Quantitation of the efflux of acylcarnitines from rat heart, brain, and liver mitochondria

The efflux of individual short-chain and medium-chain acylcarnitines from rat liver, heart, and brain mitochondria metabolizing several substrates has been measured. The acylcarnitine efflux profiles depend on the substrate, the source of mitochondria, and the incubation conditions. The largest amount of any acylcarnitine effluxing per mg of protein was acetylcarnitine produced by heart mitochondria from pyruvate. This efflux of acetylcarnitine from heart mitochondria is almost 5 times greater with 1 mM than 0.2 mM carnitine. Apparently the acetyl-CoA generated from pyruvate by pyruvate dehydrogenase is very accessible to carnitine acetyltransferase. Very little acetylcarnitine effluxes from heart mitochondria when octanoate is the substrate except in the presence of malonate. Acetylcarnitine production from some substrates peaks and then declines, indicating uptake and utilization. The unequivocal demonstration that considerable amounts of propionylcarnitine or isobutyrylcarnitine efflux from heart mitochondria metabolizing alpha-ketoisovalerate and alpha-keto-beta-methylvalerate provides evidence for a role (via removal of non-metabolizable propionyl-CoA or slowly metabolizable acyl-CoAs) for carnitine in tissues which have limited capacity to metabolize propionyl-CoA. These results also show propionyl-CoA must be formed during the metabolism of alpha-ketoisovalerate and that extra-mitochondrial free carnitine rapidly interacts with matrix short-chain aliphatic acyl-CoA generated from alpha-keto acids of branched-chain amino acids and pyruvate in the presence and absence of malate.     

Effect of carnitines on Lactobacillus plantarum subjected to osmotic stress

We investigated the effect of carnitine analogues on the physiology of Lactobacillus plantarum subjected to salt stress. Salt stressed cells of L. plantarum accumulated exogenously provided carnitine and its structural analogues acetylcarnitine and propionylcarnitine to maximum concentrations of 466, 122 and 75 μmol (g dry weight of cells)⁻¹, respectively. Addition of these carnitines to osmotically stressed medium increased growth rate. Furthermore, the intracellular amino acid pool, consisting of mainly aspartate and glutamate, was reduced when carnitine, acetylcarnitine or propionylcarnitine were included in the medium. This is the first study demonstrating a role for β-substituted acylcarnitine esters in osmoadaptation of a lactic acid bacterium.     

Seminal carnitine and acetylcarnitine content and carnitine acetyltransferase activity in young Maremmano stallions

The reproductive characteristics and seminal carnitine and acetylcarnitine content as well as carnitine acetyltransferase activity of young Maremmano stallions (n=25) are reported. The stallions were subjected to semen collection in November and January; in each trial two ejaculates were collected 1h apart. The total motile morphologically normal spermatozoa (TMMNS) and the progressively motile spermatozoa at collection and during storage at +4 degrees C were evaluated. Seminal L-carnitine (LC), acetylcarnitine (AC), pyruvate and lactate were measured using spectrophotometric methods, whereas carnitine acetyltransferase activity was measured by radioenzymatic methods. Since there were no major significant differences in seminal and biochemical characteristics between the November and January trials, data were also pooled for the first and second ejaculates. Significant differences (P<0.001) were observed between the first and second ejaculates for sperm count (0.249+/-0.025 versus 0.133+/-0.014x10(9)/ml), total number spermatozoa by ejaculate (12.81+/-1.23 versus 6.36+/-0.77x10(9)), progressively motile spermatozoa (48.6+/-3.0 versus 52.6+/-3.0%) and TMMNS (3.35+/-0.50 versus 2.02+/-0.37x10(9)). In the raw semen the LC and AC were significantly higher in the first ejaculate than in the second (P<0.001), whereas, pyruvate and pyruvate/lactate ratio were higher in the second ejaculate (P<0.05). Seminal plasma AC and LC concentrations resulted higher in the first ejaculate (P<0.001). The pyruvate/lactate ratio was higher in the second ejaculate (P<0.05). Both raw semen and seminal plasma LC and AC concentrations were positively correlated with spermatozoa concentration (P<0.01); in raw semen AC was also correlated to TMMNS (P<0.01). Lactate levels of raw semen was correlated to progressively motile spermatozoa after storage (P<0.01). In the second ejaculate, significant correlations were also observed among AC/LC ratio in raw semen and progressively motile spermatozoa after 48 and 72h of refrigeration. Furthermore, AC levels were correlated to lactate concentration. The positive correlation between LC, AC and spermatozoa concentration, and between AC and TMMNS indicated carnitine as potential semen quality marker. Moreover, the correlation between AC/LC ratio and progressive spermatozoa motility after refrigeration, suggests that carnitine may contribute towards improving the maintenance of spermatozoa viability during in vitro storage.     

Carnitine and derivatives in rat tissues

1. Free carnitine, acetylcarnitine, short-chain acylcarnitine and acid-insoluble carnitine (probably long-chain acylcarnitine) have been measured in rat tissues. 2. Starvation caused an increase in the proportion of carnitine that was acetylated in liver and kidney; at least in liver fat-feeding had the same effect, whereas a carbohydrate diet caused a very low acetylcarnitine content. 3. In heart, on the other hand, starvation did not cause an increase in the acetylcarnitine/carnitine ratio, whereas fat-feeding caused a decrease. The acetylcarnitine content of heart was diminished by alloxan-diabetes or a fatty diet, but not by re-feeding with carbohydrate. 4. Under conditions of increased fatty acid supply the acid-insoluble carnitine content was increased in heart, liver and kidney. 5. The acylation state of carnitine was capable of very rapid change. Concentrations of carnitine derivatives varied with different methods of obtaining tissue samples, and very little acid-insoluble carnitine was found in tissues of rats anaesthetized with Nembutal. In liver the acetylcarnitine (and acetyl-CoA) content decreased if freezing of tissue samples was delayed; in heart this caused an increase in acetylcarnitine. 6. Incubation of diaphragms with acetate or dl-β-hydroxybutyrate caused the acetylcarnitine content to become elevated. 7. Perfusion of hearts with fatty acids containing an even number of carbon atoms, dl-β-hydroxybutyrate or pyruvate resulted in increased contents of acetylcarnitine and acetyl-CoA. Accumulation of these acetyl compounds was prevented by the additional presence of propionate or pentanoate in the perfusion medium; this prevention was not due to extensive propionylation of CoA or carnitine. 8. Perfusion of hearts with palmitate caused a severalfold increase in the content of acid-insoluble carnitine; this increase did not occur when propionate was also present. 9. Comparison of the acetylation states of carnitine and CoA in perfused hearts suggests that the carnitine acetyltransferase reactants may remain near equilibrium despite wide variations in their steady-state concentrations. This is not the case with the citrate synthase reaction. It is suggested that the carnitine acetyltransferase system buffers the tissue content of acetyl-CoA against rapid changes.     

Effect of L-carnitine administration on the seminal characteristics of oligoasthenospermic stallions

The effect of orally administered l-carnitine on the quality of semen obtained from stallions with different semen qualities was investigated. Four stallions with proven fertility (high motility group, HM) and with normal seminal characteristics (>50% progressive motility and > 80 x 10(6) spermatozoa/ml), and four questionable breeders (low motility group, LM) with <50% of sperm progressive motility and < 80 x 10(6) spermatozoa/ml, received p.o. 20 g of l-carnitine for 60 days. Blood and semen samples were collected before treatment (T0) and after 30 (T1) and 60 days (T2). Semen evaluation were performed on five consecutive daily ejaculates (n = 120 ejaculates) and conventional semen analysis was carried out on each ejaculate, both at collection and after refrigeration for 24, 48, and 72 h. Furthermore l-carnitine, acetylcarnitine, pyruvate, and lactate concentrations, and carnitine acetyltransferase activity (CAT) were determined both in raw semen and seminal plasma. There were an increase in progressive motile spermatozoa only in the LM group (26.8 +/- 12.9, 39.1 +/- 15.5, and 48.8 +/- 8.6 for T0, T1, and T2, respectively). Free seminal plasma carnitine concentration was higher in the LM group compared to the HM one. Both pyruvate and lactate were higher in the LM group. Raw semen and seminal plasma carnitine and acetylcarnitine levels correlate positively with both sperm concentration and progressive motility; moreover, acetylcarnitine content was positively correlated with total motile morphologically normal spermatozoa. In conclusion, oral administration of l-carnitine to stallions with questionable seminal characteristics may improve spermatozoa kinetics and morphological characteristics; whereas, it seem to be ineffective in normospermic animals.     

Selective inhibition of pyruvate and lactate metabolism in bovine epididymal spermatozoa by dinitrophenol and alpha-cyano-3-hydroxycinnamate

The disparity between the effects of the uncouplers, 2,4-dinitrophenol (DNP) and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) on pyruvate metabolism in bovine spermatozoa has been characterized. In bovine epididymal spermatozoa metabolizing pyruvate, the uncouplers of oxidative phosphorylation, DNP (100 μm) and FCCP (0.4 or 5 μm), decreased the intracellular ATP concentration from 30 to ～10 nmol/10⁸ cells. Both uncouplers decreased, but did not abolish, sperm motility. DNP strongly inhibited pyruvate metabolism and stimulated the appearance of free carnitine from the acetylcarnitine pool. In contrast, FCCP enhanced the oxidation of pyruvate, diminished the reduction of pyruvate to lactate, and permitted the maintenance of the normal amount of acetylcarnitine. The effects of DNP and FCCP on mitochondrial pyruvate metabolism were examined in spermatozoa treated with filipin, which renders the plasma membrane permeable to small molecules. In these cells, DNP inhibited metabolism and respiration with pyruvate or lactate, but did not affect respiration supported by acetylcarnitine. Similarly, the pyruvate translocase inhibitor, α-cyano-3-hydroxycinnamate, markedly decreased the rate of metabolism of both pyruvate and lactate. With maximally inhibitory concentrations of DNP or α-cyano-3-hydroxycinnamate, the rates of pyruvate use and lactate use were the same. Metabolism of both lactate and pyruvate and production of ATP were inhibited by similar concentrations of DNP ($\text{I}{}_{50}\text{? 7}\text{μM}$). A common mitochondrial translocase for pyruvate and lactate in bovine spermatozoa is posited. This translocase is inhibited by minimally effective uncoupling concentrations of DNP.     

The metabolism of acetylcarnitine and acetate by bovine and hamster epididymal spermatozoa

Since acetylcarnitine has been identified in the epididymal plasma of many mammalian species, we investigated whether acetylcarnitine could serve as an energy substrate for epididymal bull and hamster spermatozoa. Intact caudal cells from both species oxidized [I-14C]acetyl-l-carnitine to 14CO2, in vitro, and the amount oxidized was dependent on time, substrate concentration, and cell number. Within each species, the rate of oxidation was the same as the rate at which free [1-14C]acetate was oxidized. Spermatozoa incubated with [3H]acetyl-L-carnitine hydrolyzed the compound and [3H]acetate accumulated in the medium. Unlabeled acetate added to the incubation medium competed with cellular uptake of [3H]acetate and resulted in further increase in [3H]acetate accumulation in the medium. Furthermore, the acetyl group of acetylcarnitine was oxidized by spermatozoa without concomitant uptake of the carnitine group. Purified plasma membrane vesicles contained an acetylcarnitine hydrolase activity that was solubilized from whole cells by detergents and that could be distinguished from acetylcholinesterase also present in the cells. The solubilized acetylcarnitine hydrolase activity was inhibited by p-hydroxymercuriphenylsulfonate, but not by the specific acetylcholinesterase inhibitors, eserine or BW63C47. The sulfhydryl blocker also inhibited the production of 14CO2 from [1-14C]acetylcarnitine by intact cells; acetylcholinesterase inhibitors did not. From estimates of sperm energy requirements, our results indicate that extracellular acetylcarnitine serves as a physiologically important energy substrate for maturing sperm cells.     

Carnitine acetyltransferase in pea cotyledon mitochondria

Purified pea cotyledon mitochondria did not oxidise acetyl-CoA in the presence of carnitine. However, acetylcarnitine was oxidised. It was concluded that acetylcarnitine passed through the mitochondrial membrane barrier but acetyl-CoA did not. Only a sensitive radioactive assay detected carnitine acetyltransferase in intact mitochondrion or intact mitoplast preparations. When the mitochondria or mitoplasts were burst, acetyl-CoA substrate was available to the matrix carnitine acetyltransferase and a high activity of the enzyme was measured. The inner mitochondrial membrane is there-fore the membrane barrier to acetyl-CoA but acetylcarnitine is suggested to be transported through this membrane via an integral carnitine: acylcarnitine translocator. Evidence is presented to indicate that when the cotyledons from 48-h-grown peas are oxidising pyruvate, acetylcarnitine formed in the mitochondrial matrix by the action of matrix carnitine acetyltransferase may be transported to extra-mitochondrial sites via the membrane translocator.     

Metabolism of pyruvate and carnitine esters in bovine epididymal sperm mitochondria

Filipin-treated bovine epididymal spermatozoa have been used to study mitochondrial l-acetylcarnitine, l-palmitoylcarnitine, and pyruvate metabolism. The cells were supplemented with malate to allow rapid rates of substrate oxidation. The rate of l-palmitoylcarnitine-supported state 3 respiration was slow. In contrast, pyruvate, acetylcarnitine, or lactate supported rapid and approximately equal respiratory rates. l-Palmitoylcarnitine was a weak inhibitor of pyruvate-supported respiration and pyruvate use and a more potent inhibitor of l-acetylcarnitine. l-Carnitine was an effective inhibitor of l-acetylcarnitine oxidation; however, it did not influence l-palmitoylcarnitine oxidation or inhibit pyruvate utilization. Pyruvate (1.4 mm) disappearance was rapid and was complete within 6–7 min; the lactate produced during pyruvate metabolism was then oxidized. ATP synthesis was constant throughout the 20-min incubation. With pyruvate plus l-acetylcarnitine as substrate, the l-acetylcarnitine concentration initially dropped and then recovered to a level that was dependent on free carnitine addition. Data obtained from experiments using [2-¹⁴C]pyruvate indicated that the ¹⁴C label from pyruvate and lactate entered the l-acetylcarnitine pool and labeling was maximal when free l-carnitine was added. The rate of citrate synthesis was maximal when pyruvate was being metabolized; the largest total accumulation occurred when all three substrates were included in the incubation. The data suggest that the high NAD⁺/ NADH maintained during pyruvate metabolism may restrict flux through the citric acid cycle. The relationships of l-carnitine and the l-carnitine esters to pyruvate metabolism are discussed.     

Metabolic changes induced by maximal exercise in human subjects following L-carnitine administration

In double-blind cross-over experiments, ten moderately trained male subjects were submitted to two bouts of maximal cycle ergometer exercise separated by a 3 day interval. Each subject was randomly given either L-carnitine (2 g) or placebo orally 1 h before the beginning of each exercise session. At rest L-carnitine supplementation resulted in an increase of plasma-free carnitine without a change in acid-soluble carnitine esters. Treatment with L-carnitine induced a significant post-exercise decrease of plasma lactate and pyruvate and a concurrent increase of acetylcarnitine. The determination of the individual carnitine esters in urine collected for 24 h after the placebo exercise trial revealed a decrease of acetyl carnitine and a parallel increase of a C4 carnitine ester, probably isobutyrylcarnitine. Conversely, acetylcarnitine was strongly increased and C4 compounds were almost suppressed in the L-carnitine loading trial. These results suggest that L-carnitine administration prior to high-intensity exercise stimulates pyruvate dehydrogenase activity, thus diverting pyruvate from lactate to acetylcarnitine formation.     

Ester composition of carnitine in the perfusate of liver and in the plasma of donor rats

When the carnitine pool of fed rats was labelled with tritium, in non-recirculating perfusate of their liver 44% of acid-soluble 3H activity was identified as free carnitine and 47% as short-chain acylcarnitine. Of the latter component acetylcarnitine accounted for 30% and propionylcarnitine for 10% of total acid-soluble. In plasma the contribution of short-chain acylcarnitines to total carnitine in fed, fasted and diabetic rats was 15.6%, 43.1% and 48.0%, respectively. Recirculating perfusion of livers from the same animals revealed that livers from fed rats released short-chain acylcarnitines as much as 56.2% of total and this proportion did not increase further in the other two groups. At the same time, ketone bodies in the perfusate increased gradually in the fed, fasted and diabetic group, paralleling the plasma ketone levels. Although liver supplies the organism with carnitine the increment of plasma short-chain acylcarnitines seen in ketosis is not a result of some extra output by the liver.     

Formation of acetylcarnitine in muscle of horse during high intensity exercise

To study the changes in carnitine in muscle with spring exercise, two Thoroughbred horses performed two treadmill exercise tests. Biopsies of the middle gluteal were taken before, after exercise and after 12 min recovery. Resting mean muscle total carnitine content was 29.5 mmol.kg-1 dry muscle (d.m.). Approximately 88% was free carnitine, 7% acetylcarnitine and acylcarnitine was estimated at 5%. Exercise did not affect total carnitine, but resulted in a marked fall in free carnitine and almost equivalent rise in acetylcarnitine. The results are consistent with a role for carnitine in the regulation of the acetyl-CoA/CoA ratio during sprint exercise in the Thoroughbred horse by buffering excess production of acetyl units.     

The effect of acute and prolonged ethanol treatment on the concentrations of coenzyme A, carnitine and their derivatives in rat liver

1. CoA, acetyl-CoA, long-chain acyl-CoA, carnitine, acetylcarnitine and long-chain acylcarnitine were measured in rat liver under various conditions. 2. Starvation caused an increase in the contents of these intermediates, except that of carnitine. 3. A single dose of ethanol had no effect on CoA content, whereas those of acetyl-CoA, acetylcarnitine and carnitine were increased and those of long-chain acyl-CoA and acylcarnitine were decreased. 4. Four weeks' adaptation to ethanol consumption did not change the effect of ethanol administration on these metabolites. 5. It is suggested that ethanol directly increases hepatic fatty acid synthesis and esterification. It is also suggested that this change is reversible and limited to the period of ethanol oxidation. 6. It is demonstrated that ethanol-induced triglyceride accumulation is not related to carnitine deficiency.     

The effect of electrical stimulation, fasting and anesthesia on the carnitine(s) and acyl-carnitines of rat white and red skeletal muscle fibres

Time courses for the effect of electrical stimulation, fasting and sampling mode (anesthesia vs decapitation) on the quantitative distribution of carnitine and its esters in fast white and fast red skeletal muscle fibres of rats were determined. Both fibre types responded similarly to electrical stimulation with respect to changes in acetyl- and propionylcarnitine, but the time course was very different. The proportion of esterified carnitine decreased with fasting and anesthesia in both fibres compared to the fed decapitated group. This shift in the acylation state of carnitine was mainly due to the decrease of acetylcarnitine levels. The data show that the use of anesthetics may induce significant quantitative changes in specific acylcarnitine levels, presumably reflecting changes in specific acylcoenzyme A levels.     

Distribution of carnitine and acylcarnitine in the hamster epididymis and in epididymal spermatozoa during maturation

The highest levels of carnitine and acylcarnitine were found in the cauda epididymidis, and spermatozoa from the cauda contained greater amounts of total carnitine (free carnitine plus acylcarnitine) than those removed from the corpus or caput epididymidis. Spermatozoa from the distal cauda contained significantly greater amounts of both free and total carnitine than those removed from the proximal cauda epididymidis. The acylcarnitine:carnitine ratio was 1.7 and 0.37 in caput and cauda spermatozoa, respectively and 1.7 and 1.3 in caput and cauda fluid, respectively. It is suggested that the accumulation of carnitine is involved in sperm maturation and that acylcarnitine serves as an energy substrate for epididymal spermatozoa.     

Quantitation of the effect of L-carnitine on the levels of acid-soluble short-chain acyl-CoA and CoASH in rat heart and liver mitochondria

The steady state levels of mitochondrial acyl-CoAs produced during the oxidation of pyruvate, alpha-ketoisovalerate, alpha-ketoisocaproate, and octanoate during state 3 and state 4 respiration by rat heart and liver mitochondria were determined. Addition of carnitine lowered the amounts of individual short-chain acyl-CoAs and increased CoASH in a manner that was both tissue- and substrate-dependent. The largest effects were on acetyl-CoA derived from pyruvate in heart mitochondria using either state 3 or state 4 oxidative conditions. Carnitine greatly reduced the amounts of propionyl-CoA derived from alpha-ketoisovalerate, while smaller effects were obtained on the branched-chain acyl-CoA levels, consistent with the latter acyl moieties being poorer substrates for carnitine acetyltransferase and also poorer substrates for the carnitine/acylcarnitine translocase. The levels of acetyl-CoA in heart and liver mitochondria oxidizing octanoate during state 3 respiration were lower than those obtained with pyruvate. The rate of acetylcarnitine efflux from heart mitochondria during state 3 (with pyruvate or octanoate as substrate, in the presence or absence of malate with 0.2 mM carnitine) shows a linear response to the acetyl-CoA/CoASH ratio generated in the absence of carnitine. This relationship is different for liver mitochondria. These data demonstrate that carnitine can modulate the aliphatic short-chain acyl-CoA/CoA ratio in heart and liver mitochondria and indicate that the degree of modulation varies with the aliphatic acyl moiety.     

Carnitine, acetylcarnitine and the activity of carnitine acyltransferases in seminal plasma and spermatozoa of men, rams and rats.

The concentration of total carnitine (i.e. carnitine plus acetylcarnitine) was measured in seminal plasma and spermatozoa of men and rams. In ram semen, there was a close correlation between the concentration of spermatozoa and that of total carnitine in the seminal plasma, indicating that the epididymal secretion was the sole source of seminal carnitine. The percentage of total carnitine present as acetylcarnitine was 40% in seminal plasma and 70-80% in spermatozoa. The acetylation state of carnitine in seminal plasma was apparently not influenced by the metabolic activity of spermatozoa in ejaculated ram semen as no change was found in the plasma concentration of carnitine or acetylcarnitine up to 45 min after ejaculation. In spermatozoa, the activity of carnitine acetyltransferase (EC 2.3.1.7) was approximately equivalent to that of carnitine palmitoyltransferase (EC 2.3.1.21); and the activity of these enzymes was similar in ram and human spermatozoa but greater in rat spermatozoa. It is concluded that there is no correlation between the content of either total carnitine or the carnitine acyltransferases and the respiratory capacity of spermatozoa.     

Effect of cell density on metabolism in isolated rat hepatocytes

Freshly isolated rat hepatocytes show many changes in metabolic activities as a function of cell density in the incubation flask. Fatty acid synthesis, cholesterol synthesis, general protein synthesis, and rates of accumulation of pyruvate, lactate, citrate, acetyl-CoA, acetoacetate and beta-hydroxybutyrate decrease as the cell density increases between 1 mg/ml and 60 mg/ml. Glucose release only decreases between 1-5 mg/ml and the concentration of ATP does not vary at any density. There is a small increase in the lactate/pyruvate ratio and a dramatic decrease in the beta-hydroxybutyrate/acetoacetate ratio with increasing cell concentration. When cells at 8 or 28 mg/ml were incubated with added lactate and pyruvate, or alanine, a two fold increase in fatty acid synthesis and 50% decrease in cholesterol synthesis were observed as compared to rates with endogenous substrate. With added glucose the synthetic rates were similar to those obtained with endogenous substrate. However, regardless of the type of substrate used, the less dense cells gave rates up to three times greater than that of the more dense cells. When conditioned medium isolated after incubation of cells at high density was added to the less dense cells, a decrease in the rates of fatty acid synthesis and cholesterol synthesis was observed in the less dense cells; however, when medium from the less dense cells after incubation for the same period was added to the more dense cells, there was no significant change in fatty acid or cholesterol synthesis. These results suggest that a factor may be released into the medium of incubating hepatocytes that progressively inhibits certain metabolic processes as the cell density increases.     

Effect of carnitine acetyltransferase inhibition on rat hepatocyte metabolism

Carnitine acetyltransferase (CAT) catalyzes the reversible transfer of short chain (less than six carbons in length) acyl groups from acyl-CoA thioesters to form the corresponding acylcarnitines. This reaction has been suggested to be of importance in decreasing cellular content of acyl-CoA under conditions characterized by accumulation of poorly metabolized, potentially toxic acyl-CoAs. To study the importance of the CAT reaction, the effect of CAT inhibitors on rat hepatocyte metabolism in the presence of propionate was examined. Acetyl-DL-aminocarnitine inhibited [14C]propionylcarnitine accumulation by isolated hepatocytes incubated with [14C]propionate (1.0-10.0 mM). Inhibition of propionylcarnitine formation by acetyl-DL-aminocarnitine was concentration dependent and was not due to non-specific cellular toxicity as [14C]glucose formation from [14C]propionate, and [1-14C]pyruvate oxidation were unaffected by the CAT inhibitor. Inhibition of propionylcarnitine formation was increased by preincubating hepatocytes with acetyl-DL-aminocarnitine, suggesting competition for cellular uptake between carnitine and the inhibitor. Hemiacetylcartinium (HAC) and meso-2,6-bis(carboxymethyl)4,4-dimethylmorpholinium bromide (CMDM), potent inhibitors of CAT in broken cell systems, did not inhibit hepatocyte propionylcarnitine formation under the conditions evaluated. Propionate (5 mM) inhibited hepatocyte pyruvate (10 mM) oxidation, and this inhibition was partially reversed by 5 mM carnitine. Addition of 5.0 mM acetyl-DL-aminocarnitine abolished the stimulatory effect of carnitine on pyruvate oxidation in the presence of propionate. These studies establish that acetyl-DL-aminocarnitine inhibits intact hepatocyte CAT activity, and thus provide a useful probe of the role of CAT in cellular metabolism. CAT activity appears to be critical for carnitine-mediated reversal of propionate-induced inhibition of pyruvate oxidation.     

Activation of bovine epididymal sperm respiration by caffeine: Its transient nature and relationship to the utilization of acetyl carnitine

Caffeine, which stimulates the motility of freshly extruded bovine epididymal spermatozoa, caused a large but transient increase in the respiratory activity of these cells incubated in a modified Ringer buffer without exogenously added substrate. In spermatozoa that were incubated without added substrate for 2 h at 30 °C or for 15 min at 37 °C, caffeine addition failed to increase respiratory activity even transiently. However, subsequent addition of pyruvate to these aged and caffeine-treated cells resulted in a rapid increase in the respiratory rate, nearly equal to that observed after caffeine addition to fresh cells or to cells stored at 4 °C. These observations indicate that the loss in metabolic response to caffeine is a result of the active metabolism of the spermatozoa.In freshly prepared sperm that were incubated without added substrate, the acetyl carnitine content declined and the free carnitine content of the sperm increased in amounts sufficient to account for the entire respiratory increment produced by caffeine addition. Respiratory stimulation by caffeine was sustained in the presence of those exogenously added substrates that are capable of entering the acetyl carnitine pool, such as acetate, pyruvate, l(+)-lactate, glucose, fructose or β-hydroxybutyrate. Tricarboxylic acid cycle intermediates were not effective.These observations clarify the relationship between the stimulatory effects of caffeine and the metabolic state of the spermatozoan and suggest the importance of the acetyl carnitine pool to the activation of sperm motility and oxidative metabolism.