Effects of cigarette smoke on in situ mitochondrial substrate oxidation of slow- and fast-twitch skeletal muscles.

Epidemiological and clinical evidence suggests that cigarette smoke exposure alters glucose and fatty acid metabolism, leading to greater susceptibility to metabolic disorders. However, the effects of cigarette smoke exposure on mitochondrial substrate oxidation in the skeletal muscle are still poorly understood. Accordingly, this study aimed to examine the acute effects of cigarette smoke on mitochondrial respiratory capacity, sensitivity, and concurrent utilization of palmitoylcarnitine (PC), a long-chain fatty acid, and pyruvate, a product of glycolysis, in permeabilized gastrocnemius and soleus muscle fibers exposed to an acute (1 h) dose (4 %) of cigarette smoke concentrate. Cigarette smoke decreased both mitochondrial respiratory capacity (CONTROL: 50.4 ± 11.8 pmolO2/s/mgwt and SMOKE: 22.3 ± 4.4 pmolO2/s/mgwt, p < 0.01) and sensitivity for pyruvate (CONTROL: 0.10 ± 0.04 mM and SMOKE: 0.11 ± 0.04 mM, p < 0.01) in the gastrocnemius muscle. In the soleus, only the sensitivity for pyruvate-stimulated mitochondrial respiration trended toward a decrease (CONTROL: 0.11 ± 0.04 mM and SMOKE: 0.23 ± 0.15 mM, p = 0.08). In contrast, cigarette smoke did not significantly alter palmitoylcarnitine-stimulated mitochondrial respiration in either muscle. In the control condition, pyruvate-supported respiration was inhibited by the concurrent addition of palmitoylcarnitine in the fast-twitch gastrocnemius muscle (-27.1 ± 19.7 %, p < 0.05), but not in the slow-twitch soleus (-9.2 ± 17.0 %). With cigarette smoke, the addition of palmitoylcarnitine augmented the maximal respiration rate stimulated by the concurrent addition of pyruvate in the gastrocnemius (+18.5 ± 39.3 %, p < 0.05). However, cigarette smoke still significantly impaired mitochondrial respiratory capacity with combined substrates compared to control (p < 0.05). Our findings underscore that cigarette smoke directly impairs mitochondrial respiration of carbohydrate-derived substrates and is a primary mechanism underlying cigarette smoke-induced muscle dysfunction, which leads to a vicious cycle involving excess glucose conversion into fatty acids and lipotoxicity.

Fatty acid oxidation: a neglected factor in understanding the adjustment of mitochondrial function to cold temperatures.

For ectothermic species, adaptation to thermal changes is of critical importance. Mitochondrial oxidative phosphorylation (OXPHOS), which leverages multiple electron pathways to produce energy needed for survival, is among the crucial metabolic processes impacted by temperature. Our aim in this study was to identify how changes in temperature affect the less-studied electron transferring flavoprotein pathway, fed by fatty acid substrates. We used the planarian Dugesia tigrina, acclimated for 4 weeks at 10°C (cold acclimated) or 20°C (normothermic). Respirometry experiments were conducted at an assay temperature of either 10 or 20°C to study specific states of the OXPHOS process using the fatty acid substrates palmitoylcarnitine (long chain), octanoylcarnitine (medium chain) or acetylcarnitine (short chain). Following cold acclimation, octanoylcarnitine exhibited increases in both the OXPHOS and electron transfer (ET, non-coupled) states, indicating that the pathway involved in medium-chain length fatty acids adjusts to cold temperatures. Acetylcarnitine only showed an increase in the OXPHOS state as a result of cold acclimation, but not in the ET state, indicative of a change in phosphorylation system capacity rather than fatty acid ß-oxidation. Palmitoylcarnitine oxidation was unaffected. Our results show that cold acclimation in D. tigrina caused a specific adjustment in the capacity to metabolize medium-chain fatty acids rather than an adjustment in the activity of the enzymes carnitine-acylcarnitine translocase, carnitine acyltransferase and carnitine palmitoyltransferase-2. Here, we provide novel evidence of the alterations in fatty acid ß-oxidation during cold acclimation in D. tigrina.

Intestinal metabolomics of juvenile lenok (Brachymystax lenok) in response to heat stress.

Changes in the metabolic profile within the intestine of lenok (Brachymystax lenok) when challenged to acute and lethal heat stress (HS) are studied using no-target HPLC-MS/MS metabonomic analysis. A total of 51 differentially expressed metabolites (VIP > 1, P < 0.05) were identified in response to HS, and 34 occurred in the positive ion mode and 17 in negative ion mode, respectively. After heat stress, changes in metabolites related to glycolysis (i.e., alpha-D-glucose, stachyose, and L-lactate) were identified. The metabolites (acetyl carnitine, palmitoylcarnitine, carnitine, and erucic acid) related to fatty acid ß-oxidation accumulated significantly, and many amino acids (L-tryptophan, D-proline, L-leucine, L-phenylalanine, L-aspartate, L-tyrosine, L-methionine, L-histidine, and L-glutamine) were significantly decreased in HS-treated lenok. The mitochondrial ß-oxidation pathway might be inhibited, while severe heat stress might activate the anaerobic glycolysis and catabolism of amino acid for energy expenditure. Oxidative damage in HS-treated lenok was indicated by the decreased glycerophospholipid metabolites (i.e., glycerophosphocholine, 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-sn-glycero-3-phosphocholine, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine, and 1, 2-dioleoyl-sn-glycero-3-phosphatidylcholine) and the increased oxylipin production (12-HETE and 9R, 10S-EpOME). The minor oxidative pathways (omega-oxidation and peroxisomal beta-oxidation) were likely to be induced in HS-treated lenok.

Protein kinase C inhibitor anchored BRD4 PROTAC PEGylated nanoliposomes for the treatment of vemurafenib-resistant melanoma.

Limited treatment options and development of resistance to targeted therapy within few months pose significant challenges in the treatment of BRAF-mutated malignant melanoma. Moreover, extensive angiogenesis and vasculogenic mimicry promote the rapid progression of disease. The purpose of this study was to develop a protein kinase C inhibitor anchored BRD4 PROTAC (ARV) loaded PEGylated nanoliposomes (LARPC). Palmitoyl-dl-carnitine chloride (PC) was used as a protein kinase C inhibitor to provide a cationic surface charge to LARPC. The formulation was characterized for particle size, zeta potential, drug release and various cell culture assays using HUVEC and vemurafenib resistant melanoma cells. The particle size of LARPC was found to be 105.25 ± 2.76 nm with a zeta potential of +26.6 ± 6.25 mV. Inhibition of angiogenesis was demonstrated by ARV and LARPC using human umbilical vein endothelial cells (HUVEC)-based matrigel basement membrane model. Additionally, LARPC demonstrated very low IC50 with promising inhibition of vasculogenic mimicry channel formation, cell migration as well as colony formation in vemurafenib-resistant melanoma cell lines. Hence, the outcome of this combination therapy indicated the suitability of LARPC as a potential and novel approach for eradicating vemurafenib-resistant melanoma.

Carnitine palmitoyltransferase 2 knockout potentiates palmitate-induced insulin resistance in C2C12 myotubes.

Saturated fatty acids (SFAs) are implicated in muscle inflammation/cell stress and insulin resistance, but the catalog of factors involved is incomplete. SFA derivatives that accumulate with mismatched FA availability and FA oxidation (FAO) are likely involved, and evidence has emerged that select acylcarnitines should be considered. To understand if excessive long-chain acylcarnitine accumulation and limited FAO associate with lipotoxicity, carnitine palmitoyltransferase 2 knockout C2C12 cells were generated (CPT2 KO). CPT2 KO was confirmed by Western blot, increased palmitoylcarnitine accumulation, and loss of FAO capacity. There was no effect of CPT2 KO on palmitic acid (PA) concentration-dependent increases in media IL-6 or adenylate kinase. PA at 200 and 500 µM did not trigger cell stress responses (phospho-Erk, -JNK, or -p38) above that of vehicle in WT or CPT2 KO cells. In contrast, loss of CPT2 exacerbated PA-induced insulin resistance (acute phospho-Akt; 10 or 100 nM insulin) by as much as ~50-96% compared with WT. Growing cells in carnitine-free media abolished differences between WT and CPT2 KO, but this did not fully rescue PA-induced insulin resistance. The results suggest that PA-induced insulin resistance stems in part from palmitoylcarnitine accumulation, further supporting the hypothesis that select acylcarnitines participate in cell signaling and, when in excess, can compromise cell function. Since carnitine-free conditions could not fully rescue insulin signaling, and CPT2 KO did not alter cell stress responses, the majority of PA-induced "lipotoxicity" in C2C12 myotubes cannot be attributed to palmitoylcarnitine alone.

Fatty Acid Oxidation and Mitochondrial Morphology Changes as Key Modulators of the Affinity for ADP in Rat Heart Mitochondria.

Fatty acids are the main respiratory substrates important for cardiac function, and their oxidation is altered during various chronic disorders. We investigated the mechanism of fatty acid-oxidation-induced changes and their relations with mitochondrial morphology and ADP/ATP carrier conformation on the kinetics of the regulation of mitochondrial respiration in rat skinned cardiac fibers. Saturated and unsaturated, activated and not activated, long and medium chain, fatty acids similarly decreased the apparent KmADP. Addition of 5% dextran T-70 to mimic the oncotic pressure of the cellular cytoplasm markedly increased the low apparent KmADP value of mitochondria in cardiac fibers respiring on palmitoyl-l-carnitine or octanoyl-l-carnitine, but did not affect the high apparent KmADP of mitochondria respiring on pyruvate and malate. Electron microscopy revealed that palmitoyl-l-carnitine oxidation-induced changes in the mitochondrial ultrastructure (preventable by dextran) are similar to those induced by carboxyatractyloside. Our data suggest that a fatty acid oxidation-induced conformational change of the adenosine diphosphate (ADP)/adenosine triphosphate (ATP) carrier (M-state to C-state, condensed to orthodox mitochondria) may affect the oxidative phosphorylation affinity for ADP.

Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging of Lipids in Experimental Model of Traumatic Brain Injury Detecting Acylcarnitines as Injury Related Markers.

Identifying new lipid markers linked to traumatic brain injury (TBI) is of major importance in characterizing their central role in the regeneration process and inflammatory response in such an injury model. In the present study, an advanced lipidomics analysis using high spectral resolution matrix-assisted laser desorption/ionization-mass spectrometry imaging was performed on different brain regions in an experimental rat model of moderate controlled cortical impact (CCI) while considering different time points (1 day, 3 days, 7 days, and 10 days) assessing the acute and subacute phase after injury. Our results revealed a new family of lipids, the acylcarnitines, as TBI-lipid related markers, with maximum expression at 3 days after impact and main colocalization within resident microglia of the brain. Furthermore, our experiments highlighted the upregulation of these acylcarnitine lipids, secreted by microglia, in the ipsilateral substantia nigra, the main region in the brain affected in Parkinson's disease (PD).

Exploring the entry route of palmitic acid and palmitoylcarnitine into myoglobin.

Myoglobin, besides its role in oxygen turnover, has gained recognition as a potential regulator of lipid metabolism. Previously, we confirmed the interaction of fatty acids and acylcarnitines with Oxy-Myoglobin, using both molecular dynamic simulations and Isothermal Titration Calorimetry studies. However, those studies were limited to testing only the binding sites derived from homology to fatty acid binding proteins and predictions using automated docking. To explore the entry mechanisms of the lipid ligands into myoglobin, we conducted molecular dynamic simulations of murine Oxy- and Deoxy-Mb structures with palmitate or palmitoylcarnitine starting at different positions near the protein surface. The simulations indicated that both ligands readily (under ∼10-20 ns) enter the Oxy-Mb structure through a dynamic area ("portal region") near heme, known to be the entry point for small molecule gaseous ligands like O2, CO and NO. The entry is not observed with Deoxy-Mb where lipid ligands move away from protein surface, due to a compaction of the entry portal and the heme-containing crevice in the Mb protein upon O2 removal. The results suggest quick spontaneous binding of lipids to Mb driven by hydrophobic interactions, strongly enhanced by oxygenation, and consistent with the emergent role of Mb in lipid metabolism.

Mass spectrometry imaging identifies palmitoylcarnitine as an immunological mediator during Salmonella Typhimurium infection.

Salmonella Typhimurium causes a self-limiting gastroenteritis that may lead to systemic disease. Bacteria invade the small intestine, crossing the intestinal epithelium from where they are transported to the mesenteric lymph nodes (MLNs) within migrating immune cells. MLNs are an important site at which the innate and adaptive immune responses converge but their architecture and function is severely disrupted during S. Typhimurium infection. To further understand host-pathogen interactions at this site, we used mass spectrometry imaging (MSI) to analyse MLN tissue from a murine model of S. Typhimurium infection. A molecule, identified as palmitoylcarnitine (PalC), was of particular interest due to its high abundance at loci of S. Typhimurium infection and MLN disruption. High levels of PalC localised to sites within the MLNs where B and T cells were absent and where the perimeter of CD169+ sub capsular sinus macrophages was disrupted. MLN cells cultured ex vivo and treated with PalC had reduced CD4+CD25+ T cells and an increased number of B220+CD19+ B cells. The reduction in CD4+CD25+ T cells was likely due to apoptosis driven by increased caspase-3/7 activity. These data indicate that PalC significantly alters the host response in the MLNs, acting as a decisive factor in infection outcome.

CLUH couples mitochondrial distribution to the energetic and metabolic status.

Mitochondrial dynamics and distribution are critical for supplying ATP in response to energy demand. CLUH is a protein involved in mitochondrial distribution whose dysfunction leads to mitochondrial clustering, the metabolic consequences of which remain unknown. To gain insight into the role of CLUH on mitochondrial energy production and cellular metabolism, we have generated CLUH-knockout cells using CRISPR/Cas9. Mitochondrial clustering was associated with a smaller cell size and with decreased abundance of respiratory complexes, resulting in oxidative phosphorylation (OXPHOS) defects. This energetic impairment was found to be due to the alteration of mitochondrial translation and to a metabolic shift towards glucose dependency. Metabolomic profiling by mass spectroscopy revealed an increase in the concentration of some amino acids, indicating a dysfunctional Krebs cycle, and increased palmitoylcarnitine concentration, indicating an alteration of fatty acid oxidation, and a dramatic decrease in the concentrations of phosphatidylcholine and sphingomyeline, consistent with the decreased cell size. Taken together, our study establishes a clear function for CLUH in coupling mitochondrial distribution to the control of cell energetic and metabolic status.

Controlling skeletal muscle CPT-I malonyl-CoA sensitivity: the importance of AMPK-independent regulation of intermediate filaments during exercise.

The obligatory role of carnitine palmitoyltransferase-I (CPT-I) in mediating mitochondrial lipid transport is well established, a process attenuated by malonyl-CoA (M-CoA). However, the necessity of reducing M-CoA concentrations to promote lipid oxidation has recently been challenged, suggesting external regulation on CPT-I. Since previous work in hepatocytes suggests the involvement of the intermediate filament fraction of the cytoskeleton in regulating CPT-I, we investigated in skeletal muscle if CPT-I sensitivity for M-CoA inhibition could be regulated by the intermediate filaments, and whether AMP-activated protein kinase (AMPK) could be involved in this process. Chemical disruption (3,3'-iminodipropionitrile, IDPN) of the intermediate filaments did not alter mitochondrial respiration or sensitivity for numerous substrates (palmitoyl-CoA, ADP, palmitoyl carnitine and pyruvate). In contrast, IDPN reduced CPT-I sensitivity for M-CoA inhibition in permeabilized muscle fibers, identifying M-CoA kinetics as a specific target for intermediate filament regulation. Importantly, exercise mimicked the effect of IDPN on M-CoA sensitivity, suggesting that intermediate filament disruption in vivo is physiologically important for CPT-I regulation. To ascertain a potential mechanism, since AMPK is activated during exercise, AMPK ß1ß2-KO mice were utilized in an attempt to ablate the observed exercise response. Unexpectedly, these mice displayed drastic attenuation in resting M-CoA sensitivity, such that exercise and IDPN could not further alter M-CoA sensitivity. These data suggest that AMPK is not required for the regulation of the intermediate filament interaction with CPT-I. Altogether, these data highlight that M-CoA sensitivity is important for regulating mitochondrial lipid transport. Moreover, M-CoA sensitivity appears to be regulated by intermediate filament interaction with CPT-I, a process that is important when metabolic homeostasis is challenged.

Efficacy of bezafibrate on fibroblasts of glutaric acidemia type II patients evaluated using an in vitro probe acylcarnitine assay.

INTRODUCTION: We evaluated the effects of bezafibrate (BEZ) on ß-oxidation in fibroblasts obtained from patients with glutaric acidemia type II (GA2) of various clinical severities using an in vitro probe (IVP) assay. METHODS: Cultured fibroblasts from 12 patients with GA2, including cases of the neonatal-onset type both with and without congenital anomalies (the prenatal- and neonatal-onset forms, respectively), the infantile-onset, and the myopathic forms, were studied. The IVP assay was performed by measuring acylcarnitines (ACs) in the cell culture medium of fibroblasts incubated with palmitic acid for 96h in the presence of 0-800µM BEZ using tandem mass spectrometry. RESULTS: The IVP assay showed that 100µM BEZ markedly reduced the level of palmitoylcarnitine (C16) in the neonatal-onset, infantile-onset, and myopathic forms of GA2, either increasing or maintaining a high level of acetylcarnitine (C2), which serves as an index of energy production via ß-oxidation. In the prenatal-onset form, although a small reduction of C16 was also observed in the presence of 100µM BEZ, the level of C2 remained low. At concentrations higher than 100µM, BEZ further decreased the level of ACs including C16, but a concentration over 400µM decreased the level of C2 in most cases. DISCUSSION: BEZ at 100µM was effective for all GA2 phenotypes except for the prenatal-onset form, as a reduction of C16 without deterioration of C2 is considered to indicate improvement of ß-oxidation. The effects of higher doses BEZ could not be estimated by the IVP assay but might be small or nonexistent.

Accumulation of Palmitoylcarnitine and Its Effect on Pro-Inflammatory Pathways and Calcium Influx in Prostate Cancer.

BACKGROUND: Acylcarnitines are intermediates of fatty acid oxidation and accumulate as a consequence of the metabolic dysfunction resulting from the insufficient integration between ß-oxidation and the tricarboxylic acid (TCA) cycle. The aim of this study was to investigate whether acylcarnitines accumulate in prostate cancer tissue, and whether their biological actions could be similar to those of dihydrotestosterone (DHT), a structurally related compound associated with cancer development. METHODS: Levels of palmitoylcarnitine (palcar), a C16:00 acylcarnitine, were measured in prostate tissue using LC-MS/MS. The effect of palcar on inflammatory cytokines and calcium (Ca(2+) ) influx was investigated in in vitro models of prostate cancer. RESULTS: We observed a significantly higher level of palcar in prostate cancerous tissue compared to benign tissue. High levels of palcar have been associated with increased gene expression and secretion of the pro-inflammatory cytokine IL-6 in cancerous PC3 cells, compared to normal PNT1A cells. Furthermore, we found that high levels of palcar induced a rapid Ca(2+) influx in PC3 cells, but not in DU145, BPH-1, or PNT1A cells. This pattern of Ca(2+) influx was also observed in response to DHT. Through the use of whole genome arrays we demonstrated that PNT1A cells exposed to palcar or DHT have a similar biological response. CONCLUSIONS: This study suggests that palcar might act as a potential mediator for prostate cancer progression through its effect on (i) pro-inflammatory pathways, (ii) Ca(2+) influx, and (iii) DHT-like effects. Further studies need to be undertaken to explore whether this class of compounds has different biological functions at physiological and pathological levels. Prostate 76:1326-1337, 2016. © 2016 The Authors. The Prostate published by Wiley Periodicals, Inc.

Trimetazidine does not alter metabolic substrate oxidation in cardiac mitochondria of target patient population.

BACKGROUND AND PURPOSE: Trimetazidine, known as a metabolic modulator, is an anti-anginal drug used for treatment of stable coronary artery disease (CAD). It is proposed to act via modulation of cardiac metabolism, shifting the mitochondrial substrate utilization towards carbohydrates, thus increasing the efficiency of ATP production. This mechanism was recently challenged; however, these studies used indirect approaches and animal models, which made their conclusions questionable. The goal of the current study was to assess the effect of trimetazidine on mitochondrial substrate oxidation directly in left ventricular myocardium from CAD patients. EXPERIMENTAL APPROACH: Mitochondrial fatty acid (palmitoylcarnitine) and carbohydrate (pyruvate) oxidation were measured in permeabilized left ventricular fibres obtained during coronary artery bypass grafting surgery from CAD patients, which either had trimetazidine included in their therapy (TMZ group) or not (Control). KEY RESULTS: There was no difference between the two groups in the oxidation of either palmitoylcarnitine or pyruvate, and in the ratio of carbohydrate to fatty acid oxidation. Activity and expression of pyruvate dehydrogenase, the key regulator of carbohydrate metabolism, were also not different. Lastly, acute in vitro exposure of myocardial tissue to different concentrations of trimetazidine did not affect myocardial oxidation of fatty acid. CONCLUSION AND IMPLICATIONS: Using myocardial tissue from CAD patients, we found that trimetazidine (applied chronically in vivo or acutely in vitro) had no effect on cardiac fatty acid and carbohydrate oxidation, suggesting that the clinical effects of trimetazidine are unlikely to be due to its metabolic effects, but rather to an as yet unidentified intracardiac mechanism.

Measurement of ß-oxidation capacity of biological samples by respirometry: a review of principles and substrates.

Oxidation of fatty acids is a major source of energy in the heart, liver, and skeletal muscle. It can be measured accurately using respirometry in isolated mitochondria, intact cells, and permeabilized cells or tissues. This technique directly measures the rate of oxygen consumption or flux at various respiratory states when appropriate substrates, uncouplers, and inhibitors are used. Acylcarnitines such as palmitoylcarnitine or octanoylcarnitine are the commonly used substrates. The ß-oxidation pathway is prone to feedforward inhibition resulting from accumulation of short-chain acyl-CoA and depletion of CoA, but inclusion of malate or carnitine prevents accumulation of these intermediaries and CoA depletion.

Transorgan fluxes in a porcine model reveal a central role for liver in acylcarnitine metabolism.

Acylcarnitines are derived from mitochondrial acyl-CoA metabolism and have been associated with diet-induced insulin resistance. However, plasma acylcarnitine profiles have been shown to poorly reflect whole body acylcarnitine metabolism. We aimed to clarify the individual role of different organ compartments in whole body acylcarnitine metabolism in a fasted and postprandial state in a porcine transorgan arteriovenous model. Twelve cross-bred pigs underwent surgery where intravascular catheters were positioned before and after the liver, gut, hindquarter muscle compartment, and kidney. Before and after a mixed meal, we measured acylcarnitine profiles at several time points and calculated net transorgan acylcarnitine fluxes. Fasting plasma acylcarnitine concentrations correlated with net hepatic transorgan fluxes of free and C2- and C16-carnitine. Transorgan acylcarnitine fluxes were small, except for a pronounced net hepatic C2-carnitine production. The peak of the postprandial acylcarnitine fluxes was between 60 and 90 min. Acylcarnitine production or release was seen in the gut and liver and consisted mostly of C2-carnitine. Acylcarnitines were extracted by the kidney. No significant net muscle acylcarnitine flux was observed. We conclude that liver has a key role in acylcarnitine metabolism, with high net fluxes of C2-carnitine both in the fasted and fed state, whereas the contribution of skeletal muscle is minor. These results further clarify the role of different organ compartments in the metabolism of different acylcarnitine species.

The effect of high-intensity training on mitochondrial fat oxidation in skeletal muscle and subcutaneous adipose tissue.

High-intensity interval training (HIT) is known to increase mitochondrial content in a similar way as endurance training [60-90% of maximal oxygen uptake (VO2peak)]. Whether HIT increases the mitochondria's ability to oxidize lipids is currently debated. We investigated the effect of HIT on mitochondrial fat oxidation in skeletal muscle and adipose tissue. Mitochondrial oxidative phosphorylation (OXPHOS) capacity, mitochondrial substrate sensitivity (K(m)(app)), and mitochondrial content were measured in skeletal muscle and adipose tissue in healthy overweight subjects before and after 6 weeks of HIT (three times per week at 298 ± 21 W). HIT significantly increased VO2peak from 2.9 ± 0.2 to 3.1 ± 0.2 L/min. No differences were seen in maximal fat oxidation in either skeletal muscle or adipose tissue. K(m)(app) for octanoyl carnitine or palmitoyl carnitine were similar after training in skeletal muscle and adipose tissue. Maximal OXPHOS capacity with complex I- and II-linked substrates was increased after training in skeletal muscle but not in adipose tissue. In conclusion, 6 weeks of HIT increased VO2peak. Mitochondrial content and mitochondrial OXPHOS capacity were increased in skeletal muscle, but not in adipose tissue. Furthermore, mitochondrial fat oxidation was not improved in either skeletal muscle or adipose tissue.

Functional analysis of iPSC-derived myocytes from a patient with carnitine palmitoyltransferase II deficiency.

INTRODUCTION: Carnitine palmitoyltransferase II (CPT II) deficiency is an inherited disorder involving ß-oxidation of long-chain fatty acids (FAO), which leads to rhabdomyolysis and subsequent acute renal failure. The detailed mechanisms of disease pathogenesis remain unknown; however, the availability of relevant human cell types for investigation, such as skeletal muscle cells, is limited, and the development of novel disease models is required. METHODS: We generated human induced pluripotent stem cells (hiPSCs) from skin fibroblasts of a Japanese patient with CPT II deficiency. Mature myocytes were differentiated from the patient-derived hiPSCs by introducing myogenic differentiation 1 (MYOD1), the master transcriptional regulator of myocyte differentiation. Using an in vitro acylcarnitine profiling assay, we investigated the effects of a hypolipidemic drug, bezafibrate, and heat stress on mitochondrial FAO in CPT II-deficient myocytes and controls. RESULTS: CPT II-deficient myocytes accumulated more palmitoylcarnitine (C16) than did control myocytes. Heat stress, induced by incubation at 38°C, leads to a robust increase of C16 in CPT II-deficient myocytes, but not in controls. Bezafibrate reduced the amount of C16 in control and CPT II-deficient myocytes. DISCUSSION: In this study, we induced differentiation of CPT II-deficient hiPSCs into mature myocytes in a highly efficient and reproducible manner and recapitulated some aspects of the disease phenotypes of CPT II deficiency in the myocyte disease models. This approach addresses the challenges of modeling the abnormality of FAO in CPT II deficiency using iPSC technology and has the potential to revolutionize translational research in this field.

Lipid metabolites and their differential pro-arrhythmic profiles: of importance in the development of a new anti-arrhythmic pharmacology.

Arrhythmias have been treated for a long time with drugs that mainly target the ionic pumps and channels. These anti-arrhythmic regimens per se introduce new arrhythmias, which can be detrimental to patients. Advances in development of novel pharmacology without introduction of iatrogenic arrhythmias are thus favorable for an effective treatment of arrhythmias. Electrophysiological stability of the heart has been shown to be closely associated with cardiac metabolism. The present effective anti-arrhythmic drugs such as beta-blockers and amiodarone have profound beneficial effects in regulating myocardial metabolism. Aiming at decreasing production of toxic metabolites or preventing accumulation of arrhythmogenic lipids perhaps is a good strategy to effectively control arrhythmias. Therefore, a better understanding of the pro-arrhythmic profiles of cardiac metabolites helps to explore a new generation of metabolically oriented anti-arrhythmic medications. In this review, we present several lipid metabolites and summarize their arrhythmogenic characteristics.

The mechanism of Intralipid®-mediated cardioprotection complex IV inhibition by the active metabolite, palmitoylcarnitine, generates reactive oxygen species and activates reperfusion injury salvage kinases.

BACKGROUND: Intralipid® administration at reperfusion elicits protection against myocardial ischemia-reperfusion injury. However, the underlying mechanisms are not fully understood. METHODS: Sprague-Dawley rat hearts were exposed to 15 min of ischemia and 30 min of reperfusion in the absence or presence of Intralipid® 1% administered at the onset of reperfusion. In separate experiments, the reactive oxygen species (ROS) scavenger N-(2-mercaptopropionyl)-glycine was added either alone or with Intralipid®. Left ventricular work and activation of Akt, STAT3, and ERK1/2 were used to evaluate cardioprotection. ROS production was assessed by measuring the loss of aconitase activity and the release of hydrogen peroxide using Amplex Red. Electron transport chain complex activities and proton leak were measured by high-resolution respirometry in permeabilized cardiac fibers. Titration experiments using the fatty acid intermediates of Intralipid® palmitoyl-, oleoyl- and linoleoylcarnitine served to determine concentration-dependent inhibition of complex IV activity and mitochondrial ROS release. RESULTS: Intralipid® enhanced postischemic recovery and activated Akt and Erk1/2, effects that were abolished by the ROS scavenger N-(2-mercaptopropionyl)glycine. Palmitoylcarnitine and linoleoylcarnitine, but not oleoylcarnitine concentration-dependently inhibited complex IV. Only palmitoylcarnitine reached high tissue concentrations during early reperfusion and generated significant ROS by complex IV inhibition. Palmitoylcarnitine (1 µM), administered at reperfusion, also fully mimicked Intralipid®-mediated protection in an N-(2-mercaptopropionyl)-glycine -dependent manner. CONCLUSIONS: Our data describe a new mechanism of postconditioning cardioprotection by the clinically available fat emulsion, Intralipid®. Protection is elicited by the fatty acid intermediate palmitoylcarnitine, and involves inhibition of complex IV, an increase in ROS production and activation of the RISK pathway.