Functional and pharmacological characterization of human Na(+)-carnitine cotransporter hOCTN2.
(65/1186)
L-Carnitine is essential for the translocation of acyl-carnitine into the mitochondria for beta-oxidation of long-chain fatty acids. It is taken up into the cells by the recently cloned Na(+)-driven carnitine organic cation transporter OCTN2. Here we expressed hOCTN2 in Xenopus laevis oocytes and investigated with two-electrode voltage- clamp and flux measurements its functional and pharmacological properties as a Na(+)-carnitine cotransporter. L-carnitine transport was electrogenic. The L-carnitine-induced currents were voltage and Na(+) dependent, with half-maximal currents at 0.3 +/- 0.1 mM Na(+) at -60 mV. Furthermore, L-carnitine-induced currents were pH dependent, decreasing with acidification. In contrast to other members of the organic cation transporter family, hOCTN2 functions as a Na(+)-coupled carnitine transporter. Carnitine transport was stereoselective, with an apparent Michaelis-Menten constant (K(m)) of 4.8 +/- 0.3 microM for L-carnitine and 98.3 +/- 38.0 microM for D-carnitine. The substrate specificity of hOCTN2 differs from rOCT-1 and hOCT-2 as hOCTN2 showed only small currents with classic OCT substrates such as choline or tetraethylammonium; by contrast hOCTN2 mediated transport of betaine. hOCTN2 was inhibited by several drugs known to induce secondary carnitine deficiency. Most potent blockers were the antibiotic emetine and the ion channel blockers quinidine and verapamil. The apparent IC(50) for emetine was 4.2 +/- 1.2 microM. The anticonvulsant valproic acid did not induce a significant inhibition of carnitine transport, pointing to a different mode of action. In summary, hOCTN2 mediates electrogenic Na(+)-dependent stereoselective high-affinity transport of L-carnitine and Na(+). hOCTN2 displays transport properties distinct from other members of the OCT family and is directly inhibited by several substances known to induce systemic carnitine deficiency. (+info)
Identification of positive and negative determinants of malonyl-CoA sensitivity and carnitine affinity within the amino termini of rat liver- and muscle-type carnitine palmitoyltransferase I.
(66/1186)
The extreme amino terminus and, in particular, residue Glu-3 in rat liver (L) carnitine palmitoyltransferase I (CPT I) have previously been shown to be essential for the sensitivity of the enzyme to inhibition by malonyl-CoA. Using the Pichia pastoris expression system, we now observe that, although mutants E3A (Glu-3 --> Ala) or Delta(3-18) of L-CPT I have markedly lowered sensitivity to malonyl-CoA compared with the wild-type protein, the mutant Delta(1-82) generated an enzyme that had regained much of the sensitivity of wild-type CPT I. This suggests that a region antagonistic to malonyl-CoA sensitivity is present within residues 19-82 of the enzyme. This was confirmed in the construct Delta(19-30), which was found to be 50-fold more sensitive than wild-type L-CPT I. Indeed, this mutant was >4-fold more sensitive than even the native muscle (M)-CPT I isoform expressed and assayed under identical conditions. This behavior was dependent on the presence of Glu-3, with the mutant E3A-Delta(19-30) having kinetic characteristics similar to those of the E3A mutant. The increase in the sensitivity of the L-CPT I-Delta(19-30) mutant was not due to a change in the mechanism of inhibition with respect to palmitoyl-CoA, nor to any marked change of the K(0.5) for this substrate. Conversely, for M-CPT I, a decrease in malonyl-CoA sensitivity was invariably observed with increasing deletions from Delta(3-18) to Delta(1-80). However, deletion of residues 3-18 from M-CPT I affected the K(m) for carnitine of this isoform, but not of L-CPT I. These observations (i) provide the first evidence for negative determinants of malonyl-CoA sensitivity within the amino-terminal segment of L-CPT I and (ii) suggest a mechanism for the inverse relationship between affinity for malonyl-CoA and for carnitine of the two isoforms of the enzyme. (+info)
Elucidation of the mechanism by which (+)-acylcarnitines inhibit mitochondrial fatty acid transport.
(67/1186)
It is well established that medium and long chain (+)-acylcarnitines (i.e. fatty acid esters of the unnatural d-isomer of carnitine) inhibit the oxidation of long chain fatty acids in mammalian tissues by interfering with some component(s) of the mitochondrial carnitine palmitoyltransferase (CPT) system. However, whether their site of action is at the level of CPT I (outer membrane), CPT II (inner membrane), carnitine-acylcarnitine translocase (CACT, inner membrane), or some combination of these elements has never been resolved. We chose to readdress this question using rat liver mitochondria and employing a variety of assays that distinguish between the three enzyme activities. The effect on each of (+)-acetylcarnitine, (+)-hexanoylcarnitine, (+)-octanoylcarnitine, (+)-decanoylcarnitine, and (+)-palmitoylcarnitine was examined. Contrary to longstanding belief, none of these agents was found to impact significantly upon the activity of CPT I or CPT II. Whereas (+)-acetylcarnitine also failed to influence CACT, both (+)-octanoylcarnitine and (+)-palmitoylcarnitine strongly inhibited this enzyme with a similar IC(50) value ( approximately 35 microm) under the assay conditions employed. Remarkably, (+)-decanoylcarnitine was even more potent (IC(50) approximately 5 microm), whereas (+)-hexanoylcarnitine was far less potent (IC(50) >200 microm). These findings resolve a 35-year-old puzzle by establishing unambiguously that medium and long chain (+)-acylcarnitines suppress mitochondrial fatty acid transport solely through the inhibition of the CACT component. They also reveal a surprising rank order of potency among the various (+)-acylcarnitines in this respect and should prove useful in the design of future experiments in which selective blockade of CACT is desired. (+info)
Kinetics of carnitine palmitoyltransferase-I are altered by dietary variables and suggest a metabolic need for supplemental carnitine in young pigs.
(68/1186)
To examine the kinetics of carnitine palmitoyltransferase-I (CPT-I) and the influence of dietary variables, young pigs (18 kg, n = 20) were fed corn-soybean meal diets supplemented with 40 g soy oil/kg and containing either 136 or 180 g crude protein/kg and either 0 or 500 mg/kg L-carnitine (2 x 2 factorial design). Diets were offered for 10 d (85% of ad libitum); CPT-I activities in liver and skeletal muscle mitochondria were determined, and enzyme kinetic constants (V:(max) and K:(m) for carnitine) were estimated. Kinetics of CPT-I in muscle were not affected by diet (P: > 0.1; carnitine K:(m) = 480 +/- 44 micromol/L). In contrast, the K:(m) for carnitine in liver was increased from 164 to 216 +/- 20 micromol/L by dietary L-carnitine supplementation (P: < 0.01) and from 169 to 211 +/- 20 micromol/L by high protein feeding (P: < 0.05). Dietary L-carnitine increased muscle and liver free carnitine concentrations by 72 and 158% over control concentrations (770 and 80 micro;mol/kg wet muscle and liver, respectively). Because tissue carnitine concentrations were within the range of the respective K:(m) for both liver and muscle tissue, it is inferred that alteration of tissue carnitine concentrations via dietary supplementation could modulate CPT-I activity in young pigs. (+info)
Inhibition by etomoxir of rat liver carnitine octanoyltransferase is produced through the co-ordinate interaction with two histidine residues.
(69/1186)
Rat peroxisomal carnitine octanoyltransferase (COT), which facilitates the transport of medium-chain fatty acids through the peroxisomal membrane, is irreversibly inhibited by the hypoglycaemia-inducing drug etomoxir. To identify the molecular basis of this inhibition, cDNAs encoding full-length wild-type COT, two different variant point mutants and one variant double mutant from rat peroxisomal COT were expressed in Saccharomyces cerevisiae, an organism devoid of endogenous COT activity. The recombinant mutated enzymes showed activity towards both carnitine and decanoyl-CoA in the same range as the wild type. Whereas the wild-type version expressed in yeast was inhibited by etomoxir in an identical manner to COT from rat liver peroxisomes, the activity of the enzyme containing the double mutation H131A/H340A was completely insensitive to etomoxir. Individual point mutations H131A and H340A also drastically reduced sensitivity to etomoxir. Taken together, these results indicate that the two histidine residues, H131 and H340, are the sites responsible for inhibition by etomoxir and that the full inhibitory properties of the drug will be shown only if both histidines are intact at the same time. Our data demonstrate that both etomoxir and malonyl-CoA inhibit COT by interacting with the same sites. (+info)
Carbohydrate ingestion prior to exercise augments the exercise-induced activation of the pyruvate dehydrogenase complex in human skeletal muscle.
(70/1186)
This study examined the effect of pre-exercise carbohydrate (CHO) ingestion on pyruvate dehydrogenase complex (PDC) activation, acetyl group availability and substrate level phosphorylation (glycogenolysis and phosphocreatine (PCr) hydrolysis) in human skeletal muscle during the transition from rest to steady-state exercise. Seven male subjects performed two 10 min treadmill runs at 70 % maximum oxygen uptake (VO2,max), 1 week apart. Each subject ingested 8 ml (kg body mass (BM))-1 of either a placebo solution (CON trial) or a 5.5 % CHO solution (CHO trial) 10 min before each run. Muscle biopsy samples were obtained from the vastus lateralis at rest and immediately after each trial. Muscle PDC activity was higher at the end of exercise in the CHO trial compared with the CON trial (1.78+/-0.18 and 1.27+/-0.16 mmol min(-1) (kg wet matter (WM))(-1), respectively; P 0.05) and this was accompanied by lower acetylcarnitine (7.1+/-1.2 and 9.1+/-1.1 mmol kg(-1) (dry matter (DM))(-1) in CHO and CON, respectively; P<0.05) and citrate concentrations (0.73+/-0.05 and 0.91+/-0.10 mmol (kg DM)(-1) in CHO and CON, respectively; P<0.05). No difference was observed between trials in the rates of muscle glycogen and PCr breakdown and lactate accumulation. This is the first study to demonstrate that CHO ingestion prior to exercise augments the exercise-induced activation of muscle PDC and reduces acetylcarnitine accumulation during the transition from rest to steady-state exercise. However, those changes did not affect the contribution of substrate level phosphorylation to ATP resynthesis. (+info)
Correlation of carnitine levels to methionine and lysine intake.
(71/1186)
Plasma carnitine levels were measured in two alternative nutrition groups--strict vegetarians (vegans) and lactoovovegetarians (vegetarians consuming limited amounts of animal products such as milk products and eggs). The results were compared to an average sample of probands on mixed nutrition (omnivores). Carnitine levels were correlated with the intake of essential amino acids, methionine and lysine (as substrates of its endogenous synthesis), since the intake of carnitine in food is negligible in the alternative nutrition groups (the highest carnitine content is in meat, lower is in milk products, while fruit, cereals and vegetables contain low or no carnitine at all). An average carnitine level in vegans was significantly reduced with hypocarnitinemia present in 52.9% of probands. Similarly, the intake of methionine and lysine was significantly lower in this group due to the exclusive consumption of plant proteins with reduced content of these amino acids. Carnitine level in lactoovovegetarians was also significantly reduced, but the incidence of values below 30 micromol/l was lower than in vegans representing 17.8% vs. 3.3% in omnivores. Intake of methionine and lysine was also significantly reduced in this group, but still higher compared to vegans (73% of protein intake covered by plant proteins). Significant positive correlation of carnitine levels with methionine and lysine intake in alternative nutrition groups indicates that a significant portion of carnitine requirement is covered by endogenous synthesis. Approximately two thirds of carnitine requirement in omnivores comes from exogenous sources. The results demonstrate the risks of alternative nutrition with respect to the intake of essential amino acids, methionine and lysine, and with respect to the intake and biosynthesis of carnitine. (+info)
Lipid oxidation is reduced in obese human skeletal muscle.
(72/1186)
The purpose of this study was to discern cellular mechanisms that contribute to the suppression of lipid oxidation in the skeletal muscle of obese individuals. Muscle was obtained from obese [body mass index (BMI), 38.3 +/- 3.1 kg/m(2)] and lean (BMI, 23.8 +/- 0.9 kg/m(2)) women, and fatty acid oxidation was studied by measuring (14)CO(2) production from (14)C-labeled fatty acids. Palmitate oxidation, which is at least partially dependent on carnitine palmitoyltransferase-1 (CPT-1) activity, was depressed (P < 0.05) by approximately 50% with obesity (6.8 +/- 2.2 vs. 13.7 +/- 1.4 nmole CO(2).g(-1).h(-1)). The CPT-1-independent event of palmitoyl carnitine oxidation was also depressed (P < 0.01) by approximately 45%. There were significant negative relationships (P < 0.05) for adiposity with palmitate (r = -0.76) and palmitoyl carnitine (r = -0.82) oxidation. Muscle CPT-1 and citrate synthase activity, an index of mitochondrial content, were also significantly (P < 0.05) reduced ( approximately 35%) with obesity. CPT-1 (r = -0.48) and citrate synthase (r = -0.65) activities were significantly (P < 0.05) related to adiposity. These data suggest that lesions at CPT-1 and post-CPT-1 events, such as mitochondrial content, contribute to the reduced reliance on fat oxidation evident in human skeletal muscle with obesity. (+info)