clonogenic tests. Pulmonary A549 cells were employed for data comparison along with silver nitrate as metal ionic form. Short-term data: (i) AgNP produced dose- and time-dependent mitochondrial metabolism changes and cell membrane damage (effects starting at 25 μg/ml after 4 h: EC50s were 40.7 ± 2.0 and 49.5 ± 2.1 μg/ml for SH-SY5Y and D384, respectively). A549 were less vulnerable; (ii) AgNP doses of ≤ 18 μg/ml were noncytotoxic; (iii) AgNO3 induced more pronounced effects compared to AgNP on cerebral cells. Long-term data: (i) low AgNP doses (≤1 μg/ml) compromised proliferative capacity of all cell types (cell sensibility: SHSY5Y > A549 > D384). Colony number decrease in SH-SY5Y and D384 was 50% and 25%, respectively, at 1 μg/ml, and lower dose (0.5 μg/ml) was significantly effective towards SH-SY5Y and pulmonary cells; (ii) cell proliferation activity was more affected by AgNO3 than AgNPs. In summary, AgNP-induced cytotoxic effects after short-term and prolonged exposure (even at low doses) were evidenced regardless of cell model types.

Contrasting metabolic effects of medium- versus long-chain fatty acids in skeletal muscle

Dietary intake of long-chain fatty acids (LCFAs) plays a causative role in insulin resistance and risk of diabetes. Whereas LCFAs promote lipid accumulation and insulin resistance, diets rich in medium-chain fatty acids (MCFAs) have been associated with increased oxidative metabolism and reduced adiposity, with few deleterious effects on insulin action. The molecular mechanisms underlying these differences between dietary fat subtypes are poorly understood. To investigate this further, we treated C2C12 myotubes with various LCFAs (16:0, 18:1n9, and 18:2n6) and MCFAs (10:0 and 12:0), as well as fed mice diets rich in LCFAs or MCFAs, and investigated fatty acid-induced changes in mitochondrial metabolism and oxidative stress. MCFA-treated cells displayed less lipid accumulation, increased mitochondrial oxidative capacity, and less oxidative stress than LCFA-treated cells. These changes were associated with improved insulin action in MCFA-treated myotubes. MCFA-fed mice exhibited increased energy expenditure, reduced adiposity, and better glucose tolerance compared with LCFA-fed mice. Dietary MCFAs increased respiration in isolated mitochondria, with a simultaneous reduction in reactive oxygen species generation, and subsequently low oxidative damage. Collectively our findings indicate that in contrast to LCFAs, MCFAs increase the intrinsic respiratory capacity of mitochondria without increasing oxidative stress. These effects potentially contribute to the beneficial metabolic actions of dietary MCFAs.

Mitochondrial activity and oxidative stress markers in peripheral blood mononuclear cells of patients with bipolar disorder, schizophrenia, and healthy subjects

Evidence suggests that mitochondrial dysfunction is involved in the pathophysiology of psychiatric disorders such as schizophrenia (SZ) and bipolar disorder (BD). However, the exact mechanisms underlying this dysfunction are not well understood. Impaired activity of electron transport chain (ETC) complexes has been described in these disorders and may reflect changes in mitochondrial metabolism and oxidative stress markers. The objective of this study was to compare ETC complex activity and protein and lipid oxidation markers in 12 euthymic patients with BD type I, in 18 patients with stable chronic SZ, and in 30 matched healthy volunteers. Activity of complexes I, II, and III was determined by enzyme kinetics of mitochondria isolated from peripheral blood mononuclear cells (PBMCs). Protein oxidation was evaluated using the protein carbonyl content (PCC) method, and lipid peroxidation, the thiobarbituric acid reactive substances (TBARS) assay kit. A significant decrease in complex I activity was observed (p = 0.02), as well as an increase in plasma levels of TBARS (p = 0.00617) in patients with SZ when compared to matched controls. Conversely, no significant differences were found in complex I activity (p = 0.17) or in plasma TBARS levels (p = 0.26) in patients with BD vs. matched controls. Our results suggest that mitochondrial complex I dysfunction and oxidative stress play important roles in the pathophysiology of SZ and may be used in potential novel adjunctive therapy for SZ, focusing primarily on cognitive impairment and disorder progression.

Role of polyhydroxybutyrate in mitochondrial calcium uptake

Polyhydroxybutyrate (PHB) is a biological polymer which belongs to the class of polyesters and is ubiquitously present in all living organisms. Mammalian mitochondrial membranes contain PHB consisting of up to 120 hydroxybutyrate residues. Roles played by PHB in mammalian mitochondria remain obscure. It was previously demonstrated that PHB of the size similar to one found in mitochondria mediates calcium transport in lipid bilayer membranes. We hypothesized that the presence of PHB in mitochondrial membrane might play a significant role in mitochondrial calcium transport. To test this, we investigated how the induction of PHB hydrolysis affects mitochondrial calcium transport. Mitochondrial PHB was altered enzymatically by targeted expression of bacterial PHB hydrolyzing enzyme (PhaZ7) in mitochondria of mammalian cultured cells. The expression of PhaZ7 induced changes in mitochondrial metabolism resulting in decreased mitochondrial membrane potential in HepG2 but not in U87 and HeLa cells. Furthermore, it significantly inhibited mitochondrial calcium uptake in intact HepG2, U87 and HeLa cells stimulated by the ATP or by the application of increased concentrations of calcium to the digitonin permeabilized cells. Calcium uptake in PhaZ7 expressing cells was restored by mimicking calcium uniporter properties with natural electrogenic calcium ionophore – ferutinin. We propose that PHB is a previously unrecognized important component of the mitochondrial calcium uptake system.

Role of Polyphosphate in Mitochondria: From Modification of Energy Metabolism to Induction of the Cell Death

Inorganic polyphosphate (polyP) is found in all living organisms ranging from bacteria to mammals. PolyP plays multiple physiological functions, which are distinct and dependent on the type of organism and the subcellular localization of the polymer. Recently we demonstrated that polyP levels are dependent on the cell metabolism and can be changed by mitochondrial substrates and inhibitors. We propose the existence of a feedback mechanism where polyP production and cell energy metabolism regulate each other. In order to investigate this we study the effect of polyP on mitochondrial oxygen consumption. We have found that application of short polyP (14 phosphate residues) or medium polyP (70 ortophosphates) significantly increase the level of respiratory coefficient by activation of state V3 and inhibition of V4 compare to control. Importantly, both short and medium polyP significantly reduced efficiency of oxidative phosphorylation (ADP/O ratio). It has previously been reported that polyP can modify membrane permeability for ions that can be a trigger for changes in mitochondrial metabolism. Furthermore polyP has been linked to activation of the mitochondrial permeability transition pore (mPTP) in different cells that also can be activated by calcium. Long, medium, and in lower degree, short polyP increase permeability of de-energised mitochondria for Ca2+. This effect was dependent on inhibitor of mPTP - cyclosporine A. We also found that long polyP (130 orthophosphates) caused cell death in primary neurons and astrocytes, while medium (70) polyP had a much smaller effect and short (14) did not cause any. Thus, polyP has a multiple action on mitochondrial function from modification of mitochondrial energy metabolism to stimulation of calcium permeability and cell death.

Skeletal muscle Nur77 expression enhances oxidative metabolism and substrate utilization

Mitochondrial dysfunction has been implicated in the pathogenesis of type 2 diabetes. Identifying novel regulators of mitochondrial bioenergetics will broaden our understanding of regulatory checkpoints that coordinate complex metabolic pathways. We previously showed that Nur77, an orphan nuclear receptor of the NR4A family, regulates the expression of genes linked to glucose utilization. Here we demonstrate that expression of Nur77 in skeletal muscle also enhances mitochondrial function. We generated MCK-Nur77 transgenic mice that express wild-type Nur77 specifically in skeletal muscle. Nur77-overexpressing muscle had increased abundance of oxidative muscle fibers and mitochondrial DNA content. Transgenic muscle also exhibited enhanced oxidative metabolism, suggestive of increased mitochondrial activity. Metabolomic analysis confirmed that Nur77 transgenic muscle favored fatty acid oxidation over glucose oxidation, mimicking the metabolic profile of fasting. Nur77 expression also improved the intrinsic respiratory capacity of isolated mitochondria, likely due to the increased abundance of complex I of the electron transport chain. These changes in mitochondrial metabolism translated to improved muscle contractile function ex vivo and improved cold tolerance in vivo. Our studies outline a novel role for Nur77 in the regulation of oxidative metabolism and mitochondrial activity in skeletal muscle.

Changes in the mitochondrial phosphoproteome during mammalian hibernation

Mammalian hibernation involves periods of substantial suppression of metabolic rate (torpor) allowing energy conservation during winter. In thirteen-lined ground squirrels (Ictidomys tridecemlineatus), suppression of liver mitochondrial respiration during entrance into torpor occurs rapidly (within 2 h) before core body temperature falls below 30°C, whereas reversal of this suppression occurs slowly during arousal from torpor. We hypothesized that this pattern of rapid suppression in entrance and slow reversal during arousal was related to changes in the phosphorylation state of mitochondrial enzymes during torpor catalyzed by temperature-dependent kinases and phosphatases. We compared mitochondrial protein phosphorylation among hibernation metabolic states using immunoblot analyses and assessed how phosphorylation related to mitochondrial respiration rates. No proteins showed torpor-specific changes in phosphorylation, nor did phosphorylation state correlate with mitochondrial respiration. However, several proteins showed seasonal (summer vs. winter) differences in phosphorylation of threonine or serine residues. Using matrix-assisted laser desorption/ionization-time of flight/time of flight mass spectrometry, we identified three of these proteins: F1-ATPase α-chain, long chain-specific acyl-CoA dehydrogenase, and ornithine transcarbamylase. Therefore, we conclude that protein phosphorylation is likely a mechanism involved in bringing about seasonal changes in mitochondrial metabolism in hibernating ground squirrels, but it seems unlikely to play any role in acute suppression of mitochondrial metabolism during torpor.

The role of mitochondria in leaf nitrogen metabolism

For optimal plant growth and development, cellular nitrogen (N) metabolism must be closely coordinated with other metabolic pathways, and mitochondria are thought to play a central role in this process. Recent studies using genetically modified plants have provided insight into the role of mitochondria in N metabolism. Mitochondrial metabolism is linked with N assimilation by amino acid, carbon (C) and redox metabolism. Mitochondria are not only an important source of C skeletons for N incorporation, they also produce other necessary metabolites and energy used in N remobilization processes. Nitric oxide of mitochondrial origin regulates respiration and influences primary N metabolism. Here, we discuss the changes in mitochondrial metabolism during ammonium or nitrate nutrition and under low N conditions. We also describe the involvement of mitochondria in the redistribution of N during senescence. The aim of this review was to demonstrate the role of mitochondria as an integration point of N cellular metabolism.

Dynamic measurements of mitochondrial hydrogen peroxide concentration and glutathione redox state in rat pancreatic β-cells using ratiometric fluorescent proteins: confounding effects of pH with HyPer but not roGFP1

Using the ROS (reactive oxygen species)-sensitive fluorescent dyes dichlorodihydrofluorescein and dihydroethidine, previous studies yielded opposite results about the glucose regulation of oxidative stress in insulin-secreting pancreatic β-cells. In the present paper, we used the ratiometric fluorescent proteins HyPer and roGFP1 (redox-sensitive green fluorescent protein 1) targeted to mitochondria [mt-HyPer (mitochondrial HyPer)/mt-roGFP1 (mitochondrial roGFP1)] to monitor glucose-induced changes in mitochondrial hydrogen peroxide concentration and glutathione redox state in adenovirus-infected rat islet cell clusters. Because of the reported pH sensitivity of HyPer, the results were compared with those obtained with the mitochondrial pH sensors mt-AlpHi and mt-SypHer. The fluorescence ratio of the mitochondrial probes slowly decreased (mt-HyPer) or increased (mt-roGFP1) in the presence of 10 mmol/l glucose. Besides its expected sensitivity to H2O2, mt-HyPer was also highly pH sensitive. In agreement, changes in mitochondrial metabolism similarly affected mt-HyPer, mt-AlpHi and mt-SypHer fluorescence signals. In contrast, the mt-roGFP1 fluorescence ratio was only slightly affected by pH and reversibly increased when glucose was lowered from 10 to 2 mmol/l. This increase was abrogated by the catalytic antioxidant Mn(III) tetrakis (4-benzoic acid) porphyrin but not by N-acetyl-L-cysteine. In conclusion, due to its pH sensitivity, mt-HyPer is not a reliable indicator of mitochondrial H2O2 in β-cells. In contrast, the mt-roGFP1 fluorescence ratio monitors changes in β-cell mitochondrial glutathione redox state with little interference from pH changes. Our results also show that glucose acutely decreases rather than increases mitochondrial thiol oxidation in rat β-cells.

Role of Polyhydroxybutyrate in Mitochondrial Calcium Transport

Polyhydroxybutyrate (PHB) is a biological polymer which belongs to polyesters class and ubiquitously present in all living organisms. It has been demonstrated that mammalian mitochondrial membranes contain PHB polymer consisting of up to 120 butyrate residues. We found before that mitochondrial PHB complexed with Ca2+ and inorganic polyphosphate forms channels with properties similar to mitochondrial permeability transition pore. It is also experimentally proven that PHB in its free form possesses ionophoretic properties and mediates Ca2+ transport through the model phospholipid membranes. We hypothesized that PHB might significantly contribute to the process of mitochondrial Ca2+ transport. To test this idea we investigated Ca2+ transport in mitochondria with decreased levels of PHB. Mitochondrial PHB was reduced enzymatically by targeted expression of specific bacterial PHB hydrolyzing enzyme (PhaZ7) in mitochondria of mammalian cultured cells. Experiments were performed using transiently transfected cultured cells: HepG2, U87 and HeLa. PHB deficiency induced changes in mitochondrial metabolism resulting in decreased mitochondrial membrane potential (measured by TMRM) in HepG2 but not in U87 and HeLa cells. Kinetics of mitochondrial Ca2+ transport was measured using mitochondrial Ca2+ sensitive fluorescent probe - x-Rhod-1 and confocal microscopy. Mitochondrial Ca2+ signal was stimulated either by addition of ATP or Histamine in experiments with intact cells or by addition of increased concentrations of calcium to the recording solution in digitonin permeabilized cells. We found that over-expression of mitochondrially targeted PHB depolymeraze in mammalian cultured cells dramatically inhibits their Ca2+ uptake. Ca2+ uptake in these cells was restored by addition of mimic of calcium uniporter with electrogenic Ca2+ ionophore properties - ferutinin. Our data suggest that PHB is previously unrecognized essential component of mitochondrial Ca2+ uptake system.

Differentiation of SH-SY5Y cells to a neuronal phenotype changes cellular bioenergetics and the response to oxidative stress

Cell differentiation is associated with changes in metabolism and function. Understanding these changes during differentiation is important in the context of stem cell research, cancer, and neurodegenerative diseases. An early event in neurodegenerative diseases is the alteration of mitochondrial function and increased oxidative stress. Studies using both undifferentiated and differentiated SH-SY5Y neuroblastoma cells have shown distinct responses to cellular stressors; however, the mechanisms remain unclear. We hypothesized that because the regulation of glycolysis and oxidative phosphorylation is modulated during cellular differentiation, this would change bioenergetic function and the response to oxidative stress. To test this, we used retinoic acid (RA) to induce differentiation of SH-SY5Y cells and assessed changes in cellular bioenergetics using extracellular flux analysis. After exposure to RA, the SH-SY5Y cells had an increased mitochondrial membrane potential, without changing mitochondrial number. Differentiated cells exhibited greater stimulation of mitochondrial respiration with uncoupling and an increased bioenergetic reserve capacity. The increased reserve capacity in the differentiated cells was suppressed by the inhibitor of glycolysis 2-deoxy- d-glucose. Furthermore, we found that differentiated cells were substantially more resistant to cytotoxicity and mitochondrial dysfunction induced by the reactive lipid species 4-hydroxynonenal or the reactive oxygen species generator 2,3-dimethoxy-1,4-naphthoquinone. We then analyzed the levels of selected mitochondrial proteins and found an increase in complex IV subunits, which we propose contributes to the increase in reserve capacity in the differentiated cells. Furthermore, we found an increase in MnSOD that could, at least in part, account for the increased resistance to oxidative stress. Our findings suggest that profound changes in mitochondrial metabolism and antioxidant defenses occur upon differentiation of neuroblastoma cells to a neuron-like phenotype.

Acetyl-L-Carnitine Modulates TP53 and IL10 Gene Expression Induced by 3-NPA Evoked Toxicity in PC12 Cells

The neurotoxicity induced by the mitochondrial inhibitor 3-nitropropionic acid (3-NPA) is associated with a decrease of ATP synthesis and an increase of free radical production which can lead to apoptosis or necrosis. We have used the PC12, neuron-like rat pheochromocytoma cell line, to study further the mechanism of 3-NPA-evoked neurotoxicity and the effects of acetyl-L-carnitine (ALC) which has neuroprotective actions against various types of mitochondrial inhibitors.Cultured PC 12 cells were exposed to a low dose of 3-NPA 50 (microM) in the presence or absence of 5 mM ALC. The dose of 3-NPA was sub toxic and no changes in pro-apoptotic Bax or anti-apoptotic Bcl-2 gene expression were observed. We followed specific genetic markers to look for changes evoked by 3-NPA toxicity and also changes associated with neuroprotection exerted by the ALC treatment, using RT-PCR arrays (delta-delta method). 3-NPA exposure evoked a decrease in expression of the Tp53 gene. This down regulation was prevented by pretreatment of the cells with ALC. The Tp53 gene responds to cellular stresses and the effects seen here are possibly associated with the 3-NPA evoked changes in mitochondrial metabolism. Other genes associated with stress and apoptosis, Parp-1, Bcl-2, and Bax were not affected by 3-NPA or ALC. The decrease of inflammatory response Il-10 gene expression due to 3-NPA was further lowered by presence of ALC. Other inflammation related genes, Il1rn, Nr3c1 and Cxcr4 were not affected. Interestingly, the glutamate transporter slc17a7, carnitine-acylcarnitine translocase Slc25a20 and heat shock proteins genes, Hsp27, Hmox1 (Hsp32, HO1) as well as Hspa 1a (Hsp 70) increased only when both ALC and small dose of 3-NPA were present. The alterations in gene expression detected in this study suggest role of several intracellular pathways in the neurotoxicity of 3-NPA and the neuroprotection against 3-NPA-induced neurotoxicity by ALC.

Fuel-Stimulated Insulin Secretion Depends upon Mitochondria Activation and the Integration of Mitochondrial and Cytosolic Substrate Cycles

The pancreatic islet β-cell is uniquely specialized to couple its metabolism and rates of insulin secretion with the levels of circulating nutrient fuels, with the mitochondrial playing a central regulatory role in this process. In the β-cell, mitochondrial activation generates an integrated signal reflecting rates of oxidativephosphorylation, Kreb's cycle flux, and anaplerosis that ultimately determines the rate of insulin exocytosis. Mitochondrial activation can be regulated by proton leak and mediated by UCP2, and by alkalinization to utilize the pH gradient to drive substrate and ion transport. Converging lines of evidence support the hypothesis that substrate cycles driven by rates of Kreb's cycle flux and by anaplerosis play an integral role in coupling responsive changes in mitochondrial metabolism with insulin secretion. The components and mechanisms that account for the integrated signal of ATP production, substrate cycling, the regulation of cellular redox state, and the production of other secondary signaling intermediates are operative in both rodent and human islet β-cells.

Oxidative stress leads to a decline in mitochondrial capacity in aged fast‐twitch, but not slow‐twitch mouse muscle

Reactive oxygen species (ROS) are implicated in cell pathology associated with many degenerative diseases, but are also important signaling molecules involved in adaptive responses in cell energy metabolism. Our goal was to determine how age and muscle fiber type influence the response of skeletal muscle to a given oxidative stress. We used paraquat (PARA) treatment to induce acute oxidative stress in vivo in young (4 month old) and old (27 month old) mice. To examine how this response varies with muscle fiber type we measured mitochondrial metabolism in permeabilized soleus (SOL, slow‐twitch) and extensor digitorum longus (EDL, fast‐twitch) muscles. In the EDL of old mice there was a significant (24%) decline in state 3 respiration one week after treatment with 30 mg/kg PARA, while in young mice PARA treatment resulted in a trend toward an increase (20%) in state 3 respiration. In contrast, there was no effect of PARA treatment on mitochondrial respiration in young or old SOL. Our results indicate that fast‐twitch muscle is more susceptible to oxidative stress induced changes in mitochondrial metabolism and that these effects are qualitatively different in young and old muscle. Supported by NIH grants AG028455, AG022385, AG029052 and the Ellison Medical Foundation.