Mitochondrial Uncoupling Activity Research Articles

Expanding the π-system of Fatty Acid-Anion Transporter Conjugates Modulates Their Mechanism of Proton Transport and Mitochondrial Uncoupling Activity.

Mitochondrial uncoupling by small molecule protonophores is a promising strategy for developing novel anticancer agents. Recently, aryl urea substituted fatty acids (aryl ureas) were identified as a new class of protonophoric anticancer agents. To mediate proton transport these molecules self-assemble into membrane-permeable anionic dimers in which intermolecular hydrogen bonds between the carboxylate and aryl-urea anion receptor delocalise the negative charge across the aromatic π-system. In this work, we extend the aromatic π-system by introducing a second phenyl substituent to the aryl urea scaffold and compare the proton transport mechanisms and mitochondrial uncoupling actions of these compounds to their monoaryl analogues. It was found that incorporation of meta-linked phenyl substituents into the aryl urea scaffold enhanced proton transport in vesicles and demonstrated superior capacity to depolarise mitochondria, inhibit ATP production and reduce the viability of MDA-MB-231 breast cancer cells. In contrast, diphenyl ureas linked through a 1,4-distribution across the phenyl ring displayed diminished proton transport activity, despite both diphenyl urea isomers possessing similar binding affinities for carboxylates. Mechanistic studies suggest that inclusion of a second aryl ring changes the proton transport mechanism, presumably due to steric factors that impose higher energy penalties for dimer formation.

Ester-stabilized phosphorus ylides as protonophores on bilayer lipid membranes, mitochondria and chloroplasts

Triphenylphosphonium ylides are commonly used as key intermediates in the Wittig reaction. Based on the known acidities of stabilized ylide precursors, we proposed that a methylene group adjacent to phosphorus in these compounds can ensure proton shuttling across lipid membranes. Here, we synthesized (decyloxycarbonylmethyl)triphenylphosphonium bromide (CMTPP-C10) by reaction of triphenylphosphine with decyl bromoacetate. This phosphonium salt precursor of the ester-stabilized phosphorus ylide along with its octyl (CMTPP-C8) and dodecyl (CMTPP-C12) analogues was found to be a carrier of protons in mitochondrial, chloroplast and artificial lipid membranes, suggesting that it can reversibly release hydrogen ions and diffuse through the membranes in both zwitterionic (ylide) and cationic forms. The CMTPP-C10-mediated electrical current across planar bilayer lipid membranes exhibited pronounced proton selectivity. Similar to conventional protonophores, known to uncouple electron transport and ATP synthesis, CMTPP-Cn (n = 8, 10, 12) stimulated mitochondrial respiration, while decreasing membrane potential, at micromolar concentrations, thereby showing the classical uncoupling activity in mitochondria. CMTPP-C12 also caused dissipation of transmembrane pH gradient on chloroplast membranes. Importantly, CMTPP-C10 exhibited substantially lower toxicity in cell culture, than C12TPP. Thus, we report the finding of a new class of ylide-type protonophores, which is of substantial interest due to promising therapeutic properties of uncouplers.

Protonophoric and mitochondrial uncoupling activity of aryl-carbamate substituted fatty acids.

Aryl-urea substituted fatty acids are protonophores and mitochondrial uncouplers that utilise a urea-based synthetic anion transport moiety to carry out the protonophoric cycle. Herein we show that replacement of the urea group with carbamate, a functional group not previously reported to possess anion transport activity, produces analogues that retain the activity of their urea counterparts. Thus, the aryl-carbamate substituted fatty acids uncouple oxidative phosphorylation and inhibit ATP production by collapsing the mitochondrial proton gradient. Proton transport proceeds via self-assembly of the deprotonated aryl-carbamates into membrane permeable dimeric species, formed by intermolecular binding of the carboxylate group to the carbamate moiety. These results highlight the anion transport capacity of the carbamate functional group.

Nitro-Deficient Niclosamide Confers Reduced Genotoxicity and Retains Mitochondrial Uncoupling Activity for Cancer Therapy.

Niclosamide is an oral anthelmintic drug, approved for use against tapeworm infections. Recent studies suggest however that niclosamide may have broader clinical applications in cancers, spurring increased interest in the functions and mechanisms of niclosamide. Previously, we reported that niclosamide targets a metabolic vulnerability in p53-deficient tumours, providing a basis for patient stratification and personalised treatment strategies. In the present study, we functionally characterised the contribution of the aniline 4′-NO2 group on niclosamide to its cellular activities. We demonstrated that niclosamide induces genome-wide DNA damage that is mechanistically uncoupled from its antitumour effects mediated through mitochondrial uncoupling. Elimination of the nitro group in ND-Nic analogue significantly reduced γH2AX signals and DNA breaks while preserving its antitumour mechanism mediated through a calcium signalling pathway and arachidonic acid metabolism. Lipidomics profiling further revealed that ND-Nic-treated cells retained a metabolite profile characteristic of niclosamide-treated cells. Notably, quantitative scoring of drug sensitivity suggests that elimination of its nitro group enhanced the target selectivity of niclosamide against p53 deficiency. Importantly, the results also raise concern that niclosamide may impose a pleiotropic genotoxic effect, which limits its clinical efficacy and warrants further investigation into alternative drug analogues that may ameliorate any potential unwanted side effects.

Compounds Isolated from the Ethyl Acetate Fraction of Canna edulis Ker Gawl Rhizomes

Three compounds were isolated from the rhizome part of Canna edulis for the first time including liquiritigenin, methyl caffeate and uracil. Their structures were elucidated by spectroscopic methods as MS and NMR.
 Keywords
 Canna edulis Ker Gawl, liquiritigenin, methyl caffeate, uracil.
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Alternative Oxidase Confers Thermogenesis and Increased Growth to Drosophila Larvae under Severe Cold Stress

Alternative oxidases (AOX) are non-proton-pumping enzymes present in most groups of organisms, but naturally absent in the mitochondria of vertebrates and insects. However, its xenotopic expression in mammalian and Drosophila models have suggested an immense therapeutic potential, as diverse phenotypes related to mitochondrial diseases have been attenuated. The expression of this enzyme creates an extra pathway for oxygen reduction when the cytochrome c segment of the electron transfer system is overloaded, relieving possible oxidative stress and providing beneficial effects. AOX is known to function in thermogenesis in plants due to its mitochondrial uncoupling activity, so understanding the inherent properties of AOX expressed in metazoans and how they interfere with the metabolism and physiology of model organisms under stress conditions is essential. We have been investigating the effects of temperature and have showed previously that D. melanogaster lines constitutively expressing AOX from Ciona intestinalis develop faster and have larval and pupal viability higher than that of control flies at low temperatures. Here we show that AOX-expressing larvae are also more active and accumulate ~12,5% more body mass when cultured at the severely stressful temperature of 12°C. Using infrared thermography, we observed that these larvae lose body heat significantly less pronounced, maintaining their body temperature ~0.2°C higher than controls. To understand these effects we have analyzed larval mitochondrial respiration and show a reconfiguration of the electron transfer pathways that sustain oxygen consumption: AOX-expressing flies have a ~30% decrease in glycerol-3-phosphate dehydrogenase (mGPDH)-driven oxygen consumption, which is compensated by a ~30% increase in complex I (CI)-driven oxygen consumption, at 12°C. AOX inhibition leads to a ~37% decrease in mGPDH-driven respiration, suggesting a functional interaction between mGPDH and AOX. Because mGPDH is also a non-proton-pumping enzyme, this interaction would ultimately uncouple mitochondria. At low temperatures, this configuration becomes highly functional, especially because mGPDH appears naturally less sensitive to cold, dissipating the energy of electron transfer as heat. Assuming that the increased CI-driven respiration may stimulate the reactions in the tricarboxylic acid cycle, we speculate the increased body mass and growth in AOX larvae may be explained by increased cataplerosis. In summary, our results suggest the increase in cold-dependent fitness observed for AOX lines is caused by a combination of heat production via mGPDH-linked mitochondrial uncoupling and increased CI-linked cataplerosis.

A Selective ERRα/γ Inverse Agonist, SLU-PP-1072, Inhibits the Warburg Effect and Induces Apoptosis in Prostate Cancer Cells.

The estrogen related receptors (ERRs) are a subgroup of nuclear receptors that play a role in regulation of cellular metabolism. Prostate cancer (PCa) cells display altered metabolic signatures, such as the Warburg effect, and the ERRs have been implicated in driving this phenotype. Despite the lack of a known endogenous ligand, synthetic ligands that target the ERRs have been discovered. For example, the ERRα inverse agonist XCT790 modulates metabolic pathways in PCa cells, but it also functions as a mitochondrial uncoupler independent of targeting ERRα. Here, we describe a novel dual ERRα/γ inverse agonist, SLU-PP-1072, derived from the GSK4716 ERRγ agonist scaffold that is distinct from the XCT790 scaffold. SLU-PP-1072 alters PCa cell metabolism and gene expression, resulting in cell cycle dysregulation and increased apoptosis without acute mitochondrial uncoupling activity. Our data suggest that inhibition of ERRα/γ may be beneficial in treatment of PCa, and SLU-PP-1072 provides a unique chemical tool to evaluate the pharmacology of ERRα and ERRγ.

Anilinopyrazines as potential mitochondrial uncouplers

Mitochondrial protonophores transport protons through the mitochondrial inner membrane into the matrix to uncouple nutrient oxidation from ATP production thereby decreasing the proton motive force. Mitochondrial uncouplers have beneficial effects of decrease reactive oxygen species generation and have the potential for treating diseases such as obesity, neurodegenerative diseases, non-alcoholic fatty liver disease (NAFLD), diabetes, and many others. In this study, we report the structure-activity relationship profile of the pyrazine scaffold bearing substituted aniline rings. Our work indicates that a trifluoromethyl group is best at the para position while the trifluoromethoxy group is preferred in the meta position of the aniline rings of 2,3-substituted pyrazines. As proton transport and cycling requires the formation of a negative charge that has to traverse the mitochondrial membrane, a stabilizing internal hydrogen bond is a key feature for efficient mitochondrial uncoupling activity.

Thiazolides promote G1 cell cycle arrest in colorectal cancer cells by targeting the mitochondrial respiratory chain.

Systemic toxicity and tumor cell resistance still limit the efficacy of chemotherapy in colorectal cancer. Therefore, alternative treatments are desperately needed. The thiazolide Nitazoxanide (NTZ) is an FDA-approved drug for the treatment of parasite-mediated infectious diarrhea with a favorable safety profile. Interestingly, NTZ and the thiazolide RM4819-its bromo-derivative lacking antibiotic activity-are also promising candidates for cancer treatment. Yet the exact anticancer mechanism(s) of these compounds still remains unclear. In this study, we systematically investigated RM4819 and NTZ in 2D and 3D colorectal cancer culture systems. Both compounds strongly inhibited proliferation of colon carcinoma cell lines by promoting G1 phase cell cycle arrest. Thiazolide-induced cell cycle arrest was independent of the p53/p21 axis, but was mediated by inhibition of protein translation via the mTOR/c-Myc/p27 pathway, likely caused by inhibition of mitochondrial respiration. While both thiazolides demonstrated mitochondrial uncoupling activity, only RM4819 inhibited the mitochondrial respiratory chain complex III. Interestingly, thiazolides also potently inhibited the growth of murine colonic tumoroids in a comparable manner with cisplatin, while in contrast to cisplatin thiazolides did not affect the growth of primary intestinal organoids. Thus, thiazolides appear to have a tumor-selective antiproliferative activity, which offers new perspectives in the treatment of colorectal cancer.

Leukotriene-B4 modulates macrophage metabolism and fat loss in type 1 diabetic mice.

Serum levels of leukotriene-B4 (LTB4) are increased in type 1 diabetes (T1D) and it mediates systemic inflammation and macrophage reprogramming associated with this condition. Herein, we investigated the involvement of LTB4 in adiposity loss, hyperlipidemia, and changes in macrophage metabolism in a mouse model of streptozotocin-induced T1D. LTB4 receptor (BLT1) antagonist u75302 was employed to block LTB4 effects. As expected, hypoinsulinemia in T1D was associated with hyperglycemia, low levels of glucagon, hyperlipidemia, significant body fat loss, and increased white adipose tissue expression of Fgf21, a marker for lipolysis. With the exception of hyperglycemia and hypoglucagonemia, blockade of LTB4 signaling reverted these parameters in T1D mice. Along with hyperlipidemia, macrophages from T1D mice exhibited higher lipid uptake and accumulation. These cells also had enhanced glycolysis and oxidative metabolism and these parameters were dependent on the mitochondrial uncoupling respiration, as evidenced by elevated expression of oxidation markers carnitine palmitoyltransferase and uncoupling protein 1. Interestingly, all these parameters were at least partially reverted in T1D mice treated with u75302. Altogether, these findings suggest that in T1D mice LTB4/BLT1 is involved in the fat loss, hyperlipidemia, and increased macrophage lipid uptake and metabolism with an important involvement of mitochondrial uncoupling activity. These previously unrecognized LTB4/BLT1 functions may be explored in future to therapeutically alleviate severity of hyperlipidemia and systemic inflammation in T1D.

N-substituted phenylbenzamides of the niclosamide chemotype attenuate obesity related changes in high fat diet fed mice.

Obesity and insulin resistance are primary risk factors for Non-Alcoholic Fatty Liver Disease (NAFLD). NAFLD is generally exhibited by non-progressive simple steatosis. However, a significant subset of patient’s progress to nonalcoholic steatohepatitis (NASH) that is defined by the presence of steatosis, inflammation and hepatocyte injury with fibrosis. Unfortunately, there are no approved therapies for NAFLD or NASH and therefore therapeutic approaches are urgently needed. Niclosamide is an U.S. Food and Drug Administration (FDA)-approved anthelmintic drug that mediates its effect by uncoupling oxidative phosphorylation. Niclosamide and its salt forms, Niclosamide Ethanolamine (NEN), and Niclosamide Piperazine (NPP) have shown efficacy in murine models of diet induced obesity characterized by attenuation of the prominent fatty liver disease phenotype and improved glucose metabolism. While the exact mechanism(s) underlying these changes remains unclear, the ability to uncouple oxidative phosphorylation leading to increased energy expenditure and lipid metabolism or attenuation of PKA mediated glucagon signaling in the liver have been proposed. Unfortunately, niclosamide has very poor water solubility, leading to low oral bioavailability. This, in addition to mitochondrial uncoupling activity and potential genotoxicity have reduced enthusiasm for its clinical use. More recently, salt forms of niclosamide, NEN and NPP, have demonstrated improved oral bioavailability while retaining activity. This suggests that development of safer more effective niclosamide derivatives for the treatment of NAFLD and Type 2 Diabetes may be possible. Herein we explored the ability of a series of N-substituted phenylbenzamide derivatives of the niclosamide salicylanilide chemotype to attenuate hepatic steatosis using a novel phenotypic in vitro model of fatty liver and the high fat diet-fed mouse model of diet induced obesity. These studies identified novel compounds with improved pre-clinical properties that attenuate hepatic steatosis in vitro and in vivo. These compounds with improved drug properties may be useful in alleviating symptoms and protection against disease progression in patients with metabolic syndrome and NAFLD.

Niclosamide piperazine prevents high-fat diet-induced obesity and diabetic symptoms in mice.

Obesity and type 2 diabetes (T2D) have become the major public health challenges globally. Mitochondrial uncoupling, which reduces intracellular lipid loads and corrects the underlying cause of insulin resistance, has emerged as a promising anti-obese and anti-diabetic intervention. Niclosamide is an anthelmintic drug approved by the US FDA with the mechanism of action that uncouples mitochondria of parasitic worms. Recently, niclosamide ethanolamine salt (NEN) was found to be a safe and effective hepatic mitochondrial uncoupler for the prevention and treatment of obesity and T2D in mouse models. The striking features of NEN prompt us to examine the anti-obese and anti-diabetic efficacy of other salt forms of niclosamide, with the ultimate goal to identify a suitable salt formulation for future clinical development. Here, we report the study with niclosamide piperazine salt (NPP), another salt form of niclosamide with documented safety profile. Mitochondrial uncoupling activity of NEN and NPP were determined by oxygen consumption assay with Seahorse XF24e Analyzer, as well as by mitochondrial membrane potential measurement in cultured cells. The in vivo anti-diabetic and anti-obesity activities were determined in C57BL/6J mice fed high-fat diet (HFD) or HFD containing 2000ppm. NPP for 11weeks. Niclosamide piperazine salt showed a comparable mitochondrial uncoupling activity to NEN. Oral administration of NPP significantly reduced HFD-induced obesity, hyperglycemia and hepatic steatosis, and sensitized the insulin responses in mice. Niclosamide piperazine salt may hold the promise to become an alternative to NEN as a drug lead for the treatment of obesity and T2D. No level of evidence Animal study.

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.

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.

Adaptation of Hepatic Mitochondrial Function in Humans with Non-Alcoholic Fatty Liver Is Lost in Steatohepatitis

The association of hepatic mitochondrial function with insulin resistance and non-alcoholic fatty liver (NAFL) or steatohepatitis (NASH) remains unclear. This study applied high-resolution respirometry to directly quantify mitochondrial respiration in liver biopsies of obese insulin-resistant humans without (n= 18) or with (n= 16) histologically proven NAFL or with NASH (n= 7) compared to lean individuals (n= 12). Despite similar mitochondrial content, obesehumans with or without NAFL had 4.3- to 5.0-fold higher maximal respiration rates in isolated mitochondria than lean persons. NASH patients featured higher mitochondrial mass, but 31%-40% lower maximal respiration, which associated with greater hepatic insulin resistance, mitochondrial uncoupling, and leaking activity. In NASH, augmented hepatic oxidative stress (H2O2, lipid peroxides) andoxidative DNAdamage (8-OH-deoxyguanosine) was paralleled by reduced anti-oxidant defense capacity and increased inflammatory response. These data suggest adaptation of the liver ("hepatic mitochondrial flexibility") at early stages of obesity-related insulin resistance, which is subsequently lost in NASH.

Synthesis of prenylated quinolinecarboxylic acid derivatives and their anti-obesity activities

Mitochondrial uncoupling is one of the therapeutic strategies used to control energy metabolism in various metabolic diseases and in obesity. Ppc-1 (1), a prenylated quinolinecarboxylic acid isolated from cellular slime molds, shows uncoupling activity in vitro and anti-obesity activity in vivo. In this study, we synthesized Ppc-1 (1) and its derivatives, and revealed the structure–activity relationship of uncoupling activities. The triprenylated compound 18 showed mitochondrial uncoupling activity that was more potent than that of Ppc-1 (1). Compound 18 also suppressed weight gain in mice without undesired effects such as lesions on tissues. These results indicate that compound 18 could be used as a seed compound for new anti-obesity drugs.

Electrogenic proton transport across lipid bilayer membranes mediated by cationic derivatives of rhodamine 19: comparison with anionic protonophores

Protonophores can be considered as candidates for anti-obesity drugs and tools to prevent excessive reactive oxygen species production in mitochondria by means of a limited decrease in the mitochondrial potential. Experimentally used protonophores are weak acids that can carry protons across a membrane in a neutral (protonated) form, and they come back in an anionic (deprotonated) form. A cationic derivative of rhodamine 19 and plastoquinone (SkQR1) was recently shown to possess uncoupling activity in mitochondria and in intact cells. In this article, we studied the mechanism of action of SkQR1 and its plastoquinone-lacking analog (C12R1) on a planar bilayer lipid membrane by applying voltage jumps. The steady-state current was proportional to the C12R1 concentration in a manner as if the monomeric form of the carrier were operative. As predicted by the carrier model, at high pH, when rhodamines were mainly deprotonated, the current changed immediately following a jump in the applied potential and then remained constant. By contrast, at low pH, the current relaxed from an initially high value to a lower value since the protonated carrier cations were redistributed in the membrane. An inverse pH dependence was revealed with the anionic protonophore CCCP. The dependence of the SkQR1 protonophorous activity on voltage exhibited an increase at high voltages, an effect that might facilitate mild (self-limited) uncoupling of mitochondria.

Role of arachidonic acid, lipoxygenase, and mitochondrial depolarization in reperfusion arrhythmias

We have sought evidence that arachidonic acid (AA) induces mitochondrial depolarization in isolated myocytes by a lipoxygenase (LOX)-dependent mechanism and that such depolarization might contribute to arrhythmogenesis following ischemia-reperfusion injury. A method was developed for measuring mitochondrial depolarization in isolated adult rat myocytes in suspension, using tetramethylrhodamine ethyl ester. The addition of AA to myocytes resulted in mitochondrial depolarization that was inhibited by the LOX inhibitor baicalein, by the reactive oxygen species (ROS) scavenger mercaptoproprionylglycine, and by the anion channel inhibitor diisothiocyanatostilbene-disulfonic acid (DIDS). AA induced mitochondrial uncoupling and mitochondrial ATPase activity in myocytes, but both were insensitive to baicalein. We conclude that the metabolic effect of AA in myocytes puts mitochondria into an energetically compromised state where membrane potential is easily changed by the DIDS-sensitive LOX/ROS-mediated opening of an inner membrane anion channel. In an in vivo anesthetized rat model of coronary artery occlusion, baicalein was found to strongly inhibit arrhythmias induced by ischemia-reperfusion injury. Arrhythmias following ischemia-reperfusion injury have been previously associated with DIDS-sensitive ROS-mediated mitochondrial depolarization, and free fatty acids including AA were previously found to accumulate during such injury. We therefore conclude that arrhythmias following ischemia-reperfusion injury might originate from mitochondrial depolarization mediated by LOX and AA.

Structural constraints and the importance of lipophilicity for the mitochondrial uncoupling activity of naturally occurring caffeic acid esters with potential for the treatment of insulin resistance

Caffeic acid phenethyl ester (CAPE) has recently been shown to potently stimulate glucose uptake in cultured skeletal muscle cells through the AMPK pathway and therefore to have anti-diabetic potential. We report here that CAPE increases glucose uptake in C2C12 muscle cells by 225±21% at 50μM, and that activation of AMPK is a consequence of the metabolic stress resulting from an uncoupling-type disruption of mitochondrial function (complete uncoupling at 50μM). We also observe that the therapeutic potential of CAPE is offset by its high potential for toxicity. The purpose of this study was therefore to identify other active caffeic acid derivatives, evaluate their ratio of activity to toxicity, and elucidate their structure–activity relationship. Twenty naturally occurring derivatives were tested for glucose-uptake stimulating activity in C2C12 cells following 18h of treatment and for uncoupling activity in isolated rat liver mitochondria. Cytotoxicity was assessed in C2C12 cells by the release of lactate dehydrogenase over 18h. In addition to CAPE, four compounds were identified to be active, both stimulating glucose uptake and uncoupling isolated mitochondria. Activity required that the caffeic acid moiety be intact and that the compound not contain a strongly ionized group. Both activity and toxicity were found to be well-correlated to predicted lipophilicity. However, two compounds exhibited little to no toxicity while still stimulating glucose uptake by 65–72%. These results support a therapeutic potential for this family of compounds and provide the framework for the design of alternatives to Metformin with an optimized balance of safety and activity.

Stimulation of cardiac fatty acid oxidation by leptin is mediated by a nitric oxide–p38 MAPK-dependent mechanism

Leptin has previously been shown to stimulate fatty acid oxidation independent of AMP-activated protein kinase (AMPK). Nitric oxide and p38 mitogen activated protein kinase (MAPK) are known effectors of leptin signaling. The aim of the present study was to determine whether nitric oxide and p38 MAPK mediate the stimulation of leptin by MAPK. Hearts from male Sprague–Dawley rats were mounted on the isolated perfused working heart in the presence or absence of leptin (1.9 nM), N-Nitro- l-Arginine Methyl Ester ( l-NAME) (3 µM), the specific p38 MAPK inhibitor 4-[4-(4-Fluorophenyl)-5-(4-pyridinyl)-1H-imidazol-2-yl] phenol (SB202190, 2 µM) and the specific STAT-3 inhibitor (E)-2-Cyano-3-(3,4-dihydrophenyl)-N-(phenylmethyl)-2-propenamide (AG490, 5 µM) for the measurement of substrate metabolism and function. AMPK and carnitine palimitoyltransferase-1 activity, nitrate/nitrite levels, STAT-3 phosphorylation and p38 MAPK phosphorylation were measured. To assess mitochondrial function, hearts were perfused with or without leptin prior to the isolation of mitochondria. Leptin stimulated fatty acid oxidation and decreased cardiac function, associated with the activation of STAT-3 and p38 MAPK and an increase in tissue nitrate/nitrite levels; the effect on function was ameliorated and the effect on fatty acid oxidation was prevented by L-NAME, B202190 and AG490. L-NAME lowered tissue nitrate/nitrite levels, and prevented the phosphorylation of p38, whereas SB202190 had no effect on tissue nitrate/nitrite levels. AG490 also lowered tissue nitrate/nitrite levels. Leptin had no effect on fatty acid-dependent mitochondrial respiration or uncoupling activity, but, surprisingly, stimulated pyruvate-dependent mitochondrial respiration. These data indicate that leptin stimulates fatty acid oxidation by a STAT-3–nitric oxide–p38 MAPK-dependent mechanism. The target of the pathway is upstream of the mitochondria.