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.

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 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.

Carnitine supplementation induces acylcarnitine production in tissues of very long-chain acyl-CoA dehydrogenase-deficient mice, without replenishing low free carnitine.

Deficiency of very long-chain acyl-CoA dehydrogenase (VLCAD) results in accumulation of C14-C18 acylcarnitines and low free carnitine. Carnitine supplementation is still controversial. VLCAD knockout (VLCAD(+/-)) mice exhibit a similar clinical and biochemical phenotype to those observed in humans. VLCAD(+/-) mice were fed with carnitine dissolved in drinking water. Carnitine, acylcarnitines, and gamma-butyrobetaine were measured in blood and tissues. Measurements were performed under resting conditions, after exercise and after 24 h of regeneration. HepG2 cells were incubated with palmitoyl-CoA and palmitoyl-carnitine, respectively, to examine toxicity. With carnitine supplementation, acylcarnitine production was significantly induced. Nevertheless, carnitine was low in skeletal muscle after exercise. Without carnitine supplementation, liver carnitine significantly increased after exercise, and after 24 h of regeneration, carnitine concentrations in skeletal muscle completely replenished to initial values. Incubation of hepatic cells with palmitoyl-CoA and palmitoyl-carnitine revealed a significantly reduced cell viability after incubation with palmitoyl-carnitine. The present study demonstrates that carnitine supplementation results in significant accumulation of potentially toxic acylcarnitines in tissues. The expected prevention of low tissue carnitine was not confirmed. The principle mechanism regulating carnitine homeostasis seems to be endogenous carnitine biosynthesis, also under conditions with increased demand of carnitine such as in VLCAD-deficiency.

Effects of dietary polyunsaturated fatty acids and hepatic steatosis on the functioning of isolated working rat heart under normoxic conditions and during post-ischemic reperfusion.

The purpose of this study was to modify the amount of 22:4 n-6, 22:5 n-6 and 20:5 n-3 in cardiac phospholipids and to evaluate the influence of these changes on the functioning of working rat hearts and mitochondrial energy metabolism under normoxic conditions and during postischemic reperfusion. The animals were fed one of these four diets: (i) 10% sunflower seed oil (SSO); (ii) 10% SSO + 1% cholesterol; (iii) 5% fish oil (FO, EPAX 3000TG, Pronova) + 5% SSO; (iv) 5% FO + 5% SSO + 1% cholesterol. Feeding n-3 PUFA decreased n-6 PUFA and increased n-3 PUFA in plasma lipids. In the phospholipids of cardiac mitochondria, this dietary modification also induced a decrease in the n-6/n-3 PUFA ratio. Cholesterol feeding induced marked hepatic steatosis (HS) characterized by the whitish appearance of the liver. It also brought about marked changes in the fatty acid composition of plasma and mitochondrial phospholipids. These changes, characterized by the impairment of deltaS- and delta6-desaturases, were more obvious in the SSO-fed rats, probably because of the presence of the precursor of the n-6 family (linoleate) in the diet whereas the FO diet contained large amounts of eicosapentaenoic and docosahexaenoic acids. In the mitochondrial phospholipids of SSO-fed rats, the (22:4 n-6 + 22:5 n-6) to 18:2 n-6 ratio was decreased by HS, without modification of the proportion of 20:4 n-6. In the mitochondrial phospholipids of FO-fed rats, the amount of 20:5 n-3 tended to be higher (+56%). Cardiac functioning was modulated by the diets. Myocardial coronary flow was enhanced by HS in the SSO-fed rats, whereas it was decreased in the FO-fed animals. The rate constant k012 representing the activity of the adenylate kinase varied in the opposite direction, suggesting that decreased ADP concentrations could cause oxygen wasting through the opening of the permeability transition pore. The recovery of the pump function tended to be increased by n-3 PUFA feeding (+22%) and HS (+45%). However, the release of ascorbyl free radical during reperfusion was not significantly modified by the diets. Conversely, energy production was increased by ischemia/reperfusion in the SSO group, whereas it was not modified in the FO group. This supports greater ischemia/reperfusion-induced calcium accumulation in the SSO groups than in the FO groups. HS did not modify the mitochondrial energy metabolism during ischemia/reperfusion. Taken together, these data suggest that HS- and n-3 PUFA-induced decrease in 22:4 and 22:5 n-6 and increase in 20:5 n-3 favor the recovery of mechanical activity during post-ischemic reperfusion.

Emotional distress induced rhabdomyolysis in an individual with carnitine palmitoly-transferase deficiency.

A 48-year-old male patient with underlying CPT II enzyme deficiency is described. Emotional stress appeared to precipitate recurrent myalgias, rhabdomyolysis and reversible renal impairment over a 40-year period. Our search of the English literature indicates this to be the first time that the emotional stress has been documented to precipitate the CPT II syndrome. Although the pathogenesis of this syndrome has yet to be established, existing knowledge is briefly reviewed and the likely metabolic and neuroendocrine mechanisms which link emotional stress to muscle metabolism are examined. These mechanisms influence the extent of lipolysis or glycolysis that occurs during the process of muscle ATP generation. It is suggested that neuroendocrine and other stress related changes which favour lipolysis over glycolysis adversely affect muscle energy metabolism in patients whose mitochondria are deficient in CPT II enzyme. Possible treatment strategies are those that favour glycolysis over fatty acid metabolism and include a variety of ways of modulating sympathetic and parasympathetic tone. The use of carbohydrate supplementation beta-blockers and anxiolytic agents is discussed.

Fatty acid oxidation in the heart.

The heart is known for its ability to produce energy from fatty acids (FA) because of its important beta-oxidation equipment, but it can also derive energy from several other substrates including glucose, pyruvate, and lactate. The cardiac ATP store is limited and can assure only a few seconds of beating. For this reason the cardiac muscle can adapt quickly to the energy demand and may shift from a 100% FA-derived energy production (after a lipid-rich food intake) or any balanced situation (e.g., diabetes, fasting, exercise). These situations are not similar for the heart in terms of oxygen requirement because ATP production from glucose is less oxygen-consuming than from FA. The regulation pathways for these shifts, which occur in physiologic as well as pathologic conditions (ischemia-reperfusion), are not yet known, although both insulin and pyruvate dehydrogenase activation are clearly involved. It becomes of strategic importance to clarify the pathways that control these shifts to influence the oxygen requirement of the heart. Excess FA oxidation is closely related to myocardial contraction disorders characterized by increased oxygen consumption for cardiac work. Such an increased oxygen cost of cardiac contraction was observed in stunned myocardium when the contribution of FA oxidation to oxygen consumption was increased. In rats, an increase in n-3 polyunsaturated FA in heart phospholipids achieved by a fish-oil diet improved the recovery of pump activity during postischemic reperfusion. This was associated with a moderation of the ischemia-induced decrease in mitochondrial palmitoylcarnitine oxidation. In isolated mitochondria at calcium concentrations close to that reported in ischemic cardiomyocytes, a futile cycle of oxygen wastage was reported, associated with energy wasting (constant AMP production). This occurs with palmitoylcarnitine as substrate but not with pyruvate or citrate. The energy wasting can be abolished by CoA-SH and other compounds, but not the oxygen wasting. Again, the calcium-induced decrease in mitochondrial ADP/O ratio was reduced by increasing the n-3 polyunsaturated FA in the mitochondrial phospholipids. These data suggest that in addition to the amount of circulating lipids, the quality of FA intake may contribute to heart energy regulation through the phospholipid composition. On the other hand, other intervention strategies can be considered. Several studies have focused on palmitoylcarnitine transferase I to achieve a reduction in beta-oxidation. In a different context, trimetazidine was suggested to exert its anti-ischemic effect on the heart by interfering with the metabolic shift, either at the pyruvate dehydrogenase level or by reducing the beta-oxidation. Further studies will be required to elucidate the complex system of heart energy regulation and the mechanism of action of potentially efficient molecules.

Influence of dietary fish oil on mitochondrial function and response to ischemia.

Dietary supplementation with marine oils may reduce the incidence of thromboembolism and decrease cardiac arrhythmias during myocardial ischemia. However, function of subcellular organelles isolated from hearts of these animals is impaired. In contrast to studies where marine oil was the sole source of dietary lipid in rats, menhaden oil was used to supplement standard canine laboratory chow. In mitochondria isolated from hearts of dogs fed this diet for 60 wk, the phospholipid content of arachidonic acid was replaced by the n-3 fatty acids, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids. Mitochondrial levels of linoleic and linolenic acids were not altered. The mitochondrial membrane phospholipid from the menhaden oil-fed dogs demonstrated increased cardiolipin. The respiratory function of heart mitochondria from the menhaden oil-supplemented dogs was not decreased from that of dogs on standard chow only. Increased succinate-supported respiration paralleled increased cytochrome oxidase in mitochondria from menhaden oil-fed dogs. The activity of the cardiolipin-dependent carnitine acylcarnitine translocase was unaffected. Myocardial ischemia decreased mitochondrial respiration in menhaden-fed dogs. Decreased palmitoylcarnitine-carnitine exchange following ischemia resulted from decreased matrix carnitine rather than decreased translocase activity. Normal levels of the essential fatty acids in the n-3-enriched mitochondrial membrane phospholipids appear to eliminate the mitochondrial dysfunction observed in essential fatty acid-deficient membranes.

The effect of cobalt on mitochondrial ATP-production in the rat myocardium and skeletal muscle.

Cobalt has been shown to accumulate in the myocardium of uraemic patients and has been suggested as a myocardial toxin inhibiting mitochondrial respiration. In order to study the cellular effects of cobalt exposure three groups of rats (n = 12 per group) were fed a diet containing 12% protein without supplementation or with 20 mg and 40 mg CoSO4 7 H2O/kg body weight/day respectively. After 8 weeks the hearts and soleus muscles were removed. Cobalt in tissues and in four cell fractions were analysed with neutron-activation analysis (ng/g wet weight and ng/mg protein respectively). Mitochondrial respiration was analysed as ATP-production rate using pyruvate + malate and palmitoyl-carnitine + malate as substrate. The ATP-production from pyruvate + malate was unchanged in both heart and skeletal muscle in the exposed animals. With palmitate as substrate, the heart muscle showed a slightly lower ATP-production rate (p less than 0.05) after the 20 mg cobalt dose, but the rate was unchanged in the group with higher cobalt intake. No changes in ATP-production rate from palmitate was observed in soleus muscle. The microsomal (100,000 g) fraction in the myocardial cells contained significantly higher cobalt concentrations compared to the mitochondrial fraction in both the unexposed (1.4 ng/mg protein vs 0.19, p less than 0.05) and exposed rats (53.4 ng/mg protein vs 13.2, p less than 0.005). In conclusion, cobalt showed a large accumulation in myocardial cells, without significant effects on mitochondrial ATP-formation rate from oxidation of pyruvate or palmitate and with the highest cobalt content contained in the microsomal (100,000 g) fraction.