malonyl-coenzyme-a and octanoic-acid

We reported previously that a substantial fraction of the acetyl groups used to synthesize malonyl-CoA in rat heart is derived from peroxisomal beta-oxidation of long-chain and very-long-chain fatty acids. This conclusion was based on the interpretation of the 13C-labelling ratio (malonyl-CoA)/(acetyl moiety of citrate) measured in the presence of substrates that label acetyl-CoA in mitochondria only (ratio < 1.0) or in both mitochondria and peroxisomes (ratio > 1.0). The goals of the present study were to test, in rat livers perfused with [1-(13C)]octanoate or [3-(13C)]octanoate, (i) whether peroxisomal beta-oxidation contributes acetyl groups for malonyl-CoA synthesis, and (ii) the degree of labelling homogeneity of acetyl-CoA proxies (acetyl moiety of citrate, acetate, beta-hydroxybutyrate, malonyl-CoA and acetylcarnitine). Our data show that (i) octanoate undergoes two cycles of peroxisomal beta-oxidation in liver, (ii) acetyl groups formed in peroxisomes contribute to malonyl-CoA synthesis, (iii) the labelling of acetyl-CoA proxies is markedly heterogeneous, and (iv) the labelling of C1+2 of beta-hydroxybutyrate does not reflect the labelling of acetyl-CoA used in the citric acid cycle.

Topics: Acetates; Acetyl Coenzyme A; Acetylcarnitine; Animals; Caprylates; Carbon Isotopes; Citric Acid; Isotope Labeling; Ketone Bodies; Liver; Male; Malonyl Coenzyme A; Mitochondria, Liver; Oxidation-Reduction; Perfusion; Peroxisomes; Rats; Rats, Sprague-Dawley

In the human heart, although all substrates compete for energy production, fatty acids (FA) represent the main substrate for ATP production. In the healthy heart, a balance between FA and carbohydrate utilization ensures that energy supply matches demand. This study was carried out to evaluate, in a model of spontaneously beating neonatal rat cardiomyocytes in culture, the hypothesis that glycerol could play a central role in the metabolic control of the routes involving long chain FAs and may then affect the balance between beta-oxidation and glucose oxidation. The intracellular-free glycerol significantly increased with extracellular glycerol concentration (0 to 660 microM). The synthesis of phospholipids was significantly increased in parallel with both extracellular glycerol (1.5 and 14.8 nmol glycerol/mg protein, at 82 and 660 microM of extracellular glycerol, respectively). The oxidation of glycerol increased proportionally to extracellular glycerol concentration (from 1 to 3 nmol glycerol/mg protein, at 82 microM and 660 microM extracellular glycerol, respectively, P<0.001). At its maximum, this oxidation represented 15% of the glucose oxidation, which was not affected by glycerol extracellular supply or intracellular availability. Conversely, extracellular glycerol significantly reduced the palmitate oxidation above (-47% at 660 microM glycerol), but not octanoate oxidation. Investigations on the mechanism of the decreased palmitate oxidation reveals a glycerol-dependent increase in malonyl-CoA associated with a significant decrease in CPT-1 activity which accounts for the difference between palmitate and octanoate. These results clearly demonstrate the importance of glycerol in regulating the cardiac metabolic pathways and energy balance.

Topics: Animals; Animals, Newborn; Caprylates; Cells, Cultured; Fatty Acids; Glucose; Glycerol; Glycogen; Humans; Malonyl Coenzyme A; Myocytes, Cardiac; Oxidation-Reduction; Oxygen; Palmitic Acid; Phospholipids; Rats; Rats, Wistar

It is generally accepted that endothelial cells generate most of their ATP by anaerobic glycolysis and that very little ATP is derived from the oxidation of fatty acids or glucose. Previously, we have reported that, in cultured human umbilical vein endothelial cells (HUVECs), activation of AMP-activated protein kinase (AMPK) by the cell-permeable activator 5-aminoimidazole-4-carboximide riboside (AICAR) is associated with an increase in the oxidation of (3)H-palmitate. In the present study, experiments carried out with cultured HUVECs revealed the following: (1) AICAR-induced increases in palmitate oxidation during a 2-hour incubation are associated with a decrease in the concentration of malonyl coenzyme A (CoA) (an inhibitor of carnitine palmitoyl transferase 1), which temporally parallels the increase in AMPK activity and a decrease in the activity of acetyl CoA carboxylase (ACC). (2) AICAR does not stimulate either palmitate oxidation when carnitine is omitted from the medium or oxidation of the medium-chain fatty acid octanoate. (3) When intracellular lipid pools are prelabeled with (3)H-palmitate, the measured rate of palmitate oxidation is 3-fold higher, and in the presence of AICAR, it accounts for nearly 40% of calculated ATP generation. (4) Incubation of HUVECs in a glucose-free medium for 2 hours causes the same changes in AMPK, ACC, malonyl CoA, and palmitate oxidation as does AICAR. (5) Under all conditions studied, the contribution of glucose oxidation to ATP production is minimal. The results indicate that the AMPK-ACC-malonyl CoA-carnitine palmitoyl transferase 1 mechanism plays a key role in the physiological regulation of fatty acid oxidation in HUVECs. They also indicate that HUVECs oxidize fatty acids from both intracellular and extracellular sources, and that when this is taken into account, fatty acids can be a major substrate for ATP generation. Finally, they suggest that AMPK is likely to be a major factor in modulating the response of the endothelium to stresses that alter its energy state.

Topics: 3-O-Methylglucose; Acetyl-CoA Carboxylase; Adenosine Triphosphate; Aminoimidazole Carboxamide; AMP-Activated Protein Kinases; Caprylates; Carnitine; Cells, Cultured; Dose-Response Relationship, Drug; Endothelium, Vascular; Energy Metabolism; Enzyme Activation; Fatty Acids; Glucose; Glycolysis; Humans; Intracellular Fluid; Malonyl Coenzyme A; Multienzyme Complexes; Oxidation-Reduction; Palmitic Acid; Protein Serine-Threonine Kinases; Ribonucleotides; Tritium; Umbilical Veins

This study is designed to investigate whether substrate preference in the myocardium during the neonatal period and hypoxia-induced stress is controlled intracellularly or by extracellular substrate availability. To determine this, the effect of exogenous L-carnitine on the regulation of carbohydrate and fatty acid metabolism was determined during cardiac stress (hypoxia) and during the postnatal period. The effect of L-carnitine on long chain (palmitate) and medium chain (octonoate) fatty acid oxidation was studied in cardiac myocytes isolated from less than 24 h old (new born; NB), 2 week old (2 week) and hypoxic 4 week old (HY) piglets. Palmitate oxidation was severely decreased in NB cells compared to those from 2 week animals (0.456+/-0.04 vs. 1.207+/-0.52 nmol/mg protein/30 min); surprisingly, cells from even older hypoxic animals appeared shifted toward the new born state (0.695+/-0.038 nmol/mg protein/30 min). Addition of L-carnitine to the incubation medium, which stimulates carnitine palmitoyl-transferase I (CPTI) accelerated palmitate oxidation 3 fold in NB and approximately 2 fold in HY and 2 week cells. In contrast, octanoate oxidation which was greater in new born myocytes than in 2 week cells, was decreased by L-carnitine suggesting a compensatory response. Furthermore, oxidation of carbohydrates (glucose, pyruvate, and lactate) was greatly increased in new born myocytes compared to 2 week and HY cells and was accompanied by a parallel increase in pyruvate dehydrogenase (PDH) activity. The concentration of malonyl-CoA, a potent inhibitor of CPTI was significantly higher in new born heart than at 2 weeks. These metabolic data taken together suggest that intracellular metabolic signals interact to shift from carbohydrate to fatty acid utilization during development of the myocardium. The decreased oxidation of palmitate in NB hearts probably reflects decreased intracellular L-carnitine and increased malonyl-CoA concentrations. Interestingly, these data further suggest that the cells remain compliant so that under stressful conditions, such as hypoxia, they can revert toward the neonatal state of increased glucose utilization.

Topics: Animals; Animals, Newborn; Caprylates; Carbohydrate Metabolism; Carnitine; Cell Hypoxia; Fatty Acids; Glycolysis; Heart; Malonyl Coenzyme A; Myocardium; Oxidation-Reduction; Swine

Fatty acid metabolism has been studied in Fao rat hepatoma cells. In basal conditions of culture, [1-14C]oleate is mainly esterified (85% of oleate uptake) in Fao cells, phospholipids being the most important esterified products (60% of oleate esterified). Addition of N6,O2'-dibutyryl-adenosine 3',5'-monophosphate (0.1 mM) in Fao cells does not change the metabolic fate of oleate whereas it induces gluconeogenesis and phosphoenolpyruvate carboxykinase mRNA accumulation. It is shown that the limitation of oleate oxidation is located at the level of the entry into mitochondria since octanoate is actively oxidized in Fao cells. Neither the activities of carnitine palmitoyltransferase (CPT) I and II nor the CPT II protein amount are affected by cAMP addition. The limitation of oleate oxidation in Fao cells results from (a) a high rate of lipogenesis and a high malonyl-CoA concentration, (b) a CPT I very sensitive to malonyl-CoA inhibition. The presence of an active oleate oxidation in mitochondria isolated from Fao cells confirms that CPT I is the limiting step of oleate oxidation. Moreover, Fao cells are unable to perform ketogenesis. This particular feature results from a specific deficiency in mitochondrial hydroxymethylglutaryl-CoA synthase protein, activity and gene expression. The metabolic characteristics observed in Fao cells could be a common feature in hepatoma cell lines with regard to the low capacity for long-chain fatty acid oxidation and ketone body production observed in the rat H4IIE and the human HepG2 cells.

Topics: Animals; Bucladesine; Caprylates; Carnitine O-Palmitoyltransferase; Esterification; Fatty Acids; Humans; Hydroxymethylglutaryl-CoA Synthase; Ketone Bodies; Kinetics; Lipids; Liver Neoplasms, Experimental; Male; Malonyl Coenzyme A; Mitochondria, Liver; Oleic Acid; Oleic Acids; Oxidation-Reduction; Rats; Rats, Wistar; Tumor Cells, Cultured

The biosynthesis of the positional isomers of the monounsaturated fatty acids of Methylococcus capsulatus (Bath) has been investigated by studying the incorporation of [2-14C]malonyl CoA into long-chain fatty acids in vitro. The major unsaturated products were delta 9 16 : 1 and delta 11 18 : 1; however, delta 8, delta 10, and delta 11, 16 : 1, as well as, delta 10, delta 12 and delta 13 18 : 1 were also synthesized. The exclusion of O2 from the reaction vessel did not affect the synthesis of unsaturated fatty acids or the double bonds positions. Cerulenin inhibited the synthesis of unsaturated fatty acid more than saturated fatty acid. The use of both [1-14C] octanoate and [1-14C] decanote as substrate resulted in the synthesis of long-chain fatty acids, however, unsaturates were only synthesized from octanoate. These results imply that the unique positional isomers of M. capsulatus are not synthesized by an aerobic mechanism.

Topics: Anaerobiosis; Antifungal Agents; Caprylates; Cerulenin; Decanoic Acids; Fatty Acids; Fatty Acids, Monounsaturated; Fatty Acids, Unsaturated; Isomerism; Malonyl Coenzyme A; Methylococcaceae; Oxygen