Inhibition Of Mitochondrial Oxidative Metabolism Research Articles

The inhibitory role of nitric oxide in encephalomyocarditis viral replication in pancreatic β-cells.

Abstract During type 1 diabetes (T1D) onset, insulin-secreting β-cells are selectively destroyed by T-cells. In addition to known genetic factors, viral infection (specifically by members of the picornavirus family) is an environmental factor that has been implicated in T1D onset. Viral infection stimulates macrophage release of IL-1, and in islets, stimulates the production of micromolar levels of nitric oxide (NO) by β-cells. NO impairs β-cell function via inhibition of mitochondrial oxidative metabolism, resulting in depletion of ATP. Since β-cells have a limited capacity for self-renewal, and are essential for organismal survival, we hypothesize that the cytokine-mediated production of NO by β-cells functions in a protective manner during a viral infection. In support of this hypothesis, we’ve shown that NO inhibits encephalomyocarditis viral (EMCV) replication and mediated lysis in a β-cell selective manner. EMCV is an inflammatory, mouse-tropic virus that induces diabetes in several mouse models. In this study, we have identified inositol hexakisphosphate kinase-1 (IP6K1, KmATP=1 mM) as an indirect target of NO (via the loss of ATP) that is responsible for the inhibition of EMCV replication in β-cells. We show that inhibition of IP6K1 attenuates EMCV replication and mediated β-cell lysis in a manner comparable to the actions of NO. Current studies are focused on the mechanism by which inhibition of IP6K activity attenuates EMCV replication and EMCV-mediated lysis in β-cells.

β-Cell-selective regulation of gene expression by nitric oxide.

Nitric oxide is produced at low micromolar levels following the induction of inducible nitric oxide synthase (iNOS) and is responsible for mediating the inhibitory actions of cytokines on glucose-stimulated insulin secretion by islets of Langerhans. It is through the inhibition of mitochondrial oxidative metabolism, specifically aconitase and complex 4 of the electron transport chain, that nitric oxide inhibits insulin secretion. Nitric oxide also attenuates protein synthesis, induces DNA damage, activates DNA repair pathways, and stimulates stress responses (unfolded protein and heat shock) in β-cells. In this report, the time- and concentration-dependent effects of nitric oxide on the expression of six genes known to participate in the response of β-cells to this free radical were examined. The genes included Gadd45α (DNA repair), Puma (apoptosis), Hmox1 (antioxidant defense), Hsp70 (heat shock), Chop (UPR), and Ppargc1α (mitochondrial biogenesis). We show that nitric oxide stimulates β-cell gene expression in a narrow concentration range of ∼0.5-1 µM or levels corresponding to iNOS-derived nitric oxide. At concentrations greater than 1 µM, nitric oxide fails to stimulate gene expression in β-cells, and this is associated with the inhibition of mitochondrial oxidative metabolism. This narrow concentration range of responses is β-cell selective, as the actions of nitric oxide in non-β-cells (α-cells, mouse embryonic fibroblasts, and macrophages) are concentration dependent. Our findings suggest that β-cells respond to a narrow concentration range of nitric oxide that is consistent with the levels produced following iNOS induction, and that these concentration-dependent actions are selective for insulin-containing cells.

Inhibition of mitochondrial oxidative metabolism attenuates EMCV replication and protects β-cells from virally mediated lysis

Viral infection is one environmental factor that may contribute to the initiation of pancreatic β-cell destruction during the development of autoimmune diabetes. Picornaviruses, such as encephalomyocarditis virus (EMCV), induce a pro-inflammatory response in islets leading to local production of cytokines, such as IL-1, by resident islet leukocytes. Furthermore, IL-1 is known to stimulate β-cell expression of iNOS and production of the free radical nitric oxide. The purpose of this study was to determine whether nitric oxide contributes to the β-cell response to viral infection. We show that nitric oxide protects β-cells against virally mediated lysis by limiting EMCV replication. This protection requires low micromolar, or iNOS-derived, levels of nitric oxide. At these concentrations nitric oxide inhibits the Krebs enzyme aconitase and complex IV of the electron transport chain. Like nitric oxide, pharmacological inhibition of mitochondrial oxidative metabolism attenuates EMCV-mediated β-cell lysis by inhibiting viral replication. These findings provide novel evidence that cytokine signaling in β-cells functions to limit viral replication and subsequent β-cell lysis by attenuating mitochondrial oxidative metabolism in a nitric oxide–dependent manner.

Inhibition of oxidative metabolism by nitric oxide restricts EMCV replication selectively in pancreatic beta-cells

Environmental factors, such as viral infection, are proposed to play a role in the initiation of autoimmune diabetes. In response to encephalomyocarditis virus (EMCV) infection, resident islet macrophages release the pro-inflammatory cytokine IL-1β, to levels that are sufficient to stimulate inducible nitric oxide synthase (iNOS) expression and production of micromolar levels of the free radical nitric oxide in neighboring β-cells. We have recently shown that nitric oxide inhibits EMCV replication and EMCV-mediated β-cell lysis and that this protection is associated with an inhibition of mitochondrial oxidative metabolism. Here we show that the protective actions of nitric oxide against EMCV infection are selective for β-cells and associated with the metabolic coupling of glycolysis and mitochondrial oxidation that is necessary for insulin secretion. Inhibitors of mitochondrial respiration attenuate EMCV replication in β-cells, and this inhibition is associated with a decrease in ATP levels. In mouse embryonic fibroblasts (MEFs), inhibition of mitochondrial metabolism does not modify EMCV replication or decrease ATP levels. Like most cell types, MEFs have the capacity to uncouple the glycolytic utilization of glucose from mitochondrial respiration, allowing for the maintenance of ATP levels under conditions of impaired mitochondrial respiration. It is only when MEFs are forced to use mitochondrial oxidative metabolism for ATP generation that mitochondrial inhibitors attenuate viral replication. In a β-cell selective manner, these findings indicate that nitric oxide targets the same metabolic pathways necessary for glucose stimulated insulin secretion for protection from viral lysis.

54-OR: Nitric Oxide Attenuates Viral Replication in Pancreatic ß-Cells through an Inhibition of Mitochondrial Oxidative Metabolism

Viral infection has been identified as an environmental factor that may play a role in initiating the destruction of pancreatic β-cells during the development of autoimmune diabetes. We have identified a pro-inflammatory response activated in pancreatic islets in response to infection with encephalomyocarditis virus (EMCV). In response to the local production of cytokines such as IL-1 by resident islet macrophages, β-cells produce nitric oxide, a free radical with known antiviral activity. Nitric oxide has been characterized both as a potent antiviral molecule and as a mediator of β-cell dysfunction and damage and so we investigated the role of nitric oxide in controlling β-cell viability in the context of EMCV infection. We find that nitric oxide, either supplied exogenously with donor compounds or produced endogenously in response to cytokine treatment, attenuates EMCV replication and protects β-cells against EMCV-mediated death. These protective actions of nitric oxide are restricted to conditions in which nitric oxide is produced at low micromolar levels (consistent with the levels of nitric oxide generated by iNOS) and are associated with the ability of nitric oxide to impair mitochondrial oxidation and decrease ATP levels in β-cells. Treatment with rotenone, an inhibitor of complex I of the electron transport chain, likewise attenuates EMCV replication in β-cells, which are reliant on oxidative metabolism for ATP generation, but not in fibroblasts, which can sustain ATP levels via glycolysis alone. These findings suggest a mechanism of protection restricted to β-cells where nitric oxide production attenuates viral replication by inhibiting oxidative metabolism and depleting cellular stores of ATP, supporting a protective role for nitric oxide in the response of β-cells to viral infection. Disclosure J. Stafford: None. J.A. Corbett: None. Funding American Heart Association (17PRE3253000); National Institutes of Health (DK052194, AI44458)

Metabolic Mechanisms and a Rational Combinational Application of Carboxyamidotriazole in Fighting Pancreatic Cancer Progression after Chemotherapy.

The anticancer and anti-inflammatory effects of carboxyamidotriazole (CAI) have been demonstrated in several studies, but the underlying mechanisms remain to be elucidated. This study showed that CAI caused metabolic reprogramming of pancreatic cancer cells. The inhibition of mitochondrial oxidative metabolism by CAI led to increased glutamine-dependent reductive carboxylation and enhanced glycolytic metabolism. The presence of environmental substances that affect cellular metabolism, such as glutamine and pyruvate, attenuated the anticancer efficacy of CAI. Based on the action of CAI: 1) when glutamine was removed, the NAD+/NADH ratio was decreased, the synthesis of cellular aspartate was reduced, and autophagy flux was blocked; and 2) when glycolysis was pharmacologically inhibited, the ATP level was significantly decreased, the cell viability was greatly inhibited, and the compensatory rescue effect of glutamine was eliminated. When combined with chemotherapy, cotreatment with CAI and the glycolysis inhibitor 2-deoxyglucose (2-DG) inhibited the pancreatic cancer progression after chemotherapy. As the inhibition of mitochondrial oxidative metabolism can explain several anticancer activities of CAI reported previously, including inhibition of calcium entry and induction of reactive oxygen species, we demonstrate that inhibition of mitochondrial oxidative phosphorylation may be the fundamental mechanism of CAI. The combination of CAI and 2-DG causes energy depletion in cancer cells, eliminating the rescue effect of the metabolic environment. Inhibiting pancreatic cancer progression after chemotherapy is a rational application of this metabolism-disturbing combination strategy.

Dual Modulation of the Mitochondrial Permeability Transition Pore and Redox Signaling Synergistically Promotes Cardiomyocyte Differentiation From Pluripotent Stem Cells

BackgroundCardiomyocytes that differentiate from pluripotent stem cells (PSCs) provide a crucial cellular resource for cardiac regeneration. The mechanisms of mitochondrial metabolic and redox regulation for efficient cardiomyocyte differentiation are, however, still poorly understood. Here, we show that inhibition of the mitochondrial permeability transition pore (mPTP) by Cyclosporin A (CsA) promotes cardiomyocyte differentiation from PSCs.Methods and ResultsWe induced cardiomyocyte differentiation from mouse and human PSCs and examined the effect of CsA on the differentiation process. The cardiomyogenic effect of CsA mainly resulted from mPTP inhibition rather than from calcineurin inhibition. The mPTP inhibitor NIM811, which does not have an inhibitory effect on calcineurin, promoted cardiomyocyte differentiation as much as CsA did, but calcineurin inhibitor FK506 only slightly increased cardiomyocyte differentiation. CsA‐treated cells showed an increase in mitochondrial calcium, mitochondrial membrane potential, oxygen consumption rate, ATP level, and expression of genes related to mitochondrial function. Furthermore, inhibition of mitochondrial oxidative metabolism reduced the cardiomyogenic effect of CsA while antioxidant treatment augmented the cardiomyogenic effect of CsA.ConclusionsOur data show that mPTP inhibition by CsA alters mitochondrial oxidative metabolism and redox signaling, which leads to differentiation of functional cardiomyocytes from PSCs.

Translocation of the Na+/H+ exchanger 1 (NHE1) in cardiomyocyte responses to insulin and energy-status signalling

The Na+/H+ exchanger NHE1 is a highly regulated membrane protein that is required for pH homoeostasis in cardiomyocytes. The activation of NHE1 leads to proton extrusion, which is essential for counteracting cellular acidity that occurs following increased metabolic activity or ischaemia. The activation of NHE1 intrinsic catalytic activity has been well characterized and established experimentally. However, we have examined in the present study whether a net translocation of NHE1 to the sarcolemma of cardiomyocytes may also be involved in the activation process. We have determined the distribution of NHE1 by means of immunofluorescence microscopy and cell-surface biotinylation. We have discovered changes in the distribution of NHE1 that occur when cardiomyocytes are stimulated with insulin that are PI3K (phosphoinositide 3-kinase)-dependent. Translocation of NHE1 also occurs when cardiomyocytes are challenged by hypoxia, or inhibition of mitochondrial oxidative metabolism or electrically induced contraction, but these responses occur through a PI3K-independent process. As the proposed additional level of control of NHE1 through translocation was unexpected, we have compared this process with the well-established translocation of the glucose transporter GLUT4. In immunofluorescence microscopy comparisons, the translocation of NHE1 and GLUT4 to the sarcolemma that occur in response to insulin appear to be very similar. However, in basal unstimulated cells the two proteins are mainly located, with the exception of some co-localization in the perinuclear region, in distinct subcellular compartments. We propose that the mechanisms of translocation of NHE1 and GLUT4 are linked such that they provide spatially and temporally co-ordinated responses to cardiac challenges that necessitate re-adjustments in glucose transport, glucose metabolism and cell pH.

Social isolation in rats inhibits oxidative metabolism, decreases the content of mitochondrial K-Ras and activates mitochondrial hexokinase

Recent observations have suggested that Ras signaling includes combinations of extracellular-signal-regulated Ras activation at the plasma membrane and endomembranes, and translocation of Ras from the plasma membrane to intracellular compartments. In this study we have shown that social isolation of rat decreases the content of Bcl-2-associated K-Ras in hippocampal mitochondria, whereas the amount of H-Ras is increased in the microsomal fraction. Furthermore, we have found that galectin 1, a binding partner of activated Ras, was increased in the soluble fractions. The redistribution of Ras isoforms was accompanied by acceleration in mitochondrial hexokinase and inhibition of mitochondrial aconitase, succinate dehydrogenase, and creatine kinase, whereas the activity of aldolase, as well as cytoplasmic creatine kinase was not changed. Our data suggest that inhibition of mitochondrial oxidative metabolism by reactive oxygen species (ROS) and compensatory elevation of glycolysis in hippocampus occurs during social isolation of rats and Ras trafficking could play an important role in switching of impaired oxidative phosphorylation to anaerobic glycolysis.

31P magnetic resonance spectroscopy suggests impaired mitochondrial function in AZT-treated HIV-infected patients.

Prompted by the report of a mitochondrial myopathy associated with chronic administration of zidovudine (AZT), an inhibitor of mitochondrial DNA synthesis, we obtained 31P magnetic resonance spectra from the calf muscles of AZT-treated patients and age-matched control subjects at rest and during an exercise protocol with a 12-second time resolution. The recovery of phosphocreatine following exercise reflects mitochondrial oxidative function and was significantly delayed in the AZT-treated patients (time constants, 43.3 +/- 12.5 seconds versus control subjects, 24.4 +/- 3.9 seconds). These findings support the hypothesis that the myopathy associated with chronic AZT results from the inhibitory effects of AZT on mitochondrial DNA synthesis and, secondarily, on the inhibition of mitochondrial oxidative metabolism.

Inhibition of mitochondrial oxidative metabolism by SKF-525a in intact cells and isolated mitochondria

We have examined the effects of various concentrations of SKF-525A (β-diethylamino-ethyldiphenylpropyl acetate · HCl) on the energy metabolism of l and two types of ascites tumor cells, as well as on ion transport in liver slices. In liver slices, 0.2 to 1.0 mM SKF-525A caused an initial stimulation of O 2 uptake which was followed, at 0.5 to 1.0 mM, by a progressive inhibition of O 2 consumption, a fall of slice ATP content, and a reduced transport of K +, Na + and Ca 2+. In isolated mitochondria, we studied the effects of SKF525A on the rate of respiration and on the oxidation-reduction responses of NAD(P) + and cytochrome b in the presence of various substrates. The results suggest that SKF-525A had three distinct actions on liver mitochondria, viz. an uncoupling action at low concentrations (0.02 to 0.17 mM); at higher concentrations (0.2 to 0.5 mM) an inhibition of the oxidation of NAD(P) +-linked substrates, exerted close to the substrate level; also at 0.2 to 0.5 mM, a less effective inhibition of electron transfer at a point between cytochrome b and O 2 in the electron-transfer chain. Experiments on O 2 consumption and cytochrome b oxidation-reduction changes in ascites cells showed only the first two of these effects in the intact tumor cells. We conclude that inhibition of mitochondrial energy-conserving reactions by SKF-525A can have a marked influence on energy-requiring aspects of liver-cell metabolism, one example of which is inhibition of cation active transport.

Metabolic inhibitors: effects on metabolism and transport in the proximal tubule.

This study examined the effects of the metabolic inhibitors rotenone and antimycin A on oxidative metabolism and transport in the proximal renal tubule of the rabbit. Measurements of oxygen consumption rate (QO2), cellular ATP content, and mitochondrial NAD redox state were made in suspensions of renal cortical tubules. Parallel experiments were conducted in isolated perfused proximal convoluted tubules to measure the absorption rates of fluid (Jv), phosphate (JlbPO4), and glucose (JlbGlc). The results indicate that rotenone and antimycin A, at doses (10(-6) M) that maximally inhibited mitochondrial oxidative phosphorylation, abolished net fluid and phosphate absorption and nearly eliminated net glucose transport. Partial inhibition of oxidative metabolism with 10(-7) M rotenone caused proportional reductions in QO2, ATP content, and Jv; however, JlbPO4 was reduced more markedly than either Jv or JlbGlc. We conclude that rotenone and antimycin A inhibit the sodium-dependent transport of fluid, phosphate, and glucose by blocking mitochondrial ATP production. Furthermore, the inhibition of mitochondrial oxidative metabolism and the inhibition of net sodium transport are closely correlated.

Ion transport and oxidative metabolism. I. The inhibition of mitochondrial oxidative metabolism by the unsubstituted aromatic sulfonamides (carbonic anhydrase inhibitors).

Ion transport and oxidative metabolism. I. The inhibition of mitochondrial oxidative metabolism by the unsubstituted aromatic sulfonamides (carbonic anhydrase inhibitors).