Defective muscle ketone body oxidation disrupts BCAA catabolism by altering mitochondrial branched-chain aminotransferase.

Ketone bodies are an endogenous fuel source generated primarily by the liver to provide alternative energy for extrahepatic tissues during prolonged fasting and exercise. Skeletal muscle is an important site of ketone body oxidation that occurs through a series of reactions requiring the enzyme succinyl-CoA:3-ketoacid-CoA transferase (SCOT/Oxct1). We have previously shown that deleting SCOT in the skeletal muscle protects against obesity-induced insulin resistance by increasing pyruvate dehydrogenase (PDH) activity, the rate-limiting enzyme of glucose oxidation. However, it remains unclear whether inhibiting muscle ketone body oxidation causes hypoglycemia and affects fuel metabolism in the absence of obesity. Here, we show that lean mice lacking skeletal muscle SCOT (SCOTSkM-/-) exhibited no overt phenotypic differences in glucose and fat metabolism from their human α-skeletal actin-Cre (HSACre) littermates. Of interest, we found that plasma and muscle branched-chain amino acid (BCAA) levels are elevated in SCOTSkM-/- lean mice compared with their HSACre littermates. Interestingly, this alteration in BCAA catabolism was only seen in SCOTSkM-/- mice under low-fat feeding and associated with decreased expression of mitochondrial branched-chain aminotransferases (BCATm/Bcat2), the first enzyme in BCAA catabolic pathway. Loss- and gain-of-function studies in C2C12 myotubes demonstrated that suppressing SCOT markedly diminished BCATm expression, whereas overexpressing SCOT resulted in an opposite effect without influencing BCAA oxidation enzymes. Furthermore, SCOT overexpression in C2C12 myotubes significantly increased luciferase activity driven by a Bcat2 promoter construct. Together, our findings indicate that SCOT regulates the expression of the Bcat2 gene, which, through the abundance of its product BCATm, may influence circulating BCAA concentrations.NEW & NOTEWORTHY Most studies investigated ketone body metabolism under pathological conditions, whereas the role of ketone body metabolism in regulating normal physiology has been relatively understudied. To address this gap, we used lean mice lacking muscle ketone body oxidation enzyme SCOT. Our work demonstrates that deleting muscle SCOT has no impact on glucose and fat metabolism in lean mice, but it disrupts muscle BCAA catabolism and causes an accumulation of BCAAs by altering BCATm.

Pimozide Alleviates Hyperglycemia in Diet-Induced Obesity by Inhibiting Skeletal Muscle Ketone Oxidation.

Perturbations in carbohydrate, lipid, and protein metabolism contribute to obesity-induced type 2 diabetes (T2D), though whether alterations in ketone body metabolism influence T2D pathology is unknown. We report here that activity of the rate-limiting enzyme for ketone body oxidation, succinyl-CoA:3-ketoacid-CoA transferase (SCOT/Oxct1), is increased in muscles of obese mice. We also found that the diphenylbutylpiperidine pimozide, which is approved to suppress tics in individuals with Tourette syndrome, is a SCOT antagonist. Pimozide treatment reversed obesity-induced hyperglycemia in mice, which was phenocopied in mice with muscle-specific Oxct1/SCOT deficiency. These actions were dependent on pyruvate dehydrogenase (PDH/Pdha1) activity, the rate-limiting enzyme of glucose oxidation, as pimozide failed to alleviate hyperglycemia in obese mice with a muscle-specific Pdha1/PDH deficiency. This work defines a fundamental contribution of enhanced ketone body oxidation to the pathology of obesity-induced T2D, while suggesting pharmacological SCOT inhibition as a new class of anti-diabetes therapy.

Myeloperoxidase-mediated oxidation of edaravone produces an apparent non-toxic free radical metabolite and modulates hydrogen peroxide-mediated cytotoxicity in HL-60 cells.

Edaravone is considered to be a potent antioxidant drug known to scavenge free radical species and prevent free radical-induced lipid peroxidation. In this study, we investigated the effect of edaravone on the myeloperoxidase (MPO) activity, an enzyme responsible for the production of an array of neutrophil-derived oxidants that can cause cellular damage. The addition of edaravone to the reaction of MPO and hydrogen peroxide (H2O2) significantly enhanced the reduction of MPO Compound II back to native MPO. Interestingly, the MPO-mediated production of toxic hypochlorous acid exhibited a concentration-dependent biphasic effect, with the apparent optimal edaravone concentration at 10 µM. Oxidation of edaravone by MPO was examined by various analytical methods. An MPO-catalyzed product(s) of edaravone was identified at 350 nm by kinetic analysis of UV-Vis spectroscopy. Several MPO-catalyzed metabolites of edaravone were proposed from the LC-MS analyses, including oxidized dimers from edaravone radicals. Electron spin resonance (ESR) spin trapping detected a carbon-centred radical metabolite of edaravone. NMR studies revealed that there are two exchangeable hydrogens, one of which is on the α-carbon, justifying the carbon-centred edaravone radical produced from MPO. Despite the formation of an edaravone carbon-radical metabolite, it did not appear to effectively oxidize GSH (in comparison with phenoxyl radicals). Viability (ATP) and cytotoxicity (LDH release) assays showed a concentration-dependent effect of edaravone on HL-60 cells treated with either a bolus concentration of 30 µM H2O2 or a flux of H2O2 generated by 5 mM glucose and 10 mU/mL glucose oxidase. The H2O2-induced toxicity was ameliorated at high edaravone concentrations (100-200 µM). In contrast, low concentrations of edaravone (1-10 µM) exacerbated the H2O2-induced toxicity. However, the effect of edaravone at low concentration (0-10 µM) appeared more prominent with the LDH assay only. The cellular findings correlated with the biochemical studies with respect to hypochlorous acid formation. These findings provide interesting perspectives regarding the duality of edaravone as an antioxidant drug.

Untying the knot of transcription factor druggability: Molecular modeling study of FOXM1 inhibitors.

The FOXM1 protein is a relevant transcription factor involved in cancer cell proliferation. The direct or indirect inhibition of this protein's transcriptional activity by small molecule drugs correlates well with a potentially significant anti-cancer profile, making this macro molecule a promising drug target. There are a few drug molecules reported to interact with (and inhibit) the FOXM1 DNA binding domain (FOXM1-BD), causing downregulation of protein expression and cancer cell proliferation inhibition. Among these drug molecules are the proteasome inhibitor thiostrepton, the former antidiabetic drug troglitazone, and the new FDI-6 molecule. Despite their structural differences, these drugs exert a similar inhibitory profile, and this observation prompted us to study a possible similar mechanism of action. Using a series of molecular dynamics simulations and docking protocols, we identified essential binding interactions exerted by all three classes of drugs, among which, a π-sulfur interaction (between a His287 and a sulfur-containing heterocycle) was the most important. In this report, we describe the preliminary evidence suggesting the presence of a drug-binding pocket within FOXM1 DNA binding domain, in which inhibitors fit to dissociate the protein-DNA complex. This finding suggests a common mechanism of action and a basic framework to design new FOXM1 inhibitors.