Depolarized Mitochondrial Membrane Potential and Protection with Duroquinone in Isolated Perfused Lungs from Rats Exposed to Hyperoxia

Dissipation of mitochondrial membrane potential (Δψm) is a hallmark of mitochondrial dysfunction. our objective was to use a previously developed experimental-computational approach to estimate tissue Δψm in intact lungs of rats exposed to hyperoxia, and to evaluate the ability of duroquinone (DQ) to reverse any hyperoxia-induced depolarization of lung Δψm. Rats were exposed to hyperoxia (>95% O2) or normoxia (room air) for 48 hrs, after which lungs were excised and connected to a ventilation-perfusion system. The experimental protocol consisted of measuring the concentration of the fluorescent dye rhodamine 6G (R6G) during three single-pass phases: loading, washing, and uncoupling, in which the lungs were perfused with and without R6G, and with the mitochondrial uncoupler FCCP, respectively. For normoxic lungs, the protocol was repeated with 1) rotenone (complex I inhibitor), 2) rotenone and either DQ or its vehicle (DMSO), and 3) rotenone, glutathione (GSH), and either DQ or DMSO added to the perfusate. Hyperoxic lungs were studied with and without DQ and GSH added to the perfusate. Computational modeling was used to estimate lung Δψm from R6G data. Rat exposure to hyperoxia resulted in partial depolarization (-33 mV) of lung Δψm, and complex I inhibition depolarized lung Δψm by -83 mV. Results also demonstrate the efficacy of DQ to fully reverse both rotenone-induced and hyperoxia-induced lung Δψm depolarization. This study demonstrates hyperoxia-induced Δψm depolarization in intact lungs, and the utility of this approach for assessing the impact of potential therapies such as exogenous quinones that target mitochondria in intact lungs.

Reprogramming the neuroblastoma epigenome with a mitochondrial uncoupler

Dysregulated DNA methylation is associated with poor prognosis in cancer patients, promoting tumorigenesis and therapeutic resistance1. DNA methyltransferase inhibitors (DNMTi) reduce DNA methylation and promote cancer cell differentiation, with two DNMTi already approved for cancer treatment2. However, these drugs rely on cell division to dilute existing methylation, thus the ‘demethylation’ effects are achieved in a passive manner, limiting their application in slow-proliferating tumor cells. In this study we use a mitochondrial uncoupler, niclosamide ethanolamine (NEN), to actively achieve global DNA demethylation. NEN treatment promotes DNA demethylation by activating electron transport chain (ETC) to produce α-ketoglutarate (α-KG), a substrate for the DNA demethylase TET. In addition, NEN inhibits reductive carboxylation, a key metabolic pathway to support growth of cancer cells with defective mitochondria or under hypoxia. Importantly, NEN treatment reduces 2-hydroxyglutarate (2-HG) generation and blocks DNA hypermethylation under hypoxia. Together, these metabolic reprogramming effects of NEN actively alter the global DNA methylation landscape and promote neuroblastoma differentiation. These results not only support Warburg’s original hypothesis that inhibition of ETC causes cell de-differentiation and tumorigenesis, but also suggest that mitochondrial uncoupling is an effective metabolic and epigenetic intervention that remodels the tumor epigenome for better prognosis.

OXPHOS deficiencies affect peroxisome proliferation by downregulating genes controlled by the SNF1 signaling pathway

How environmental cues influence peroxisome proliferation, particularly through other organelles, remains largely unknown. Yeast peroxisomes metabolize all fatty acids (FA), and methylotrophic yeasts also metabolize methanol. NADH and acetyl-CoA, the products of these pathways enter mitochondria for ATP production, and for anabolic reactions. During the metabolism of FA and/or methanol, the mitochondrial oxidative phosphorylation (OXPHOS) pathway accepts NADH for ATP production and maintains cellular redox balance. Remarkably, peroxisome proliferation in Pichia pastoris was abolished in NADH shuttling and OXPHOS mutants affecting complex I or III, or by the mitochondrial uncoupler, 2,4-dinitrophenol (DNP), indicating ATP depletion causes the phenotype. We show that mitochondrial OXPHOS deficiency inhibits the expression of several peroxisomal proteins implicated in FA and methanol metabolism, as well as in peroxisome division and proliferation. These genes are regulated by the Snf1 complex (SNF1), a pathway generally activated by high AMP and low ATP. Consistent with this mechanism, in OXPHOS mutants, Snf1 is activated by phosphorylation, but Gal83, its interacting subunit, fails to translocate to the nucleus. Phenotypic defects in peroxisome proliferation observed in the OXPHOS mutants, and phenocopied by the Δgal83 mutant, were rescued by deletion of three transcriptional repressor genes (MIG1, MIG2 and NRG1) controlled by SNF1 signaling. We uncovered here the mechanism by which peroxisomal and mitochondrial metabolites influence redox and energy metabolism, while also influencing peroxisome biogenesis and proliferation, thereby exemplifying interorganellar communication and interplay involving peroxisomes, mitochondria, cytosol and the nucleus. We discuss the physiological relevance of this work in view of human OXPHOS deficiencies.

Metabolic PCOS Features Are Ameliorated by Mitochondrial Uncoupler BAM15 in a PCOS Mouse Model

Polycystic ovary syndrome (PCOS) is a prevalent endocrine condition characterized by endocrine, reproductive and metabolic dysfunction. At present, there is no cure for PCOS and current treatments are suboptimal. Obesity and adverse metabolic features are prevalent in women with PCOS, with weight loss having a beneficial effect on PCOS features. The use of dietary interventions aimed at weight loss have low long-term compliance in women suffering from PCOS. Recent data from animal studies has shown that a small molecule mitochondrial uncoupler, BAM15, is an effective method to pharmacologically treat obesity and metabolic diseases. Therefore, the aim of this study was to investigate the efficacy of BAM15 to ameliorate PCOS-traits in a hyperandrogenic PCOS mouse model. As expected, exposure of female mice to dihydrotestosterone (DHT) induced the PCOS metabolic features of increased body weight (P<0.05), lean mass (P<0.001), increased parametrial and mesenteric fat pad weights (both P<0.05) and adipocyte hypertrophy (P<0.05). Additionally, DHT-induced PCOS mice exhibited insulin resistance measured by HOMA-IR, increased cholesterol and fasting triglyceride levels and hepatic steatosis (all P<0.05). In contrast, DHT-induced PCOS females treated with BAM15 displayed body weights which were comparable with controls, a significant decrease in parametrial and mesenteric fat depot weights (P<0.05) and reduced adipocyte hypertrophy. Furthermore, BAM15 treatment decreased insulin resistance, cholesterol and fasting triglyceride levels, as well as the degree of hepatic steatosis observed in PCOS females, to levels comparable with controls. PCOS mice presented the reproductive PCOS traits of irregular cycles and ovulatory dysfunction, however BAM15 did not improve these PCOS traits. These findings demonstrate that the pharmacologic mitochondrial uncoupler BAM15 is able to ameliorate metabolic PCOS features in a hyperandrogenic PCOS mouse model. These data provide compelling evidence to support BAM15 as a potential innovative and viable therapeutic approach to manage metabolic traits associated with PCOS.

The mitochondria-targeted derivative of the classical uncoupler of oxidative phosphorylation carbonyl cyanide m-chlorophenylhydrazone is an effective mitochondrial recoupler

The synthesis of a mitochondria-targeted derivative of the classical mitochondrial uncoupler carbonyl cyanide-m-chlorophenylhydrazone (CCCP) by alkoxy substitution of CCCP with n-decyl(triphenyl)phosphonium cation yielded mitoCCCP, which was able to inhibit the uncoupling action of CCCP, tyrphostin A9 and niclosamide on rat liver mitochondria, but not that of 2,4-dinitrophenol, at a concentration of 1–2 μM. MitoCCCP did not uncouple mitochondria by itself at these concentrations, although it exhibited uncoupling action at tens of micromolar concentrations. Thus, mitoCCCP appeared to be a more effective mitochondrial recoupler than 6-ketocholestanol. Both mitoCCCP and 6-ketocholestanol did not inhibit the protonophoric activity of CCCP in artificial bilayer lipid membranes, which might compromise the simple proton-shuttling mechanism of the uncoupling activity on mitochondria.