Changes in the Subcellular Distribution of Free Carnitine and Its Acyl Derivatives in Diabetic Rat Hearts Following Treatment with L-Carnitine.
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Carnitine deficiency has been demonstrated in diabetic hearts, and it is also well known that L-carnitine administration has a beneficial effect on cardiac function. Carnitine treatment would be expected to reduce the accumulation of long-chain acylcarnitine. However, many reports have shown that myocardial long-chain acylcarnitine levels were increased following treatment with L-carnitine in whole-heart studies. Since acylcarnitine exists in both the mitochondrial and cytosolic compartments, it is difficult to investigate changes in subcellular distribution by studying whole-heart preparations. The present study investigated the myocardial subcellular distribution of carnitine and its acyl derivatives in diabetic rats with or without L-carnitine treatment. Approximately 90% of total cellular carnitine was located in the cytosol in the diabetic hearts. Both mitochondrial and cytosolic levels of free carnitine and short-chain acylcarnitine were significantly decreased in the diabetic heart. However, the mitochondrial level of long-chain acylcarnitine was significantly increased. L-carnitine treatment reduced the mitochondrial level of long-chain acylcarnitine, but the cytosolic level of long-chain acylcarnitine was significantly increased. These results show that L-carnitine treatment prevents the accumulation of long-chain acylcarnitine in the mitochondrial space and then reduces the detergent effect of long-chain acylcarnitine on the mitochondrial membrane. We suggest that it is a possible mechanism of the beneficial effect of L-carnitine treatment on the diabetic heart.Keywords:
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Mg(2+) plays important roles in numerous cellular functions. Mitochondria take part in intracellular Mg(2+) regulation and the Mg(2+) concentration in mitochondria affects the synthesis of ATP. However, there are few methods to observe Mg(2+) in mitochondria in intact cells. Here, we have developed a novel Mg(2+)-selective fluorescent probe, KMG-301, that is functional in mitochondria. This probe changes its fluorescence properties solely depending on the Mg(2+) concentration in mitochondria under physiologically normal conditions. Simultaneous measurements using this probe together with a probe for cytosolic Mg(2+), KMG-104, enabled us to compare the dynamics of Mg(2+) in the cytosol and in mitochondria. With this method, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP)-induced Mg(2+) mobilization from mitochondria to the cytosol was visualized. Although a FCCP-induced decrease in the Mg(2+) concentration in mitochondria and an increase in the cytosol were observed both in differentiated PC12 cells and in hippocampal neurons, the time-courses of concentration changes varied with cell type. Moreover, the relationship between mitochondrial Mg(2+) and Parkinson's disease was analyzed in a cellular model of Parkinson's disease by using the 1-methyl-4-phenylpyridinium ion (MPP(+)). A gradual decrease in the Mg(2+) concentration in mitochondria was observed in response to MPP(+) in differentiated PC12 cells. These results indicate that KMG-301 is useful for investigating Mg(2+) dynamics in mitochondria. All animal procedures to obtain neurons from Wistar rats were approved by the ethical committee of Keio University (permit number is 09106-(1)).
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The compartmentation and distribution of metabolites between mitochondria and the rest of the cell is a key parameter of cell signalling and pathology. Here, we have developed a rapid fractionation procedure that enables us to take mouse heart and liver from in vivo and within ~ 30 s stabilise the distribution of metabolites between mitochondria and the cytosol by rapid cooling, homogenisation and dilution. This is followed by centrifugation of mitochondria through an oil layer to separate mitochondrial and cytosolic fractions for subsequent metabolic analysis. Using this procedure revealed the in vivo compartmentation of mitochondrial metabolites and will enable the assessment of the distribution of metabolites between the cytosol and mitochondria during a range of situations in vivo .
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Valproic acid enhances renal losses of carnitine esters and leads to decreased plasma free carnitine concentrations in many patients receiving valproic acid therapy. However, decreased serum carnitine levels are of unclear pathologic significance, and most children manifest no symptoms of carnitine deficiency. We report a child with valproic acid-associated carnitine deficiency who had severe cardiac dysfunction develop that resolved with carnitine replacement therapy. (J Child Neurol 1992;7:413-416).
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Carnitine deficiency has been demonstrated in diabetic hearts, and it is also well known that L-carnitine administration has a beneficial effect on cardiac function. Carnitine treatment would be expected to reduce the accumulation of long-chain acylcarnitine. However, many reports have shown that myocardial long-chain acylcarnitine levels were increased following treatment with L-carnitine in whole-heart studies. Since acylcarnitine exists in both the mitochondrial and cytosolic compartments, it is difficult to investigate changes in subcellular distribution by studying whole-heart preparations. The present study investigated the myocardial subcellular distribution of carnitine and its acyl derivatives in diabetic rats with or without L-carnitine treatment. Approximately 90% of total cellular carnitine was located in the cytosol in the diabetic hearts. Both mitochondrial and cytosolic levels of free carnitine and short-chain acylcarnitine were significantly decreased in the diabetic heart. However, the mitochondrial level of long-chain acylcarnitine was significantly increased. L-carnitine treatment reduced the mitochondrial level of long-chain acylcarnitine, but the cytosolic level of long-chain acylcarnitine was significantly increased. These results show that L-carnitine treatment prevents the accumulation of long-chain acylcarnitine in the mitochondrial space and then reduces the detergent effect of long-chain acylcarnitine on the mitochondrial membrane. We suggest that it is a possible mechanism of the beneficial effect of L-carnitine treatment on the diabetic heart.
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L-carnitine plays a major role in the transport of the long chain fatty acids for the beta-oxidation in the mitochondria. The deficiency of the carnitine is associated by the deficit of the beta-oxidation of the long chain fatty acids and so a different syndromes. The purpose of this study was to determine the plasmatic concentrations of the carnitine and its metabolites in patients with neuromuscular pathologies within the group of mitochondrial myopathies by the comparison with a control group. The study comprised 11 healthy volunteers and 11 patients with neuromuscular mitochondrial diseases. The plasmatic concentrations of the free carnitine, total carnitine and the short and long chain acilcarnitine were determined by using the radio enzymatic method with Acetyl Co A 14C. The values were calculated by the standard curve in increased concentrations of the L-carnitine in aquous solution.The values were expressed as mean +/- S.D. The control group gave the following values: For free carnitine (32.6 +/- 4.95 microM), total carnitine (38.48 +/- 5.8 microM), short chain acylcarnitine (4.12 +/- 0.95 microM) and for long chain acylcarnitine (1.73 +/- 0.15 microM). The patients were divided in two groups according to their values: The 1st group gave the values within the control group: For free carnitine (31.44 +/- 3.72 microM), total carnitine (36.6 +/- 3.86 microM), short chain acylcarnitine (3.52 +/- 1.56 microM) and long chain acylcarnitine (1.68 +/- 0.08 microM). The 2nd group gave values bellow the control group: For free carnitine (16.8 +/- 6.3 microM), total carnitine (20.88 +/- 6.26 microM), short chain acylcarnitine (2.98 +/- 0.81 microM) and long chain acylcarnitine (0.92 +/- 0.41 microM) respectively. In conclusion, this method showed appropriate and accurate for the determinations of the carnitine and its metabolites in plasma, and must be useful for clinical support.
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