Peroxisomal fatty acid oxidation is a substantial source of the acetyl moiety of malonyl-CoA in rat heart

Little is known about the sources of acetyl-CoA used for the synthesis of malonyl-CoA, a key regulator of mitochondrial fatty acid oxidation in the heart. In perfused rat hearts, we previously showed that malonyl-CoA is labeled from both carbohydrates and fatty acids. This study was aimed at assessing the mechanisms of incorporation of fatty acid carbons into malonyl-CoA. Rat hearts were perfused with glucose, lactate, pyruvate, and a fatty acid (palmitate, oleate or docosanoate). In each experiment, substrates were (13)C-labeled to yield singly or/and doubly labeled acetyl-CoA. The mass isotopomer distribution of malonyl-CoA was compared with that of the acetyl moiety of citrate, which reflects mitochondrial acetyl-CoA. In the presence of labeled glucose or lactate/pyruvate, the (13)C labeling of malonyl-CoA was up to 2-fold lower than that of mitochondrial acetyl-CoA. However, in the presence of a fatty acid labeled in its first acetyl moiety, the (13)C labeling of malonyl-CoA was up to 10-fold higher than that of mitochondrial acetyl-CoA. The labeling of malonyl-CoA and of the acetyl moiety of citrate is compatible with peroxisomal beta-oxidation forming C(12) and C(14) acyl-CoAs and contributing >50% of the fatty acid-derived acetyl groups that end up in malonyl-CoA. This fraction increases with the fatty acid chain length. By supplying acetyl-CoA for malonyl-CoA synthesis, peroxisomal beta-oxidation may participate in the control of mitochondrial fatty acid oxidation in the heart. In addition, this pathway may supply some acyl groups used in protein acylation, which is increasingly recognized as an important regulatory mechanism for many biochemical processes.     

Contribution of malonyl-CoA decarboxylase to the high fatty acid oxidation rates seen in the diabetic heart

Myocardial glucose oxidation is markedly reduced in the uncontrolled diabetic. We determined whether this was due to direct biochemical changes in the heart or whether this was due to altered circulating levels of insulin and substrates that can be seen in the diabetic. Isolated working hearts from control or diabetic rats (streptozotocin, 55 mg/kg iv administered 6 wk before study) were aerobically perfused with either 5 mM [(14)C]glucose and 0.4 mM [(3)H]palmitate (low-fat/low-glucose buffer) or 20 mM [(14)C]glucose and 1.2 mM [(3)H]palmitate (high-fat/high-glucose buffer) +/-100 microU/ml insulin. The presence of insulin increased glucose oxidation in control hearts perfused with low-fat/low-glucose buffer from 553 +/- 85 to 1,150 +/- 147 nmol x g dry wt(-1) x min(-1) (P < 0. 05). If control hearts were perfused with high-fat/high-glucose buffer, palmitate oxidation was significantly increased by 112% (P < 0.05), but glucose oxidation decreased to 55% of values seen in the low-fat/low-glucose group (P < 0.05). In diabetic hearts, glucose oxidation was very low in hearts perfused with low-fat/low-glucose buffer (9 +/- 1 nmol x g dry wt(-1) x min(-1)) and was not altered by insulin or high-fat/high-glucose buffer. These results suggest that neither circulating levels of substrates nor insulin was responsible for the reduced glucose oxidation in diabetic hearts. To determine if subcellular changes in the control of fatty acid oxidation contribute to these changes, we measured the activity of three enzymes involved in the control of fatty acid oxidation; AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), and malonyl-CoA decarboxylase (MCD). Although AMPK and ACC activity in control and diabetic hearts was not different, MCD activity and expression in all diabetic rat heart perfusion groups were significantly higher than that seen in corresponding control hearts. These results suggest that an increased MCD activity contributes to the high fatty acid oxidation rates and reduced glucose oxidation rates seen in diabetic rat hearts.     

Synthesis of fatty acids from malonyl-CoA and NADPH by pigeon liver fatty acid synthetase

Pigeon liver fatty acid synthetase has been found to catalyze the formation of palmitic acid from malonyl-CoA and NADPH in the absence of acetyl-CoA. Radio-chemical and spectral assays show that the activity of the complex in the absence of acetyl-CoA is about 25–30% of the activity in the presence of this compound. Initial velocities were determined for a series of reactions in which the malonyl-CoA concentration was varied over a range of 5–200 μm at a fixed NADPH concentration of 100μm and vice versa. No inhibitory effects of one substrate over the other were found. However, when the synthesis of fatty acids was studied in the presence of acetyl-CoA, a significant inhibitory effect of malonyl-CoA was observed. It has also been shown that the fatty acid synthetase synthesizes triacetic lactone from malonyl-CoA in the absence of NADPH and acetyl-CoA. No evidence was obtained for the direct decarboxylation of malonyl-CoA to acetyl-CoA in this reaction. Hence it is proposed that decarboxylation of the malonyl moiety bound covalently to 4′-phosphopantetheine occurs to yield acetyl-4′-phosphopantetheine. Further, it is proposed that the acetyl moiety of the latter compound is transferred to the cysteine site of the enzyme complex and that fatty acid synthesis proceeds in the presence of NADPH as proposed by Phillips et al. [Arch. Biochem. Biophys.138, 380 (1970)]. In the absence of NADPH triacetic lactone is formed.     

Production of 13C polyunsaturated fatty acids from the microalga Phaeodactylum tricornutum

An integrated process for the indoor production of ¹³C labelled PUFA from Phaeodactylum tricornutum is presented. The core of the process is a bubble column photobioreactor from which the exhaust gas from the reactor is returned to the culture by a low pressure compressor. To avoid accumulation of dissolved oxygen in the culture medium, the exhaust gas is bubbled through a sodium sulphite solution before returning it to the reactor. Carbon is removed from the medium before inoculating the alga, then labelled ¹³CO₂ is injected for pH control and carbon supply. The reactor has been operated in semicontinuous mode at a dilution rate of 0.01 h^–1, a biomass productivity of 0.1 g L^–1 d^–1 being obtained. Under this conditions both pH and dissolved oxygen were correctly controlled and the adequacy of the system for autotrophic production of labelled biomass was demonstrated. Analysis by GC-MS revealed that the fatty acids content of the biomass obtained was 10% d.wt., the content of eicosapentaenoic acid was 2.5% d.wt. All the fatty acids were labelled, more that 90% of the carbon present in these fatty acids was ¹³C. Element analysis of biomass and supernatant showed that 59.5% of injected carbon was assimilated into the biomass whereas 33% remained in the supernatant, and 7.5% remained undetected. Due to the high cost of ¹³CO₂ different strategies for the optimisation of labelled carbon use are proposed.     

LKB1 and the regulation of malonyl-CoA and fatty acid oxidation in muscle

5'-AMP-activated protein kinase (AMPK), by way of its inhibition of acetyl-CoA carboxylase (ACC), plays an important role in regulating malonyl-CoA levels and the rate of fatty acid oxidation in skeletal and cardiac muscle. In these tissues, LKB1 is the major AMPK kinase and is therefore critical for AMPK activation. The purpose of this study was to determine how the lack of muscle LKB1 would affect malonyl-CoA levels and/or fatty-acid oxidation. Comparing wild-type (WT) and skeletal/cardiac muscle-specific LKB1 knockout (KO) mice, we found that the 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR)-stimulated decrease in malonyl-CoA levels in WT heart and quadriceps muscles was entirely dependent on the presence of LKB1, as was the AICAR-induced increase in fatty-acid oxidation in EDL muscles in vitro, since these responses were not observed in KO mice. Likewise, the decrease in malonyl-CoA levels after muscle contraction was attenuated in KO gastrocnemius muscles, suggesting that LKB1 plays an important role in promoting the inhibition of ACC, likely by activation of AMPK. However, since ACC phosphorylation still increased and malonyl-CoA levels decreased in KO muscles (albeit not to the levels observed in WT mice), whereas AMPK phosphorylation was entirely unresponsive, LKB1/AMPK signaling cannot be considered the sole mechanism for inhibiting ACC during and after muscle activity. Regardless, our results suggest that LKB1 is an important regulator of malonyl-CoA levels and fatty acid oxidation in skeletal muscle.     

Stimulation of oxidation of mitochondrial fatty acids and of acetate by acetylcarnitine

1. Acetylcarnitine added in catalytic amounts to kidney mitochondria produces an active oxidation of endogenous fatty acids. 2. In conditions of mitochondrial ;aging', under which acetate is not oxidized, acetylcarnitine also promotes the oxidation of this exogenous substrate. 3. Dinitrophenol completely abolishes the action of acetylcarnitine. 4. Carnitine is ineffective both in the oxidation of endogenous fatty acids and of exogenous acetate. 5. The action of acetylcarnitine is shared, though to a smaller extent, by pyruvate. 6. The mechanism of acetylcarnitine action has been interpreted by considering that the readily oxidizable acetyl group of acetylcarnitine can supply the initial investment of energy needed to start fatty acid oxidation.     

Effect of palmitoyl-CoA and beta-oxidation of fatty acids on the kinetics of mitochondrial citrate transporter.

The kinetics of the hepatic mitochondrial citrate transporter were studied using 1,2,3-benzene tricarboxylate and the inhibitor-stop technique at 8 degrees C. The apparent Km for this transporter was 250 muM and the maximum velocity was 2 nmol of citrate transported per minute per milligram of mitochondrial protein. This apparent Km was increased when hepatic mitochondria were preincubated with both L-palmitoylcarnitine and CoA-SH but not with either alone. This rise in apparent Km was accompanied by a rise in the acid insoluble CoA-SH content. Removal of mitochondrial acid insoluble CoA by "defatted albumin" resulted in a parallel decrease in the apparent Km. The apparent Km for the citrate transporter was increased after coupled beta-oxidation of L-palmitoylcarnitine or octanoate without a detectable increase in acid insoluble CoA. Inhibition of beta-oxidation of L-palmitoylcarnitine by the D-derivative prevented the rise in the apparent Km. Preincubation with ATP resulted in an increase in this apparent Km. When L-palmitoylcarnitine oxidation occurred without ATP accumulation (hexokinase, glucose, ADP, and inorganic phosphate) the apparent Km for the citrate transporter increased two- to threefold. Therefore, the apparent Km for the citrate transporter varied directly with the acid insoluble CoA content. In addition, this Km was increased as a result of beta-oxidation of fatty acids but the mechanism was not solely attributable to a rise in acid insoluble CoA or ATP. The physiological implications of these findings are discussed.     

Storage and oxidation of long-chain fatty acids in the C57/BL6 mouse heart as measured by NMR spectroscopy

Triglyceride turnover in the isolated C57/BL6 mouse heart was measured by dynamic 13C edit-(1)H observe NMR and the rate of fatty acid oxidation was determined by 13C NMR isotopomer analysis. In the presence of a physiological mixture of substrates, energy was produced in the citric acid cycle by oxidation of long-chain fatty acids (18%), ketones (34%), lactate (24%), pyruvate (7%), and other sources (17%). Exogenous fatty acids appeared in the triglyceride pool at 0.24 micromol/g dry wt/min, similar to the rate of oxidation of long-chain fatty acids, 0.16 micromol/g dry wt/min. Isoproterenol decreased the rate of de novo triglyceride synthesis and increased the rate of fatty acid oxidation.     

Estimating high-affinity methanotrophic bacterial biomass, growth, and turnover in soil by phospholipid fatty acid 13C labeling

A time series phospholipid fatty acid (PLFA) 13C-labeling study was undertaken to determine methanotrophic taxon, calculate methanotrophic biomass, and assess carbon recycling in an upland brown earth soil from Bronydd Mawr (Wales, United Kingdom). Laboratory incubations of soils were performed at ambient CH4 concentrations using synthetic air containing 2 parts per million of volume of 13CH4. Flowthrough chambers maintained a stable CH4 concentration throughout the 11-week incubation. Soils were analyzed at weekly intervals by gas chromatography (GC), GC-mass spectrometry, and GC-combustion-isotope ratio mass spectrometry to identify and quantify individual PLFAs and trace the incorporation of 13C label into the microbial biomass. Incorporation of the 13C label was seen throughout the experiment, with the rate of incorporation decreasing after 9 weeks. The delta13C values of individual PLFAs showed that 13C label was incorporated into different components to various extents and at various rates, reflecting the diversity of PLFA sources. Quantitative assessments of 13C-labeled PLFAs showed that the methanotrophic population was of constant structure throughout the experiment. The dominant 13C-labeled PLFA was 18:1omega7c, with 16:1omega5 present at lower abundance, suggesting the presence of novel type II methanotrophs. The biomass of methane-oxidizing bacteria at optimum labeling was estimated to be about 7.2 x 10(6) cells g(-1) of soil (dry weight). While recycling of 13C label from the methanotrophic biomass must occur, it is a slower process than initial 13CH4 incorporation, with only about 5 to 10% of 13C-labeled PLFAs reflecting this process. Thus, 13C-labeled PLFA distributions determined at any time point during 13CH4 incubation can be used for chemotaxonomic assessments, although extended incubations are required to achieve optimum 13C labeling for methanotrophic biomass determinations.     

Biotransformation of C20- and C22-polyunsaturated fatty acids to 11S- and 13S-hydroxy fatty acids by Escherichia coli expressing 11S-lipoxygenase from Enhygromyxa salina

Biotechnology Letters - Peroxidation and reduction of 11S- and 13S-positions on C20 and C22 polyunsaturated fatty acids (PUFAs) by Escherichia coli expressing highly active arachidonate (ARA)...