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Mitochondrial reactive oxygen species (ROS) cause kidney damage in diabetes. We investigated the source and site of ROS production by kidney cortical tubule mitochondria in streptozotocin-induced type 1 diabetes in rats. In diabetic mitochondria, the increased amounts and activities of selective fatty acid oxidation enzymes is associated with increased oxidative phosphorylation and net ROS production with fatty acid substrates (by 40% and 30%, respectively), whereas pyruvate oxidation is decreased and pyruvate-supported ROS production is unchanged. Oxidation of substrates that donate electrons at specific sites in the electron transport chain (ETC) is unchanged. The increased maximal production of ROS with fatty acid oxidation is not affected by limiting the electron flow from complex I into complex HI. The maximal capacity of the ubiquinol oxidation site in complex HI in generating ROS does not differ between the control and diabetic mitochondria. In conclusion, the mitochondrial ETC is neither the target nor the site of ROS production in kidney tubule mitochondria in short-term diabetes. Mitochondrial fatty acid oxidation is the source of the increased net ROS production, and the site of electron leakage is located proximal to coenzyme Q at the electron transfer flavoprotein that shuttles electrons from acyl-CoA dehydrogenases to coenzyme Q. Diabetes 61:2074-2083, 2012, Diabetic nephropathy (DN) is the leading cause of end-stage renal disease (ESRD) (1). Hyperlipidemia is an independent factor in renal injury in animals (2) and humans (3). Increased fatty acid [...]

One of the most frequently diagnosed congenital metabolic malfunctions is the genetic disorder known as medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency. Some cases of sudden infant death syndrome (SIDS) are possibly caused by MCAD deficiency. Siblings of SIDS victims may also have this enzyme deficiency, and it has been suggested that they be screened for decreased MCAD production. The symptoms of MCAD deficiency resemble those of Reye's syndrome and the condition may be set off by viral infections and fasting. When the child is fasting or experiencing an acute episode of symptoms, the diagnosis of MCAD deficiency may be strongly suspected when there is an increase of an organic acid (carnitine) in the urine. When there are no symptoms, diagnosis is difficult because the carnitine levels are not elevated. A case study of a 17-month-old boy previously diagnosed with MCAD deficiency reports a new method to screen for MCAD deficiency by measuring levels of carnitine concentrations. The screening was done when the child was free of symptoms. He was not fasting or receiving treatment for MCAD, both of which are known to raise carnitine levels. Highly sensitive and specific radioisotopic exchange-high-pressure liquid chromatography was used in this screening process. Two carnitine esters, octanoylcarnitine and hexanoylcarnitine, could be detected. The finding in this case suggests that there may be a new technique for screening for MCAD deficiency.

Peroxisome proliferator-activated receptor α (PPARα) is a transcriptional regulator of the expression of mitochondrial thioesterase I (MTE-I) and uncoupling protein 3 (UCP3), which are induced in the heart at the mRNA level in response to diabetes. Little is known about the regulation of protein expression of MTE-I and UCP3 or about MTE-I activity; thus, we investigated the effects of diabetes and treatment with a PPARα agonist on these parameters. Rats were either made diabetic with streptozotocin (55 mg/kg ip) and maintained for 10–14 days or treated with the PPARα agonist fenofibrate (300 mg/kg/day) for 4 weeks. MTE-I and UCP3 protein expression, MTE-1 activity, palmitate export, and oxidative phosphorylation were measured in isolated cardiac mitochondria. Diabetes and fenofibrate increased cardiac MTE-I mRNA, protein, and activity (∼4-fold compared with controls). This increase in activity was matched by a 6-fold increase in palmitate export in fenofibrate-treated animals, despite there being no effect in either group on UCP3 protein expression. Both diabetes and fenofibrate caused significant decreases in state III respiration of isolated mitochondria with pyruvate + malate as the substrate, but only diabetes reduced state III rates with palmitoylcarnitine. Both diabetes and specific PPARα activation increased MTE-I protein, activity, and palmitate export in the heart, with little effect on UCP3 protein expression.

Summary. Carnitine palmitoyltransferase-I catalyzes the regulated step in overall mitochondrial fatty acid oxidation. The enzyme is controlled by the steady state level of malonyl-CoA and the enzyme’s sensitivity to inhibition by malonyl-CoA. The former is established by the activities of acetyl-CoA carboxylase 2 and malonyl-CoA decarboxylase, while the latter is influenced by post-translational modification (phosphorylation) of CPT-I and/or by changes in the lipid environment of CPT-I.

In hepatic mitochondria, the outer membrane enzyme, carnitine palmitoyltransferase-I (CPT-I), appears to colocalize with contact sites. We have prepared contact sites that are essentially devoid of noncontact site membranes. The contact site fraction has a high specific activity for CPT-I and contains a protein at 88 kDa that is recognized by antibodies directed at two different peptide epitopes on CPT-I. Similarly long-chain acyl-CoA synthetase (LCAS) specific activity is high in this fraction; a protein at 79 kDa is recognized by an antibody against LCAS. Although activity of carnitine palmitoyltransferase-II (CPT-II) is present, it is not enriched in the contact site fraction, and a protein of 68 kDa weakly reacted with anti-CPT-II antibody. Likewise, carnitine–acylcarnitine translocase (CACT) protein is present, but at a somewhat reduced level. Using an analytical continuous sucrose gradient, we demonstrate that the activities of CPT-I and LCAS and their associated immunoreactive proteins are present in a constant amount throughout the contact site subfractions. The enzymatic activity of CPT-II and its associated immunoreactive protein, as well as immunoreactive CACT, is absent in the lighter density gradient subfractions and is present in the higher density subfractions only in trace amounts. This heterogeneity of the contact site fraction is due to unvarying amounts of outer membrane and increasing amounts of attached inner membrane with increasing density of the subfractions.

Mitochondria contribute to cardiac dysfunction and myocyte injury via a loss of metabolic capacity and by the production and release of toxic products. This article discusses aspects of mitochondrial structure and metabolism that are pertinent to the role of mitochondria in cardiac disease. Generalized mechanisms of mitochondrial-derived myocyte injury are also discussed, as are the strengths and weaknesses of experimental models used to study the contribution of mitochondria to cardiac injury. Finally, the involvement of mitochondria in the pathogenesis of specific cardiac disease states (ischemia, reperfusion, aging, ischemic preconditioning, and cardiomyopathy) is addressed.

The transport of activated fatty acids across the mitochondrial outer membrane has not been fully addressed. A polyanion (Mn=22 kDa) inhibited the ADP‐stimulated carnitine‐dependent oxidation of both palmitoyl‐CoA and palmitate plus CoA as well as mitochondrial hexokinase binding. In contrast, the oxidation of palmitoylcarnitine plus malate, as well as glutamate oxidation, was essentially unaffected. Mitochondrial carnitine palmitoyltransferase‐1 was not inhibited by the polyanion. The data suggest an additional component in carnitine‐dependent mitochondrial fatty acid oxidation, possibly porin.

The data used to support the idea that malonyl-coenzyme A (CoA)-sensitive carnitine palmitoyltransferase (CPT-I) is localized on the outer mitochondrial membrane are based on harsh techniques that disrupt mitochondrial physiology. We have turned to the use of the French press, which produces a shearing force that denudes mitochondria of their outer membrane without the physiologically disruptive effects characteristic of phosphate swelling. Our results indicate that the mitoplasts contain just 15–19% of the outer membrane marker enzyme activity while retaining 85% of the total CPT activity and 50% of both CPT-I, as well as long-chain acyl-CoA synthase activity, the latter two supposed outer membrane enzymes. These mitoplasts were shown by electron microscopy to have the configuration of mitochondria that merely have been divested of their outer membranes. Carnitine-dependent fatty acid oxidation was retained in the mitoplasts, showing that they were physiologically intact. Moreover, protein immunoblotting analysis showed that CPT-I, as well as the inner CPT-II, was localized in the mitoplast fraction. The outer membrane fraction, which consisted of membrane “ghosts,” contained most (50–60%) of marker enzyme activity, monoamine oxidase-B and porin proteins, but only about 27–29% CPT-I activity. Because CPT-I and long-chain acyl-CoA synthetase appear to be associated with both inner and outer membranes, we postulate that these enzymes reside in contact sites, which represent a melding of both limiting membranes.

Rats with obesity-producing, hypothalamic knife cuts (KC) were fed a purified high fat diet for 9 wk. KC rats consumed more energy (+70–100%) and retained energy with a much higher efficiency than control rats. Adrenalectomy of KC rats 1 wk (before gross obesity was evident) or 5 wk (when KC rats were 70% overweight) after KC surgery caused a reduction in energy intake to levels approximating those of control rats. Furthermore, energy retention in adrenalectomized KC rats was depressed more than could be explained on the basis of the reduction in energy intake. Two factors associated with the reduction in energy retention, urinary excretion of norepinephrine, an indicator of sympathetic nervous system activity, and GDP binding to brown adipose tissue mitochondria, an indicator of the thermogenic capacity of the tissue, were higher in vadrenalectomized KC rats than in pair-fed KC rats. Removal of the adrenals not only suppressed hyperphagia in KC rats fed a high fat diet, but also increased energy expenditure per kilocalorie consumed.

ABSTRACT: Carnitine plasma concentrations and the excretion of carnitine and individual carnitine esters were determined in 25 children and adolescents with gastrointestinal diseases receiving carnitine-free parenteral nutrition for at least 1 mo using radiochemical and radioisotopic exchange HPLC methods. Children <12-y-old usually had carnitine plasma concentrations <-2 SD from the normal mean for age, whereas patients >12-y-old had carnitine plasma concentrations within the normal range. Age was the only variable to correlate significantly with plasma carnitine concentrations during parenteral nutrition. Free carnitine (FC) excretion was closely correlated with plasma FC concentrations and minimal at values <25 µmol/L. The excretion of FC and short-chain acylcarnitines was reduced by an order of magnitude in younger compared with older patients and controls, but the excretion of “other” acylcarnitines was less affected. Some of the latter were tentatively identified using gas-liquid chromatographic and mass spectroscopic techniques as unsaturated and/or branched medium-chain carnitine esters with a carbon chain of C8-C10. The results suggest that FC and short-chain acylcarnitine are conserved by the kidney in nutritional carnitine deficiency but that there may be an obligatory renal excretion of other carnitine esters that contributes to the development of hypocarnitinemia in the younger age group.

The concentration of total carnitine in human breast milk remained at a constant mean level near 62.9 range: 56.0–69.8/nmoles/ml during the first 21 days postpartum. The carnitine level fell significantly to 35.2 ± 1.26 nmoles/ml until the 40–50th day. The carnitine concentrations did not depend on the secreted volume of milk. In comparison, fresh and commercial pasteurized cow's milk contained 206.2 (range: 192–269) nmoles/ml and 160.0 (range: 158–200) nmoles/ml carnitine, respectively. In the sera of nursing mothers, the concentration of total carnitine was lower on the first day after delivery (27.2 ± 1.19 nmoles/ml) but returned to normal by the 21st day postpartum (38.8 ± 2.97 nmoles/ml).

Carnitine plasma concentrations and the excretion of carnitine and individual carnitine esters were determined in 25 children and adolescents with gastrointestinal diseases receiving carnitinefree parenteral nutrition for at least 1 mo using radiochemical and radioisotopic exchange HPLC methods. Children <12yold usually had carnitine plasma concentrations <2 SD from the normal mean for age, whereas patients >12yold had carnitine plasma concentrations within the normal range. Age was the only variable to correlate significantly with plasma carnitine concentrations during parenteral nutrition. Free carnitine FC excretion was closely correlated with plasma FC concentrations and minimal at values <25 molL. The excretion of FC and shortchain acylcarnitines was reduced by an order of magnitude in younger compared with older patients and controls, but the excretion of “other” acylcarnitines was less affected. Some of the latter were tentatively identified using gasliquid chromatographic and mass spectroscopic techniques as unsaturated andor branched mediumchain carnitine esters with a carbon chain of C8C10. The results suggest that FC and shortchain acylcarnitine are conserved by the kidney in nutritional carnitine deficiency but that there may be an obligatory renal excretion of other carnitine esters that contributes to the development of hypocarnitinemia in the younger age group. Pediatr Res28 158165, 1990