malonyl-CoA Concentration Research Articles

Malonyl-CoA and the regulation of fatty acid oxidation in soleus muscle.

1. Rat soleus strips were incubated with 5 mM glucose, after which tissue metabolites were measured. Alternatively, muscle strips were incubated with 5 mM glucose and 0.2 mM palmitate, and the formation of 14CO2 from exogenous palmitate or from fatty acids released from prelabelled glycerolipids was measured. 2. Etomoxir, which inhibits the mitochondrial overt form of carnitine palmitoyltransferase (CPT1), increased the tissue content of long-chain fatty acyl-CoA esters and decreased the ratio of fatty acylcarnitine to fatty acyl-CoA, suggesting that such changes could be a diagnostic for the inhibition of CPT1 3. Over a range of incubation conditions there was a positive correlation between the tissue contents of malonyl-CoA and long-chain fatty acyl-CoA esters. Under conditions in which these two metabolites increased in content (i.e. with insulin or with 3 mM dichloroacetate) there was a corresponding decrease in the ratio of fatty acylcarnitine to fatty acyl-CoA and a decrease in beta-oxidation. Isoprenaline or palmitate (0.5 mM) opposed the effect of insulin, decreasing the contents of malonyl-CoA and long-chain fatty acyl-CoA, increasing the ratio of fatty acylcarnitine to fatty acyl-CoA and increasing beta-oxidation. These findings are consistent with the notion that all of these agents can cause the acute regulation of CPT1 in Type I skeletal muscle. 4. The addition of 5-amino-4-imidazolecarboxamide ribonucleoside (AICAriboside) to cause activation of the AMP-activated protein kinase decreased the tissue content of malonyl-CoA. AICAriboside also had an antilipolytic effect in the muscle strips. 5. Measurements were made of the activities of ATP-citrate lyase, acetyl-CoA carboxylase, fatty acid synthase and malonyl-CoA decarboxylase in soleus muscle and in representative Type IIa and Type IIb muscles. A cytosolic activity of malonyl-CoA decarboxylase would seem to offer a feasible route for the disposal of malonyl-CoA in skeletal muscle.

Skeletal muscle malonyl-CoA content at the onset of exercise at varying power outputs in humans

To investigate the regulation of intramuscular fuel selection, we measured the malonyl-CoA (M-CoA) content in human skeletal muscle at three exercise power outputs [35, 65, and 90% maximal rate of O2 consumption (VO2 max)]. Four males and four females cycled for 10 min at one power output on three separate occasions with muscle biopsies sampled at rest and at 1 and 10 min. The respiratory exchange ratio was 0.84 +/- 0.03, 0.92 +/- 0.02, and >1.0 at 35, 65 and 90% VO2 max, respectively. Muscle lactate content increased and phosphocreatine content decreased as a function of power output. Pyruvate dehydrogenase a activity increased from 0.40-0.64 mmol . kg wet muscle-1 . min-1 at rest to 1.57 +/- 0.28, 2.80 +/- 0.41, and 3. 28 +/- 0.27 mmol . kg wet muscle-1 . min-1 after 1 min of cycling at the three power outputs, respectively. Mean resting M-CoA contents were similar at all power outputs (1.85-1.98 micromol/kg dry muscle). During exercise at 35% VO2 max, M-CoA decreased from rest at 1 min (1.85 +/- 0.29 to 1.20 +/- 0.12 micromol/kg dry muscle) but returned to rest level by 10 min (1.86 +/- 0.25 micromol/kg dry muscle). M-CoA content did not decrease during cycling at 65% VO2 max. At 90% VO2 max, M-CoA did not increase despite significant acetyl-CoA accumulation (the substrate for M-CoA synthesis). The data suggest that a decrease in M-CoA content is not required for the increase in free fatty acid uptake and oxidation that occurs during exercise at 35 and 65% VO2 max. Furthermore, M-CoA content does not increase during exercise at 90% VO2 max and does not contribute to the lower rate of fat oxidation at this power output.

Glucose-fatty acid interactions in health and disease

It is widely held that although obesity and type 2 diabetes are polygenic in origin, the primary defect causing both conditions is insulin resistance, which in turn gives rise to a constellation of other abnormalities, including hyperinsulinemia, dyslipidemia, glucose intolerance, and (in the genetically predisposed) frank hyperglycemia. Explored here is an alternative, albeit speculative, scenario in which hyperinsulinemia and insulin resistance arise either simultaneously or sequentially from some preexisting defect within the leptin signaling pathway. In either case, a central component of the model is that the breakdown of glucose homeostasis that is characteristic of the condition of obesity with type 2 diabetes is secondary to disturbances in lipid dynamics. The possibility is raised that abnormally high concentrations of malonyl-CoA in liver and skeletal muscle suppress the activity of mitochondrial carnitine palmitoyltransferase I and thus fatty acid oxidation in both sites. It is suggested that the buildup of fat within the muscle cell (caused in part by excessive delivery of VLDLs from the liver) interferes with glucose transport or metabolism or both, producing insulin resistance. Elevated circulating concentrations of fatty acids are also implicated in the etiology of type 2 diabetes by virtue of 1) their powerful acute insulinotropic effect, 2) their ability to exacerbate insulin resistance in muscle, and 3) their long-term detrimental action on pancreatic beta-cell function.

Malonyl CoA as a metabolic switch and a regulator of insulin sensitivity.

Malonyl CoA is a regulator of carnitine palmitoyl transferase 1 (CPT1), the enzyme that controls the transfer of long chain fatty acyl CoA into mitochondria where it is oxidized. Recent studies indicate that in skeletal muscle the concentration of malonyl CoA is acutely (minutes) regulated by changes in its fuel supply and energy expenditure. In response to changes in fuel supply, regulation appears to be due to alterations in the cytosolic concentration of citrate, which is both an allosteric activator of acetyl CoA carboxylase (ACC), the enzyme that catalyzes malonyl CoA synthesis and a source of its precursor, cytosolic acetyl CoA. During exercise and immediately thereafter regulation by citrate appears to be lost and malonyl CoA levels diminish as the result of a decrease in ACC activity secondary to phosphorylation. Sustained increases in the concentration of malonyl CoA have been observed in muscle of a number of insulin-resistant rodents including the Zucker (fa/fa) and GK rats, KKAgy mice, glucose-infused rats and rats in which muscle has been made insulin resistant by denervation. Available data suggest that malonyl CoA could be linked to insulin resistance in these rodents by virtue of its effects on the cytosolic concentration of long chain fatty acyl CoA (LCFA CoA) and one or more protein kinase C isozymes. Whether similar alterations occur in other tissues and contribute to the pathophysiology of the insulin resistance syndrome remains to be determined.

Regulation of carbohydrate and fatty acid utilization by L-carnitine during cardiac development and hypoxia.

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.

Regulation of fat/carbohydrate interaction in human skeletal muscle during exercise.

There has been continued interest in the regulation of fat and carbohydrate utilization in muscle tissue, as they are the main substrates for energy production during exercise in well fed humans. Many investigations have demonstrated that increasing fat availability increases fat oxidation and decreases carbohydrate use in the whole body and skeletal muscle. However, little work has been performed in skeletal muscle to identify the mechanisms, and specific biochemical signals which mediate these shifts in fuel selection. The classic work in this area, using contracting heart and resting diaphragm muscle, led to a theory that could explain reciprocal changes in fat and carbohydrate use from a biochemical perspective (glucose-fatty acid cycle). Using this information, we embarked on a number of studies demonstrating that the biochemical regulation of fat/carbohydrate interaction in human skeletal muscle during exercise is different than exists in the continually active heart and diaphragm muscles. By increasing the availability of free fatty acids to the working muscles, it was demonstrated that carbohydrate downregulation during moderate and intense aerobic exercise occurred mainly at glycogen phosphorylase, the enzyme that regulates the degradation of muscle glycogen. There was also coordinate downregulation of pyruvate dehydrogenase activity at low and moderate exercise intensities. We have also investigated the roles that carnitine palmitoyl-transferase I and its regulator malonyl-CoA play in governing the transport of long-chain fatty acids into the mitochondria for oxidation, and therefore the impact on carbohydrate use in human skeletal muscle. The regulation of Carnitine Palmitoyltransferase I (CPTI) activity appears to involve more than changes in the concentration of malonyl-CoA. Several additional mechanisms may exist that interact with or override the effects of malonyl-CoA during exercise, although this work is just beginning. The present work has identified several regulatory sites in the pathways of carbohydrate and fat metabolism that help explain the regulation of fat/carbohydrate interaction in human skeletal muscle during exercise.

Differential regulation in the heart of mitochondrial carnitine palmitoyltransferase-I muscle and liver isoforms.

Carnitine palmitoyltransferase-I (CPT-I) plays a crucial role in regulating cardiac fatty acid oxidation which provides the primary source of energy for cardiac muscle contraction. CPT-I catalyzes the transfer of long chain fatty acids into mitochondria and is recognized as the primary rate controlling step in fatty acid oxidation. Molecular cloning techniques have demonstrated that two CPT-I isoforms exist as genes encoding the 'muscle' and 'liver' enzymes. Regulation of fatty acid oxidation rates depends on both short-term regulation of enzyme activity and long-term regulation of enzyme synthesis. Most early investigations into metabolic control of fatty acid oxidation at the CPT-I step concentrated on the hepatic enzyme which can be inhibited by malonyl-CoA and can undergo dramatic amplification or reduction of its sensitivity to inhibition by malonyl-CoA. The muscle CPT-I is inherently more sensitive to malonyl-CoA inhibition but has not been found to undergo any alteration of its sensitivity. Short-term control of activity of muscle CPT-I is apparently regulated by malonyl-CoA concentration in response to fuel supply (glucose, lactate, pyruvate and ketone bodies). The liver isoform is the only CPT-I enzyme present in the mitochondria of liver, kidney, brain and most other tissues while muscle CPT-I is the sole isoform expressed in skeletal muscle as well as white and brown adipocytes. The heart is unique in that it contains both muscle and liver isoforms. Liver CPT-I is highly expressed in the fetal heart, but at birth its activity begins to decline whereas the muscle isoform, which is very low at birth, becomes the predominant enzyme during postnatal development. In this paper, the differential regulation of the two CPT-I isoforms at the protein and the gene level will be discussed.

Changes in size of intracellular pools of coenzyme A and its thioesters in Escherichia coli K-12 cells to various carbon sources and stresses.

Intracellular pools of three CoA molecular species of coenzyme A, CoASH, acetyl-CoA, and malonyl-CoA, in Escherichia coli K-12 cells were studied by acyl-CoA cycling method in replacement culture. The sizes and compositions of CoA pools starved for a carbon source changed within minutes after the addition of one of various carbon sources. A large acetyl-CoA pool formed after the addition of D-glucose, D-fructose, D-mannose, glycerol, or sorbitol, but there was little change when L-glucose, sucrose, maltose, succinate, or acetate was added. The beta-anomer of D-glucose was assimilated 10 times faster than the alpha-anomer. Intracellular CoA pools also changed with stress: in the pH, incubation temperature, or with osmotic stress. The sizes and compositions of CoA pools were not affected by pH changing between 4 and 8, but the breakdown of acetyl-CoA and CoASH was greater at pH 9 than at pH 4 to 8. Production of acetyl-CoA was greatest at 40 degrees C, and at 50 degrees C, an acetyl-CoA pool did not form at all and the size of the CoASH pool declined. When the organism was stressed by the addition of NaCl at concentrations of more than 0.6 M, little acetyl-CoA was produced. The total CoA pool (the sum of the concentrations of CoASH, acetyl-CoA, and malonyl-CoA) remained within the limits of 0.83-1.40 nmol/mg of dry cell weight (0.30-0.52 mM). Whenever acetyl-CoA increased, CoASH decreased. Therefore, the acetyl-CoA/CoASH ratio is an important index of facultative anaerobes that reflects the state of carbon and energy metabolism in vivo.

AICA riboside increases AMP-activated protein kinase, fatty acid oxidation, and glucose uptake in rat muscle.

5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) has previously been reported to be taken up into cells and phosphorylated to form ZMP, an analog of 5'-AMP. This study was designed to determine whether AICAR can activate AMP-activated protein kinase (AMPK) in skeletal muscle with consequent phosphorylation of acetyl-CoA carboxylase (ACC), decrease in malonyl-CoA, and increase in fatty acid oxidation. Rat hindlimbs were perfused with Krebs-Henseleit bicarbonate containing 4% bovine serum albumin, washed bovine red blood cells, 200 microU/ml insulin, and 10 mM glucose with or without AICAR (0.5-2.0 mM). Perfusion with medium containing AICAR was found to activate AMPK in skeletal muscle, inactivate ACC, and decrease malonyl-CoA. Hindlimbs perfused with 2 mM AICAR for 45 min exhibited a 2.8-fold increase in fatty acid oxidation and a significant increase in glucose uptake. No difference was observed in oxygen uptake in AICAR vs. control hindlimb. These results provide evidence that decreases in muscle content of malonyl-CoA can increase the rate of fatty acid oxidation.

Stromal concentrations of coenzyme A and its esters are insufficient to account for rates of chloroplast fatty acid synthesis: evidence for substrate channelling within the chloroplast fatty acid synthase.

Concentrations of total CoAs in chloroplasts freshly isolated from spinach and peas were 10-20 microM, assuming a stromal volume of 66 microl per mg of chlorophyll. Acetyl-CoA and CoASH constituted at least 90% of the total CoA in freshly isolated chloroplasts. For a given chloroplast preparation, the concentration of endogenous acetyl-CoA was the same when extractions were performed using HClO4, trichloroacetic acid, propan-2-ol or chloroform/methanol, and the extracts analysed by quantitative HPLC after minimal processing. During fatty acid synthesis from acetate, concentrations of CoASH within spinach and pea chloroplasts varied from less than 0.1 to 5.0 microM. Malonyl-CoA concentrations were also very low (<0.1-3.0 microM) during fatty acid synthesis but could be calculated from radioactivity incorporated from [1-14C]acetate. Concentrations of CoASH in chloroplasts synthesizing fatty acids could be doubled in the presence of Triton X-100, suggesting that the detergent stimulates fatty acid synthesis by increasing the turnover rate of acyl-CoA. However, although taken up, exogenous CoASH (1 microM) did not stimulate fatty acid synthesis by permeabilized spinach chloroplasts. Calculated rates for acetyl-CoA synthetase, acetyl-CoA carboxylase and malonyl-CoA-acyl-carrier protein transacylase reactions at the concentrations of metabolites measured here are < 0.1-4% of the observed rates of fatty acid synthesis from acetate by isolated chloroplasts. The results suggest that CoA and its esters are probably confined within, and channelled through, the initial stages of a fatty acid synthase multienzyme complex.

Contraction-induced Changes in Acetyl-CoA Carboxylase and 5′-AMP-activated Kinase in Skeletal Muscle

The concentration of malonyl-CoA, a negative regulator of fatty acid oxidation, diminishes acutely in contracting skeletal muscle. To determine how this occurs, the activity and properties of acetyl-CoA carboxylase beta (ACC-beta), the skeletal muscle isozyme that catalyzes malonyl-CoA formation, were examined in rat gastrocnemius-soleus muscles at rest and during contractions induced by electrical stimulation of the sciatic nerve. To avoid the problem of contamination of the muscle extract by mitochondrial carboxylases, an assay was developed in which ACC-beta was first purified by immunoprecipitation with a monoclonal antibody. ACC-beta was quantitatively recovered in the immunopellet and exhibited a high sensitivity to citrate (12-fold activation) and a Km for acetyl-CoA (120 microM) similar to that reported for ACC-beta purified by other means. After 5 min of contraction, ACC-beta activity was decreased by 90% despite an apparent increase in the cytosolic concentration of citrate, a positive regulator of ACC. SDS-polyacrylamide gel electrophoresis of both homogenates and immunopellets from these muscles showed a decrease in the electrophoretic mobility of ACC, suggesting that phosphorylation could account for the decrease in ACC activity. In keeping with this notion, citrate activation of ACC purified from contracting muscle was markedly depressed. In addition, homogenization of the muscles in a buffer free of phosphatase inhibitors and containing the phosphatase activators glutamate and MgCl2 or treatment of immunoprecipitated ACC-beta with purified protein phosphatase 2A abolished the decreases in both ACC-beta activity and electrophoretic mobility caused by contraction. The rapid decrease in ACC-beta activity after the onset of contractions (50% by 20 s) and its slow restoration to initial values during recovery (60-90 min) were paralleled temporally by reciprocal changes in the activity of the alpha2 but not the alpha1 isoform of 5'-AMP-activated protein kinase (AMPK). In conclusion, the results suggest that the decrease in ACC activity during muscle contraction is caused by an increase in its phosphorylation, most probably due, at least in part, to activation of the alpha2 isoform of AMPK. They also suggest a dual mechanism for ACC regulation in muscle in which inhibition by phosphorylation takes precedence over activation by citrate. These alterations in ACC and AMPK activity, by diminishing the concentration of malonyl-CoA, could be responsible for the increase in fatty acid oxidation observed in skeletal muscle during exercise.

The influence of dietary fatty acids on the activity and metabolic control of peroxisomal carnitine palmitoyltransferase in the liver

Peroxisomes play an important role in the β-oxidation of fatty acids and they possess a membrane-bound carnitine palmitoyltransferase (CPT). Whether this enzyme is regulated by the amount and type of fat consumed in the diet has not been investigated. Therefore weanling male Lewis rats were fed for various times (2 days to 4 weeks) on a low-fat (2.5% w/w) diet (LF) or on high-fat diets containing 20% (w/w) of either hydrogenated coconut oil (HCO), olive oil (OO), safflower oil (SO), evening primrose oil (EPO) or menhaden (fish) oil (MO). Hepatic peroxisomal CPT activity and sensitivity to inhibition by malonyl CoA were examined. In general, feeding the high-fat diets resulted in higher CPT activity per mg peroxisomal protein (specific activity) and per g liver (capacity) than feeding the LF diet throughout the 4 week feeding period. The most rapid induction of CPT specific was seen in the livers of animals fed the SO, EPO and MO diets. Induction of CPT specific activity was maximal after one week of feeding each of the diets. Feeding the MO diet resulted in the highest CPT specific activity at most time points examined and the highest CPT capacity at all time points. In relation to feeding the LF diet, the HCO and EPO diets increased CPT specific activity and capacity by approximately 2-fold while the OO, SO and MO diets increased the CPT specific activity by 3- to 4-fold; these diets increased CPT capacity by 3.5-fold (OO), 4.5-fold (SO) and 7.4-fold (MO). These observations are consistent with previous observations that dietary fish oils increase peroxisomal β-oxidation. Peroxisomal CPT from animals fed the LF diet was the most sensitive to inhibition by malonyl CoA at each time point ([I 50] = approx. 0.5 μM). Each of the high-fat diets decreased the sensitivity of peroxisomal CPT to inhibition by malonyl CoA. The decrease in sensitivity was apparent after feeding the OO, SO or MO diets for 2 days. At all time points peroxisomal CPT from MO-fed rats was the least sensitive to malonyl CoA inhibition. At its least sensitive (after 7 days of feeding) peroxisomal CPT from MO-fed rats required a 5.7-fold higher malonyl CoA concentration to induce 50% inhibition than did the enzyme from LF-fed rats. These data indicate that both the level of fat in the diet and the fatty acid composition of high-fat diets influence CPT activity and its sensitivity to the inhibitor malonyl CoA.

Control of Hepatic Fatty Acid Oxidation by 5′-AMP-Activated Protein Kinase Involves a Malonyl-CoA-Dependent and a Malonyl-CoA-Independent Mechanism

Incubation of rat hepatocytes with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), an activator of the 5′-AMP-activated protein kinase (AMPK), produced a twofold stimulation of palmitate oxidation and of the activity of carnitine palmitoyltransferase I (CPT-I), together with a profound decrease of the activity of acetyl-CoA carboxylase and of the intracellular level of malonyl-CoA. AICAR-induced CPT-I stimulation progressively blunted with time after cell permeabilization, pointing to reversal of conformational constraints of the enzyme in control cells due to the permeabilization-triggered dilution of intracellular malonyl-CoA. The stimulation stabilized at a steady 20–25%. This 20–25% increase in CPT-I activity survived upon complete removal of malonyl-CoA from the permeabilized cells, indicating that it was not dependent on the malonyl-CoA concentration of the cell. This malonyl-CoA-independent activation of CPT-I was not evident when mitochondria were isolated for assay of enzyme activity or when cells were disrupted by vigorous sonication. In addition, the microtubule stabilizer taxol prevented the malonyl-CoA-independent stimulation of CPT-I induced by AICAR. Hence, stimulation of hepatic fatty acid oxidation by AMPK seems to rely on the activation of CPT-I by two different mechanisms: deinhibition of CPT-I induced by depletion of intracellular malonyl-CoA levels and malonyl-CoA-independent stimulation of CPT-I, which might involve modulation of interactions between CPT-I and cytoskeletal components.

Increased cardiac fatty acid uptake with dobutamine infusion in swine is accompanied by a decrease in malonyl CoA levels

Malonyl CoA is an important regulator of fatty acid oxidation in the heart secondary to its ability to inhibit carnitine palmitoyltransferase 1 (CPT 1). Malonyl CoA is produced from acetyl CoA in a reaction catalyzed by acetyl CoA carboxylase (ACC). In this study we determined if alterations in malonyl CoA regulation of fatty acid metabolism are involved in the increase in energy transduction seen following an increase in cardiac work. Anesthetized, open-chest, domestic swine were subjected to a 30 min control period followed by a 30 min treatment period with either dobutamine (15 micrograms.kg-1. min-1 i.v.) (n = 6) or saline (n = 6). Heart rate, left ventricular peak dp/dt, and MVO2, were significantly increased in the dobutamine group compared to the saline group during the treatment period. Free fatty acid and glucose uptake were increased 210 and 248%, respectively, in the dobutamine group during the treatment period. Malonyl CoA content was decreased by 55% (from 0.40 +/- 0.05 to 0.18 +/- 0.12 nmol/g wet wt; P < 0.05) with dobutamine treatment, but was not affected by saline treatment. ACC activity was not significantly different between groups (0.31 +/- 0.02 vs. 0.30 +/- 0.04 nmol. min-1. mg protein-1, respectively). The activity of AMP-dependent protein kinase (AMPK), which phosphorylates and inactivates ACC, was also not significantly different in the dobutamine hearts compared to the saline hearts (322 +/- 26 vs. 338 +/- 39 pmol. min-1. mg protein-1, respectively). The increased cardiac work following dobutamine infusion is accompanied by a decrease in malonyl CoA levels and an increase in fatty acid uptake. However, the decrease in malonyl CoA cannot be explained by a decrease in ACC activity.

Increased cardiac fatty acid uptake with dobutamine infusion in swine is accompanied by a decrease in malonyl CoA levels

Malonyl CoA is an important regulator of fatty acid oxidation in the heart secondary to its ability to inhibit carnitine palmitoyltransferase 1 (CPT 1). Malonyl CoA is produced from acetyl CoA in a reaction catalyzed by acetyl CoA carboxylase (ACC). In this study we determined if alterations in malonyl CoA regulation of fatty acid metabolism are involved in the increase in energy transduction seen following an increase in cardiac work. Methods: Anesthetized, open-chest, domestic swine were subjected to a 30 min control period followed by a 30 min treatment period with either dobutamine (15 μg · kg−1 · min−1 i.v.) (n = 6) or saline (n = 6). Results: Heart rate, left ventricular peak dp/dt, and MVO2, were significantly increased in the dobutamine group compared to the saline group during the treatment period. Free fatty acid and glucose uptake were increased 210 and 248%, respectively, in the dobutamine group during the treatment period. Malonyl CoA content was decreased by 55% (from 0.40 ± 0.05 to 0.18 ± 0.12 nmol/g wet wt; P < 0.05) with dobutamine treatment, but was not affected by saline treatment. ACC activity was not significantly different between groups (0.31 ± 0.02 vs. 0.30 ± 0.04 nmol · min−1 · mg protein−1, respectively). The activity of AMP-dependent protein kinase (AMPK), which phosphorylates and inactivates ACC, was also not significantly different in the dobutamine hearts compared to the saline hearts (322 ± 26 vs. 338 ± 39 pmol · min−1 · mg protein−1, respectively). Conclusion: The increased cardiac work following dobutamine infusion is accompanied by a decrease in malonyl CoA levels and an increase in fatty acid uptake. However, the decrease in malonyl CoA cannot be explained by a decrease in ACC activity.

Flux control exerted by overt carnitine palmitoyltransferase over palmitoyl-CoA oxidation and ketogenesis is lower in suckling than in adult rats.

We examined the potential of overt carnitine palmitoyltransferase (CPT I) to control the hepatic catabolism of palmitoyl-CoA in suckling and adult rats, using a conceptually simplified model of fatty acid oxidation and ketogenesis. By applying top-down control analysis, we quantified the control exerted by CPT I over total carbon flux from palmitoyl-CoA to ketone bodies and carbon dioxide. Our results show that in both suckling and adult rat, CPT I exerts very significant control over the pathways under investigation. However, under the sets of conditions we studied, less control is exerted by CPT I over total carbon flux in mitochondria isolated from suckling rats than in those isolated from adult rats. Furthermore the flux control coefficient of CPT I changes with malonyl-CoA concentration and ATP turnover rate.

Effects of ranolazine on oxidative substrate preference in epitrochlearis muscle.

Ranolazine is an novel investigational antianginal agent that stimulates glucose oxidation in isolated rat hearts. This study determined its effects on metabolic substrate and O2 utilization in an in vitro skeletal muscle preparation, the rat epitrochlearis muscle. Muscles were superfused with Krebs-Henseleit buffer containing 3% albumin, 0.4 mM palmitate, 5.5 mM glucose, 0.5 mM lactate, and a physiological amino acid mixture. Perfusate also contained either 1) [U-14C]glucose for measurement of glucose oxidation or 2) [9,10-3H]palmitate and [U-14C]lactate for measurement of palmitate and lactate oxidation. Addition of ranolazine (10 microM) significantly stimulated glucose oxidation and decreased palmitate oxidation but had no effect on lactate oxidation. Overall, the calculated relative contribution of glucose oxidation to aerobic ATP production increased from 12 to 33%, whereas from palmitate it decreased from 55 to 26%. Ranolazine did not alter tissue malonyl-CoA contents, making it unlikely that the decrease in palmitate oxidation caused by ranolazine is due to a decrease in the activity of acetyl-CoA carboxylase. These data demonstrate that ranolazine can shift energy substrate preference in skeletal muscle, which could potentially prove useful in ischemic disorders of skeletal muscle.

A malonyl-CoA fuel sensing mechanism in muscle: effects of insulin, glucose and denervation: A. K. Saha, T. G. Kurowski, N. B. Ruderman.Am J Physiol 1995; 269: E283–E289

Increases in the concentration of malonyl-CoA in skeletal musclehave been observed in the KKA Y mouse, an obese rodent with high plasma insulin and glucose levels (1). To assess whether insulin and glucose directly regulate malonyl-CoA in muscle, soleus muscles from young rats were incubated with insulin and glucose at various concentrations, and their content of malonyl-CoA was determined. In addition, the effect on malonyl-CoA of denervation and electrically-induced muscle contractions was assessed. The concentration of malonyl-CoA in the soleus, taken directly from a rat fed ad libitum, was 2.0 ± 0.2 nmol/g. In muscles incubated for 20 min in a medium devoid of added insulin and glucose, the concentration was decreased to 0.8 ± 0.2 nmol/g. When the medium contained 0.5, 7.5, or 30 mM glucose, malonyl-CoA concentrations were 1.3 ± 0.1, 1.8 ± 0.1, or 2.4 ± 0.2 nmol/g, respectively, in the absence of insulin and 1.7 ± 0.1, 4.6 ± 0.3 and 5.5 ± 0.6 nmol/g. in its presence (10 mU/ml). Compared with its level in a control muscle, the concentration of malonyl-CoA increased 3-fold in the soleus 6–8 h after denervation and remained 2-fold higher for ≥ 48 h. In contrast, muscle contractions induced by sciatic nerve stimulation, in vivo, acutely decreased the concentration of malonyl-CoA by 30–35%. The results indicate that insulin and glucose, and probably contractile activity, regulate the concentration of malonyl-CoA in muscle. They suggest that malonyl-CoA is a component of a fuel-sensing and signaling mechanism that responds to changes in the fuel milieu and possibly the energy expenditure of the muscle cell.

HUMAN SKELETAL MUSCLE MALONYL-CoA AT REST AND DURING PROLONGED SUBMAXIMAL EXERCISE 445

Previous literature has indicated that malonyl-CoA content in rat skeletal muscle decreases with prolonged submaximal exercise. It has been suggested that this contraction-induced decrease in malonyl-CoA is instrumental in the increase of fatty acid oxidation during exercise. The purpose of this study was to measure malonyl-CoA content in human vastus lateralis muscle at rest and during submaximal exercise. Eight males and 1 female cycled for 70 min (10 min at 40% and 60 min at 65% VO2 max). Needle biopsies were obtained at rest, 10 min, 20 min, and 70 min of exercise. Malonyl-CoA content in pre-exercise biopsy samples determined by HPLC was 1.53 ± 0.18μmol·kg-1 dry weight. Malonyl-CoA content did not change significantly during exercise (1.39 ± 0.21 at 10 min, 1.46 ± 0.14 at 20 min and 1.22 ± 0.15 μmol·kg-1 dry wt at 70 min). This was in spite of a significant decrease in RER from 0.95 ± 0.02 to 0.89 ± 0.02 during 60 min at 65% VO2 max. In contrast, malonyl-CoA content determined by HPLC in perfused rat red gastrocnemius muscle decreased significantly during 20 min of stimulation at 0.7 Hz (3.44± 0.54 and 1.64 ± 0.23 nmol·g-1 dry wt, pre and post stimulation, respectively, n=9). We conclude that human skeletal muscle malonyl-CoA content 1) is less than reported in rat skeletal muscle at rest, 2) does not decrease with prolonged submaximal exercise, and 3) is not predictive of increased fatty acid oxidation during exercise.

Pyruvate Dehydrogenase Activity and Malonyl CoA Levels in Normal and Ischemic Swine Myocardium: Effects of Dichloroacetate

The pruposes of this study were to: (1) assess myocardial pyruvate dehydrogenase (PDH) activity and substrate exchange under well-perfused and eschemic conditions; (2) determine the metabolic effects of an intra-coronary infusion of the PDH activator, dichloroacetate (DCA); and (3) measure the effects of ischemia and DCA on malonyl CoA levels. Experiments were performed in anesthetised open-chest swine under non-ischemic conditions, followed by 40 min with a 60% reduction in left anterior descending coronary artery (LAD) blood flow. Myocardial needle biopsies for measurement of PDH activity were taken after an intracoronary infusion of either saline or DCA (1mM in LAD blood) under aerobic conditions, and after 37 min of ischemia. Pyruvate dehydrogenase activity was measured with and without maximal activation by swine PDH phosphatase. Malonyl CoA and acetyl CoA were measured after 40 min of LAD ischemia in myocardium from the ischemic DCA- or saline-treated LAD bed, and the non-ischemic untreated left circumflex coronary artery (CFX) perfusion bed. Net glucose, lactate and free fatty acid (FFA) uptakes were measured across the LAD perfusion bed throughout the study. Dichloroacetate treatment increased the amount of active dephosphorylated PDH to 88% of the total activity under aerobic conditions, compared to 55% with saline ( P<0.01). Ischemia did not signifiacantly change PDH activation state in either group. Acetyl CoA and malonyl CoA contents were significantly elevated in ischemic DCA-treated myocardium compared to saline-treated ischemic myocardium. Dichloroacetate treatment significantly lowered rates of myocardial FFA uptake under both aerobic and ischemic conditions, but did not effect glucase uptake ofr lactate exchange. Free fatty acid uptake was negatively correlated to malonyl CoA levels ( r=−0.68) during ischemia. It is proposed that the inhibition of FFA uptake observed with DCA in ischemic myocardium is due to malonyl CoA inhibition of carnitine palmitoyl transferase I.