Isolated Mouse Liver Mitochondria Research Articles

Drug-induced impairment of mitochondrial fatty acid oxidation and steatosis: assessment of causal relationship with 45 pharmaceuticals.

Drug-induced liver injury (DILI) represents a major issue for pharmaceutical companies, being a potential cause of black-box warnings on marketed pharmaceuticals, or drug withdrawal from the market. Lipid accumulation in the liver also referred to as steatosis, may be secondary to impaired mitochondrial fatty acid oxidation (mtFAO). However, an overall causal relationship between drug-induced mtFAO inhibition and the occurrence of steatosis in patients has not yet been established with a high number of pharmaceuticals. Hence, 32 steatogenic and 13 nonsteatogenic drugs were tested for their ability to inhibit mtFAO in isolated mouse liver mitochondria. To this end, mitochondrial respiration was measured with palmitoyl-l-carnitine, palmitoyl-CoA + l-carnitine, or octanoyl- l-carnitine. This mtFAO tri-parametric assay was able to predict the occurrence of steatosis in patients with a sensitivity and positive predictive value above 88%. To get further information regarding the mechanism of drug-induced mtFAO impairment, mitochondrial respiration was also measured with malate/glutamate or succinate. Drugs such as diclofenac, methotrexate, and troglitazone could inhibit mtFAO secondary to an impairment of the mitochondrial respiratory chain, whereas dexamethasone, olanzapine, and zidovudine appeared to impair mtFAO directly. Mitochondrial swelling, transmembrane potential, and production of reactive oxygen species were also assessed for all compounds. Only the steatogenic drugs amiodarone, ketoconazole, lovastatin, and toremifene altered all these 3 mitochondrial parameters. In conclusion, our tri-parametric mtFAO assay could be useful in predicting the occurrence of steatosis in patients. The combination of this assay with other mitochondrial parameters could also help to better understand the mechanism of drug-induced mtFAO inhibition.

Choline derivatives as natural ligands of mitochondrial nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) regulate mitochondria-driven apoptosis; however, their intracellular ligands are unknown. In the present paper, we show that choline and its derivatives (phosphocholine (PC), L-α-glycerophosphocholine (G-PC) and 1-palmitoyl-sn-glycero-3-phosphocholine (P-GPC)) dose-dependently influence cytochrome c release from isolated mouse liver mitochondria. Choline inhibited Ca2+-stimulated cytochrome c release, while PC attenuated wortmannin-induced cytochrome c release. Small doses of G-PC and P-GPC (up to 0.1 µM) were protective against either Ca2+ or wortmannin, while larger doses (up to 1 µM) stimulated cytochrome c release by themselves. Choline and PC disrupted interaction of VDAC1, Bax and Bcl-2 with mitochondrial α7 nAChRs and favored their interaction with α9 nAChR subunits. It is concluded that choline metabolites can regulate apoptosis by affecting mitochondrial nAChRs. Keywords: apoptosis, choline, choline derivatives, cytochrome c, mitochondria, nicotinic acetylcholine receptor

SYNERGIC EFFECT OF PREPARATION WITH COORDINATION COMPLEX “TRIMETHYDRAZINIUM PROPIONATE+ETHYMTH METHYLHYDROXYPIRIDINE SUCCINATE” ON ENERGY METABOLISM AND CELL RESPIRATION

The article presents the results of an in vitro study of the synergetic effect evaluation of the combined preparation based on coordination complex ethylmethylhydroxypyridine succinate and trimethylhydrazinium propionate on energy metabolism and cell respiration.The aim of the study was to evaluate the mitochondria-directed action of the metabolic and antioxidant preparation based on succinic acid coordination complex with trimethylhydrazinium in relation to optimizing the energy metabolism in the cells under the oxidative stress conditions, as well as against the background of ischemic processes.Materials and methods. The study of the hydroxysuccinate complex effect of the drug Brainmax® components was carried out on isolated mouse liver mitochondria. In the course of the study, the potential of mitochondria, the generation rate of hydrogen peroxide during the respiration, the respiration rate were evaluated in the following positions: a) unstimulated by malate and pyruvate, b) stimulated by malate and pyruvate (complex I substrates), by succinate (complex II substrates), c) against the background of the initial section of the electron transport chain blockade by rotenone, d) in phosphorylation blockade by oligomycin, e) against the background of the FCCP-induced uncoupling, and f) in cyanide-blocked complex IV (cytochrome C oxidase).Results. It has been shown that the succinic acid coordination complex with trimethylhydrazinium, which is the active principle of the Brainmax® drug, significantly reduced the transmembrane potential of mitochondria (IC50=197±5 µM), compared with the widely used preparations of ethylmethylhydroxypyridine succinate and trimethylhydrazinium propionate, which facilitates the transfer of the produced ATP into the cell and preserves a vital activity of mitochondria even under stress. In the study of the mitochondrial respiration stimulated by the substrates of complex I (NADP-coenzyme Q-oxidoreductase), pyruvate and malate, the studied drug led to a more pronounced increase in the oxygen consumption with IC50=75±6 µМ. When evaluating the effect of the complex on the production of ATP by mitochondria, the most pronounced effect was observed with the addition of studied complex, which indicated to the uncoupling of respiration and oxidative phosphorylation at the given concentrations of the studied compounds. When assessing the effect of the complex on the production of hydrogen peroxide by isolated mitochondria, a significant decrease in the peroxide production was shown in the samples containing the complex of trimethylhydrazinium propionate and EMHPS.Conclusion. Based on totality of the results obtained, it can be assumed that a favorable conformation of the pharmacophore groups of ethylmethylhydroxypyridine succinate and trimethylhydrozinium propionate coordination complex included in the composition of Brainmax® leads to a synergetic interaction and more pronounced pharmacological effects on target cells. This complex provides stabilization of a mitochondrial function, intensification of the adenosine triphosphate energy production and the optimization of energy processes in the cell, reduces the severity of the oxidative stress and eliminates undesirable effects of an ischemic-hypoxic tissue damage.

768-P: MSDC-0602 Is a Direct Mitochondrial Pyruvate Carrier Inhibitor That Modulates Central Carbon Metabolism in Mice and Humans

Mitochondrial pyruvate carrier (MPC) disruption attenuates type 2 diabetes NAFLD in mice. MSDC-0602, a PPARγ-sparing, TZD-like molecule that inhibits the MPC, was recently tested in a phase 2B trial for treating NASH, which reported beneficial effects on glucose and insulin homeostasis. Yet, the broad, in vivo metabolomic effects of MSDC-0602 treatment have remained undefined. Here, we tested MSDC-062 metabolic mechanisms of action in vitro and in vivo, spanning from mitochondrial pyruvate uptake assays in isolated mouse mitochondria, through cultured mouse liver cells, in vivo in mice, and in humans participating in the EMMINENCE trial. First, we examined MSDC-0602’s effect in isolated mouse liver mitochondria: Pyruvate-oxidation was decreased by MSDC-0602, and 2.5 µM MSDC-0602 was sufficient to decrease mitochondrial pyruvate uptake by >50%. We extended these findings to AML12 mouse liver cells and mouse liver in vivo. In AML12 cells, MSDC-0602 treatment increased pyruvate and lactate and decreased alanine. These results were comparable but not identical to results from parallel testing of the specific MPC inhibitor UK5099. 14-day treatment of NCD and HFD fed mice with MSDC-0602 decreased liver alanine and branched chain amino acids. As we previously observed with MPC liver-specific knockout mice, the HFD-induced liver TCA intermediate pool size expansion was mitigated by MSDC-0602. Accordingly, MSDC-0602-dependent metabolomic changes were more pronounced in HFD- versus NCD-fed mice. Human EMMINENCE trial participants receiving MSDC-0602 showed decreased circulating alanine, ketone bodies, and glucose. Similarly, circulating and liver glucose were decreased in HFD mice treated with MSDC-0602. Together, these data are consistent with MSDC-0602 modulating in vivo metabolism in a partially MPC-dependent manner and by additional mechanisms that remain to be understood. Disclosure A. J. Rauckhorst: None. D. J. Pape: None. J. R. Colca: Board Member; Self; Metabolic Solutions Development Company, LLC, Employee; Self; Cirius Therapeutics. E. B. Taylor: None. Funding American Diabetes Association (1-18-PDF-060 to A.J.R.); National Institute of Diabetes and Digestive and Kidney Diseases (DK104998); Cirius Therapeutics

Dictyostelium Differentiation-Inducing Factor-1 Promotes Glucose Uptake, at Least in Part, via an AMPK-Dependent Pathway in Mouse 3T3-L1 Cells.

Differentiation-inducing factor-1 (DIF-1) is a chlorinated alkylphenone (a polyketide) found in the cellular slime mold Dictyostelium discoideum. DIF-1 and its derivative, DIF-1(3M) promote glucose consumption in vitro in mammalian cells and in vivo in diabetic rats; they are expected to be the leading antiobesity and antidiabetes compounds. In this study, we investigated the mechanisms underlying the actions of DIF-1 and DIF-1(3M). In isolated mouse liver mitochondria, these compounds at 2–20 μM promoted oxygen consumption in a dose-dependent manner, suggesting that they act as mitochondrial uncouplers, whereas CP-DIF-1 (another derivative of DIF-1) at 10–20 μM had no effect. In confluent mouse 3T3-L1 fibroblasts, DIF-1 and DIF-1(3M) but not CP-DIF-1 induced phosphorylation (and therefore activation) of AMP kinase (AMPK) and promoted glucose consumption and metabolism. The DIF-induced glucose consumption was reduced by compound C (an AMPK inhibitor) or AMPK knock down. These data suggest that DIF-1 and DIF-1(3M) promote glucose uptake, at least in part, via an AMPK-dependent pathway in 3T3-L1 cells, whereas cellular metabolome analysis revealed that DIF-1 and DIF-1(3M) may act differently at least in part.

Abstract 1025: Aberrant TCR activation and altered metabolism drive decreased T cell cytokine production in IDH mutated acute myeloid leukemia

Abstract Isocitrate dehydrogenase 1 or 2 (IDH1/2) mutations are recurrent in AML and introduce neomorphic function in this metabolic enzyme. The resulting accumulation of the oncometabolite R-2-hydroxyglutarate (2-HG) in leukemic cells leads to epigenetic changes, abnormal gene expression, proliferation, and differentiation arrest. Moreover, studies in solid tumors have shown that 2-HG impacts T cells in the microenvironment. In the context of IDH1 mutated gliomas, elevated levels of 2-HG interfere with T cell activation and suppress chemotaxis. We therefore hypothesized that 2-HG directly decreases TCR activation via disruption of metabolic homeostasis leading to T cell dysfunction in AML. Using sorted healthy T cells, we studied the effect of 2-HG on T cell cytokine expression, proliferation, and phenotype. TNF-α expression was significantly decreased after 48-hour treatment with 10 mM 2-HG versus control. Using a co-culture assay with TCR-stimulated T cells and gene-edited AML cell line expressing the IDH2R140Q mutation (E:T 1:1), there was no change in proliferation of T cells compared with T cells expanded without co-culture. However, there was greater expansion of effector memory T cells (CCR7-CD45RA-) cells and decreased expansion of naïve T cells (CCR7+CD45RA+) in the co-culture system compared with T cells in culture alone. Next, we evaluated the effect of 2-HG on activation of NFAT and NF-κB, transcription factors involved in cytokine expression. We created an NFAT-GFP reporter Jurkat cell line that showed significantly decreased NFAT activation after treatment with 2-HG versus control using both PMA/ionomycin and direct TCR stimulation. This was similarly seen in 2-HG treated healthy T cells with decreased nuclear NFAT by Western blot. NF-κB was shown to alter TNF-α expression as treatment with IKK16, an inhibitor of the IKK complex, showed a decrease in TNF-α expression and log fold decrease at pNF-kB. However, treatment with 2-HG showed no difference in response at pNF-κB compared with control. Lastly, we examined alterations of metabolism in the presence of 2-HG. Treatment with 2-HG reduced oxidative phosphorylation in vitro while increasing fatty acid uptake. Contrary to previous reports, 2-HG did not inhibit ATP synthase activity with acute exposure to 2-HG and assay against purified yeast F1 ATPase. Moreover, there was minimal effect on ATP production in isolated mouse liver mitochondria. Our data showed that 2-HG produced in IDH-mutated AML caused decreased cytokine expression by inhibiting NFAT activation without affecting NF-κB phosphorylation. Additionally, 2-HG decreased oxidative phosphorylation in T cells which may directly affect T cell differentiation and TCR activation. This novel immunosuppressive mechanism of IDH-mutated AML demonstrates how oncogenic mutations contribute to immune evasion and has large implications for the use of immune-directed therapies in AML. Citation Format: Zhuoyan Li, Bradley I. Reinfeld, Benjamin J. Reisman, Matthew Z. Madden, Chad R. Potts, Wendy K. Rathmell, Jeffrey C. Rathmell, Paul B. Ferrell. Aberrant TCR activation and altered metabolism drive decreased T cell cytokine production in IDH mutated acute myeloid leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1025.

Betaine protects mice against acetaminophen hepatotoxicity possibly via mitochondrial complex II and glutathione availability

Overdose of acetaminophen (APAP) is a common cause of acute liver failure. Oxidative stress and mitochondrial dysfunction are related to APAP-induced hepatotoxicity. This study investigated the protective role of betaine as a methyl donor and S-adenosylmethionine precursor against APAP hepatotoxicity in isolated mice liver mitochondria. We treated male NMRC mice with 125, 250, 500, and 1000 mg/kg betaine for 5 days followed by 300 mg/kg APAP 1 h later. At 24 h after APAP administration, animals were sacrificed and liver mitochondria were isolated by different centrifugation methods. Biochemical, histological and immunohistochemical analyses were then conducted. Pretreatment of mice with 250, 500, and 1000 mg/kg betaine ameliorated hepatotoxicity (serum ALT and AST activity and histopathology finding); reversed mitochondrial complex II activity, glutathione level, superoxide dismutase, glutathione peroxidase and catalase activity, glutamate cysteine ligase catalytic protein expression, and mitochondrial membrane potential; and suppressed mitochondrial lipid peroxidation and reactive oxygen species. A contradictory effect was observed in mice treated with APAP. Betaine (500 mg/kg) was chosen as the most effective dose to rescue APAP hepatotoxicity in mice. These findings confirmed that betaine plays a protective role against APAP hepatotoxicity via protecting mitochondrial complex II and regenerating mitochondrial GSH levels by increasing GCLC expression. Betaine displayed antioxidant actions different from other antioxidants via modifying cysteine supply in the transsulfuration pathway in the liver. These findings suggested the potential of betaine as an alternative agent for the treatment of hepatotoxicity induced by APAP.

Effect of the Catechol-O-Methyltransferase Inhibitors Tolcapone and Entacapone on Fatty Acid Metabolism in HepaRG Cells.

Tolcapone and entacapone are catechol-O-methyltransferase inhibitors used in patients with Parkinson's disease. For tolcapone, patients with liver failure have been reported with microvesicular steatosis observed in the liver biopsy of 1 patient. We therefore investigated the impact of tolcapone and entacapone on fatty acid metabolism in HepaRG cells exposed for 24 h and on acutely exposed mouse liver mitochondria. In HepaRG cells, tolcapone induced lipid accumulation starting at 100 µM, whereas entacapone was ineffective up to 200 µM. In HepaRG cells, tolcapone-inhibited palmitate metabolism and activation starting at 100 µM, whereas entacapone did not affect palmitate metabolism. In isolated mouse liver mitochondria, tolcapone inhibited palmitate metabolism starting at 5 µM and entacapone at 50 µM. Inhibition of palmitate activation could be confirmed by the acylcarnitine pattern in the supernatant of HepaRG cell cultures. Tolcapone-reduced mRNA and protein expression of long-chain acyl-CoA synthetase 1 (ACSL1) and protein expression of ACSL5, whereas entacapone did not affect ACSL expression. Tolcapone increased mRNA expression of the fatty acid transporter CD36/FAT, impaired the secretion of ApoB100 by HepaRG cells and reduced the mRNA expression of ApoB100, but did not relevantly affect markers of fatty acid binding, lipid droplet formation and microsomal lipid transfer. In conclusion, tolcapone impaired hepatocellular fatty acid metabolism at lower concentrations than entacapone. Tolcapone increased mRNA expression of fatty acid transporters, inhibited activation of long-chain fatty acids and impaired very low-density lipoprotein secretion, causing hepatocellular triglyceride accumulation. The findings may be relevant in patients with a high tolcapone exposure and preexisting mitochondrial dysfunction.

Protective effect of metformin on toxicity of butyric acid and arsenic in isolated liver mitochondria and langerhans islets in male mice: an in vitro study.

Objective(s):Arsenic, a toxic metal in drinking water and butyric acid (BA) is a free fatty acid found in many foods. These two can induce oxidative stress in some tissues. The present study investigated the protective effect of metformin against toxicity induced by Arsenic (As) and BA in isolated mice liver mitochondria and pancreatic islets.Materials and Methods:In this study, liver mitochondria were isolated by adopting different centrifugation methods and pancreatic islets isolated by a collagenase method. Mitochondria were incubated by BA (75 μM), As (100 μM) and metformin (0, 0.5, 1, 3, 10 mM) and the islets also incubated by BA (1000 μM), As (100 μM) and metformin (0, 1, 3, 10 mM) for 1 hr. At the end of study, mitochondrial viability (MTT), mitochondrial membrane potential (MMP), reactive oxygen species (ROS), malondial- dehyde (MDA), glutathione (GSH) and islets insulin secretion were measured employing specific relevant methods.Results:As and BA significantly increased ROS, MDA and ΔΨm levels and decreased GSH level, succinate dehydrogenase activity and insulin secretion. On the other hand, pretreatment with metformin, returned mitochondrial complex II activity, reduced ROS, MDA and ΔΨm levels and increased GSH level and insulin secretion of pancreatic islets.Conclusion:As and BA in combination or in isolation induce oxidative stress in liver mitochondria and decrease insulin secretion of pancreatic islets. Metformin has a protective effect probably caused by its antioxidant feature. The findings suggest the potential role of metformin in mitochondria therapy and insulin secretion in many diseases.

Hepatocellular Toxicity of Imidazole and Triazole Antimycotic Agents.

Hepatotoxicity has been described for all antimycotic azoles currently marketed. A possible mechanism involving mitochondrial dysfunction has been postulated for ketoconazole, but not for the other azoles. The aim of the current investigations was to study the toxicity of different azoles in human cell models and to find out mechanisms of their toxicity. In HepG2 cells, posaconazole and ketoconazole were cytotoxic starting at 20 and 50 µM and decreased the cellular ATP content starting at 5 and 10 µM, respectively. In HepaRG cells, cytotoxicity started at 20 and 100 µM for posaconazole and ketoconazole, respectively, and was slightly accentuated by cytochrome P450 3A4 induction with rifampicin and 1A2 with 3-methylcholantrene. Voriconazole and fluconazole were not cytotoxic. In isolated mouse liver mitochondria, ketoconazole impaired membrane potential and complex I activity, whereas the other azoles were not toxic. In HepG2 cells exposed for 24 h, both posaconazole and ketoconazole (but not fluconazole or voriconazole) decreased the mitochondrial membrane potential, impaired the function of enzyme complexes of the electron transport chain, were associated with mitochondrial superoxide accumulation, decreased mitochondrial DNA and induced apoptosis. In HepG2 cells with mitochondrial dysfunction induced by the vitamin B12 antagonist hydroxy-cobalamin[c-lactam], cytotoxicity and/or ATP depletion was more accentuated than in untreated cells. We conclude that ketoconazole and posaconazole are mitochondrial toxicants starting at concentrations, which can be reached in vivo. Cytotoxicity and ATP depletion are more accentuated in cells with mitochondrial damage, suggesting that preexisting mitochondrial dysfunction is a susceptibility factor for hepatotoxicity associated with these drugs.

Abstract 220: SR4 uncouples mitochondrial oxidative-phosphorylation, modulates AMPK-mTOR signaling, and inhibits proliferation of HepG2 hepatocarcinoma cells

Abstract Mitochondrial oxidative phosphorylation produces most of the energy in aerobic cells by coupling respiration with the production of ATP. Mitochondrial uncouplers, which reduce the proton gradient across the mitochondrial inner membrane, create a futile cycle of nutrient oxidation without generating ATP. Regulation of mitochondrial dysfunction and associated cellular bioenergetics has been recently identified as promising targets for anticancer therapy. Here, we show that SR4 is a novel mitochondrial uncoupler that causes dose-dependent increase in mitochondrial respiration and dissipation of mitochondrial membrane potential (MMP) in HepG2 hepatocarcinoma cells. These effects were reversed by the recoupling agent 6-ketocholestanol, but not cyclosporin A, and were non-existent in mitochondria-DNA depleted HepG2 (po) cells. In isolated mouse liver mitochondria, SR4 similarly increased oxygen consumption independent of adenine nucleotide translocase (ANT) and uncoupling proteins, decreases MMP, and promotes swelling of valinomycin-treated mitochondria in potassium acetate media. Mitochondrial uncoupling in HepG2 cells by SR4 results in the reduction of cellular ATP production, increased ROS production, activation of the energy-sensing enzyme AMPK, and inhibition of acetyl-CoA carboxylase and mammalian target of rapamycin (mTOR) signaling pathways, leading to cell cycle arrest and apoptosis. Global analysis of SR4-associated differential gene expression confirms these observations, including significant induction of apoptotic genes and down regulation of cell cycle, mitochondrial and oxidative-phosphorylation pathway transcripts at 24 h post treatment. Collectively, our studies demonstrate that SR4's previously reported indirect activation of AMPK and in-vitro anticancer properties, as well as its beneficial effects in both animal xenograft and obese mice models could be a direct consequence of its mitochondrial uncoupling activity (Supported by NIH grant CA 77495 and Beckman Research Institute of the City of Hope). Citation Format: Sharad S. Singhal, James Figarola, Jyotsana Singhal, Joshua Tompkins, David Horne, Sanjay Awasthi, Arthur Riggs. SR4 uncouples mitochondrial oxidative-phosphorylation, modulates AMPK-mTOR signaling, and inhibits proliferation of HepG2 hepatocarcinoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 220.

Asiatic acid uncouples respiration in isolated mouse liver mitochondria and induces HepG2 cells death

Asiatic acid, one of the triterpenoid components isolated from Centella asiatica, has received increasing attention due to a wide variety of biological activities. To date, little is known about its mechanisms of action. Here we examined the cytotoxic effect of asiatic acid on HepG2 cells and elucidated some of the underlying mechanisms. Asiatic acid induced rapid cell death, as well as mitochondrial membrane potential (MMP) dissipation, ATP depletion and cytochrome c release from mitochondria to the cytosol in HepG2 cells. In mitochondria isolated from mouse liver, asiatic acid treatment significantly stimulated the succinate-supported state 4 respiration rate, dissipated the MMP, increased Ca2+ release from Ca2+-loaded mitochondria, decreased ATP content and promoted cytochrome c release, indicating the uncoupling effect of asiatic acid. Hydrogen peroxide (H2O2) produced by succinate-supported mitochondrial respiration was also significantly inhibited by asiatic acid. In addition, asiatic acid inhibited Ca2+-induced mitochondrial swelling but did not induce mitochondrial swelling in hyposmotic potassium acetate medium which suggested that asiatic acid may not act as a protonophoric uncoupler. Inhibition of uncoupling proteins (UCPs) or blockade of adenine nucleotide transporter (ANT) attenuated the effect of asiatic acid on MMP dissipation, Ca2+ release, mitochondrial respiration and HepG2 cell death. When combined inhibition of UCPs and ANT, asiatic acid-mediated uncoupling effect was noticeably alleviated. These results suggested that both UCPs and ANT partially contribute to the uncoupling properties of asiatic acid. In conclusion, asiatic acid is a novel mitochondrial uncoupler and this property is potentially involved in its toxicity on HepG2 cells.

SR4 Uncouples Mitochondrial Oxidative Phosphorylation, Modulates AMP-dependent Kinase (AMPK)-Mammalian Target of Rapamycin (mTOR) Signaling, and Inhibits Proliferation of HepG2 Hepatocarcinoma Cells

Mitochondrial oxidative phosphorylation produces most of the energy in aerobic cells by coupling respiration to the production of ATP. Mitochondrial uncouplers, which reduce the proton gradient across the mitochondrial inner membrane, create a futile cycle of nutrient oxidation without generating ATP. Regulation of mitochondrial dysfunction and associated cellular bioenergetics has been recently identified as a promising target for anticancer therapy. Here, we show that SR4 is a novel mitochondrial uncoupler that causes dose-dependent increase in mitochondrial respiration and dissipation of mitochondrial membrane potential in HepG2 hepatocarcinoma cells. These effects were reversed by the recoupling agent 6-ketocholestanol but not cyclosporin A and were nonexistent in mitochondrial DNA-depleted HepG2 cells. In isolated mouse liver mitochondria, SR4 similarly increased oxygen consumption independent of adenine nucleotide translocase and uncoupling proteins, decreased mitochondrial membrane potential, and promoted swelling of valinomycin-treated mitochondria in potassium acetate medium. Mitochondrial uncoupling in HepG2 cells by SR4 results in the reduction of cellular ATP production, increased ROS production, activation of the energy-sensing enzyme AMPK, and inhibition of acetyl-CoA carboxylase and mammalian target of rapamycin signaling pathways, leading to cell cycle arrest and apoptosis. Global analysis of SR4-associated differential gene expression confirms these observations, including significant induction of apoptotic genes and down-regulation of cell cycle, mitochondrial, and oxidative phosphorylation pathway transcripts at 24 h post-treatment. Collectively, our studies demonstrate that the previously reported indirect activation of AMPK and in vitro anticancer properties of SR4 as well as its beneficial effects in both animal xenograft and obese mice models could be a direct consequence of its mitochondrial uncoupling activity.

Identification of mitochondrial deficits and melatonin targets in liver of septic mice by high-resolution respirometry

AimsPrevious data showed that melatonin maintains liver mitochondrial homeostasis during sepsis, but neither the mechanisms underlying mitochondrial dysfunction nor the target of melatonin are known. Main methodsHere, we analyzed mitochondrial respiration in isolated mouse liver mitochondria with different substrate combinations (glutamate/malate, glutamate/malate/sucinate or succinate/rotenone) to identify mitochondrial defects and melatonin targets during sepsis. Other bioenergetic parameters including a+a3, b, and c+c1 content, mitochondrial mass, and mitochondrial supercomplexes formation were analyzed. Mitochondrial function was assessed during experimental sepsis induced by cecal ligation and puncture (CLP) in livers of 3mo. C57BL/6 mice at early and late phases of sepsis, i.e., at 8 and 24h after sepsis induction. Key findingsSeptic mice showed mitochondrial injury with a decrease in state 3, respiratory control rate, mitochondrial mass, and cytochrome b and c+c1 content, which was prevented by melatonin treatment. Mitochondrial dysfunction in sepsis was mainly linked to complex I damage, because complex II was far less impaired. These mitochondria preserved the respiratory supramolecular organization, maintaining their electron transport system capacity. SignificanceThis work strengthens the use of substrate combinations to identify specific respiratory defects and selective melatonin actions in septic mitochondria. Targeting mitochondrial complex I should be a main therapeutical approach in the treatment of sepsis, whereas the use of melatonin should be considered in the therapy of clinical sepsis.

Hepatocellular toxicity of benzbromarone: effects on mitochondrial function and structure.

Benzbromarone is an uricosuric structurally related to amiodarone and a known mitochondrial toxicant. The aim of the current study was to improve our understanding in the molecular mechanisms of benzbromarone-associated hepatic mitochondrial toxicity. In HepG2 cells and primary human hepatocytes, ATP levels started to decrease in the presence of 25-50μM benzbromarone for 24-48h, whereas cytotoxicity was observed only at 100μM. In HepG2 cells, benzbromarone decreased the mitochondrial membrane potential starting at 50μM following incubation for 24h. Additionally, in HepG2 cells, 50μM benzbromarone for 24h induced mitochondrial uncoupling,and decreased mitochondrial ATP turnover and maximal respiration. This was accompanied by an increased lactate concentration in the cell culture supernatant, reflecting increased glycolysis as a compensatory mechanism to maintain cellular ATP. Investigation of the electron transport chain revealed a decreased activity of all relevant enzyme complexes. Furthermore, treatment with benzbromarone was associated with increased cellular ROS production, which could be located specifically to mitochondria. In HepG2 cells and in isolated mouse liver mitochondria, benzbromarone also reduced palmitic acid metabolism due to an inhibition of the long-chain acyl CoA synthetase. In HepG2 cells, benzbromarone disrupted the mitochondrial network, leading to mitochondrial fragmentation and a decreased mitochondrial volume per cell. Cell death occurred by both apoptosis and necrosis. The study demonstrates that benzbromarone not only affects the function of mitochondria in HepG2 cells and human hepatocytes, but is also associated with profound changes in mitochondrial structure which may be associated with apoptosis.

α7 nicotinic acetylcholine receptors control cytochrome c release from isolated mitochondria through kinase-mediated pathways

Nicotinic acetylcholine receptors are ligand-gated ion channels found in the plasma membrane of both excitable and non-excitable cells. Previously we reported that nicotinic receptors containing α7 subunits were present in the outer membranes of mitochondria to regulate the early apoptotic events like cytochrome c release. Here we show that signaling of mitochondrial α7 nicotinic receptors affects intramitochondrial protein kinases. Agonist of α7 nicotinic receptors PNU 282987 (30nM) prevented the effect of phosphatidyl inositol-3-kinase inhibitor wortmannin, which stimulated cytochrome c release in isolated mouse liver mitochondria, and restored the Akt (Ser 473) phosphorylation state decreased by either 90μM Ca2+ or wortmannin. The effect of PNU 282987 was similar to inhibition of calcium-calmodulin-dependent kinase II (upon 90μM Ca2+) or of Src kinase(s) (upon 0.5mM H2O2) and of protein kinase C. Cytochrome c release from mitochondria could be also attenuated by α7 nicotinic receptor antagonist methyllicaconitine or α7-specific antibodies. Allosteric modulator PNU 120526 (1μM) did not improve the effect of agonist PNU 282987. Acetylcholine (1μM) and methyllicaconitine (10nM) inhibited superoxide release from mitochondria measured according to alkalization of Ca2+-containing medium. It is concluded that α7 nicotinic receptors regulate mitochondrial permeability transition pore formation through ion-independent mechanism involving activation of intramitochondrial PI3K/Akt pathway and inhibition of calcium-calmodulin-dependent or Src-kinase-dependent signaling pathways.

Bypassing the compromised mitochondrial electron transport with methylene blue alleviates efavirenz/isoniazid-induced oxidant stress and mitochondria-mediated cell death in mouse hepatocytes

Efavirenz (EFV) is an anti-retroviral drug frequently combined with isoniazid (INH) to treat HIV-1/tuberculosis co-infected patients. Both drugs have been associated with idiosyncratic liver injury (DILI), but combined anti-retroviral and anti-tubercular therapy can increase the risk for DILI as compared to either drug class alone. Because both EFV and INH have been implicated in targeting mitochondria, we aimed at exploring whether the two drugs might cause synergistic effects on the electron transport chain. We found that EFV inhibited complex I activity in isolated mouse liver mitochondria (IC50 ˜30 μM), whereas hydrazine, a major metabolite of INH generated by acylamidase-mediated hydrolytic cleavage, inhibited complex II activity (IC50 ˜30 μM). Neither INH alone (≤1000 μM) nor EFV alone (≤30 μM) was able to induce cell injury in cultured mouse hepatocytes. However, combined EFV/INH exposure resulted in increased superoxide formation and peroxynitrite stress, leading to the opening of the cyclosporine A-insensitive mode of the mitochondrial permeability transition (mPT), and necrotic cell death. The peroxynitrite scavengers, CBA or Fe-TMPyP, protected against mPT induction and alleviated cell injury. The acylamidase inhibitor bis-p-nitrophenyl phosphate prevented cell injury, suggesting that hydrazine greatly contributed to the toxicity. Methylene blue, a redox-active alternative electron acceptor/donor that bypasses complex I/II, effectively protected against EFV/INH-induced toxicity. These data demonstrate that, in murine hepatocytes, the mitochondrial electron transport chain is a critical target of combined EFV/INH exposure, and that this drug combination can lead to peroxynitrite stress-induced mPT and hepatocellular necrosis. These results are compatible with the concept that underlying silent mitochondrial dysfunction may be a key susceptibility factor contributing to idiosyncratic drug-induced liver injury.

Formation of electrophilic oxidation products from mitochondrial cardiolipin in vitro and in vivo in the context of apoptosis and atherosclerosis

Emerging evidence indicates that mitochondrial cardiolipins (CL) are prone to free radical oxidation and this process appears to be intimately associated with multiple biological functions of mitochondria. Our previous work demonstrated that a significant amount of potent lipid electrophiles including 4-hydroxy-nonenal (4-HNE) was generated from CL oxidation through a novel chemical mechanism. Here we provide further evidence that a characteristic class of CL oxidation products, epoxyalcohol-aldehyde-CL (EAA-CL), is formed through this novel mechanism in isolated mice liver mitochondria when treated with the pro-apoptotic protein t-Bid to induce cyt c release. Generation of these oxidation products are dose-dependently attenuated by a peroxidase inhibitor acetaminophen (ApAP). Using a mouse model of atherosclerosis, we detected significant amount of these CL oxidation products in liver tissue of low density lipoprotein receptor knockout (LDLR −/−) mice after Western diet feeding. Our studies highlight the importance of lipid electrophiles formation from CL oxidation in the settings of apoptosis and atherosclerosis as inhibition of CL oxidation and lipid electrophiles formation may have potential therapeutic value in diseases linked to oxidant stress and mitochondrial dysfunctions.

Effect of garlic-derived organosulfur compounds on mitochondrial function and integrity in isolated mouse liver mitochondria

The objectives of this work were to evaluate the direct effects of diallysulfide (DAS) and diallyldisulfide (DADS), two major organosulfur compounds of garlic oil, on mitochondrial function and integrity, by using isolated mouse liver mitochondria in a cell-free system. DADS produced concentration-dependent mitochondrial swelling over the range 125–1000μM, while DAS was ineffective. Swelling experiments performed with de-energized or energized mitochondria showed similar maximal swelling amplitudes. Cyclosporin A (1μM), or ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA, 1mM) were ineffective in inhibiting DADS-induced mitochondrial swelling. DADS produced a minor (12%) decrease in mitochondrial membrane protein thiols, but did not induce clustering of mitochondrial membrane proteins. Incubation of mitochondria with DADS (but not DAS) produced an increase in the oxidation rate of 2′,7′ dichlorofluorescein diacetate (DCFH-DA), together with depletion of reduced glutathione (GSH) and increased lipid peroxidation. DADS (but not DAS) produced a concentration-dependent dissipation of the mitochondrial membrane potential, but did not induce cytochrome c release. DADS-dependent effects, including mitochondrial swelling, DCFH-DA oxidation, lipid peroxidation and loss of mitochondrial membrane potential, were inhibited by antioxidants and iron chelators. These results suggest that DADS causes direct impairment of mitochondrial function as the result of oxidation of the membrane lipid phase initiated by the GSH- and iron-dependent generation of oxidants.

Prediction of Liver Injury Induced by Chemicals in Human With a Multiparametric Assay on Isolated Mouse Liver Mitochondria

Drug-induced liver injury (DILI) in humans is difficult to predict using classical in vitro cytotoxicity screening and regulatory animal studies. This explains why numerous compounds are stopped during clinical trials or withdrawn from the market due to hepatotoxicity. Thus, it is important to improve early prediction of DILI in human. In this study, we hypothesized that this goal could be achieved by investigating drug-induced mitochondrial dysfunction as this toxic effect is a major mechanism of DILI. To this end, we developed a high-throughput screening platform using isolated mouse liver mitochondria. Our broad spectrum multiparametric assay was designed to detect the global mitochondrial membrane permeabilization (swelling), inner membrane permeabilization (transmembrane potential), outer membrane permeabilization (cytochrome c release), and alteration of mitochondrial respiration driven by succinate or malate/glutamate. A pool of 124 chemicals (mainly drugs) was selected, including 87 with documented DILI and 37 without reported clinical hepatotoxicity. Our screening assay revealed an excellent sensitivity for clinical outcome of DILI (94 or 92% depending on cutoff) and a high positive predictive value (89 or 82%). A highly significant relationship between drug-induced mitochondrial toxicity and DILI occurrence in patients was calculated (p < 0.001). Moreover, this multiparametric assay allowed identifying several compounds for which mitochondrial toxicity had never been described before and even helped to clarify mechanisms with some drugs already known to be mitochondriotoxic. Investigation of drug-induced loss of mitochondrial integrity and function with this multiparametric assay should be considered for integration into basic screening processes at early stage to select drug candidates with lower risk of DILI in human. This assay is also a valuable tool for assessing the mitochondrial toxicity profile and investigating the mechanism of action of new compounds and marketed compounds.