Palmitate Oxidation Rates Research Articles

Acute regulation of fatty acid oxidation and amp-activated protein kinase in human umbilical vein endothelial cells.

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.

Glucose metabolism in perfused mouse hearts overexpressing human GLUT-4 glucose transporter.

Glucose and fatty acid metabolism was assessed in isolated working hearts from control C57BL/KsJ-m+/+db mice and transgenic mice overexpressing the human GLUT-4 glucose transporter (db/+-hGLUT-4). Heart rate, coronary flow, cardiac output, and cardiac power did not differ between control hearts and hearts overexpressing GLUT-4. Hearts overexpressing GLUT-4 had significantly higher rates of glucose uptake and glycolysis and higher levels of glycogen after perfusion than control hearts, but rates of glucose and palmitate oxidation were not different. Insulin (1 mU/ml) significantly increased glycogen levels in both groups. Insulin increased glycolysis in control hearts but not in GLUT-4 hearts, whereas glucose oxidation was increased by insulin in both groups. Therefore, GLUT-4 overexpression increases glycolysis, but not glucose oxidation, in the heart. Although control hearts responded to insulin with increased rates of glycolysis, the enhanced entry of glucose in the GLUT-4 hearts was already sufficient to maximally activate glycolysis under basal conditions such that insulin could not further stimulate the glycolytic rate.

Dietary L-carnitine suppresses mitochondrial branched-chain keto acid dehydrogenase activity and enhances protein accretion and carcass characteristics of swine.

A trial was conducted to biochemically explain the decreased lipid deposition and increased protein accretion observed in pigs fed carnitine. Our hypothesis was that an increase in the ratio of acetyl CoA:CoA-SH produced by stimulation of fatty acid oxidation by supplemental L-carnitine may decrease branched-chain alpha-keto acid dehydrogenase activity and increase pyruvate carboxylase activity. Such changes could reduce oxidative loss of branched-chain amino acids and provide more carbons for amino acid biosynthesis. Yorkshire gilts (n = 36; 12 per treatment) were fed a control diet or diets containing either 50 or 125 ppm of added L-carnitine during growth from 56 to 120 kg. After slaughter, the semitendinosus muscle and liver were collected for isolation of mitochondria and hepatocytes. Increasing dietary L-carnitine did not influence growth performance (P > 0.10) but linearly decreased (P < 0.05) 10th rib backfat thickness and increased (linear, P < 0.05) percentages of lean and muscle. The rates of [1-(14)G]palmitate oxidation in isolated hepatocytes and isolated mitochondria, and incorporation of [35S]methionine into the acid insoluble fraction of isolated hepatocytes were increased (linear, P < 0.01) in pigs fed L-carnitine. Flux through branched-chain alpha-keto acid dehydrogenase linearly decreased (P < 0.01) in isolated liver and muscle mitochondria with increasing dietary carnitine. Flux through pyruvate carboxylase was increased (linear, P < 0.01) in isolated mitochondria from liver of pigs fed carnitine, and assays with particle-free extracts indicated that the amount of mitochondrial pyruvate carboxylase was tripled by feeding carnitine (linear, P < 0.01). The association of increased protein accretion and reduced backfat thickness with greater rates of palmitate oxidation, more rapid flux through pyruvate carboxylase, and reduced flux through branched-chain alpha-keto acid dehydrogenase suggests pigs fed carnitine are more able to use fat for energy, divert carbon toward synthesis of amino acids, and spare branched-chain amino acids for protein synthesis.

Insulin-like effects of a physiologic concentration of carnitine on cardiac metabolism.

Pharmacologic (millimolar) levels of carnitine have been reported to increase myocardial glucose oxidation, but whether physiologically relevant concentrations of carnitine affect cardiac metabolism is not known. We employed the isolated, perfused rat heart to compare the effects of physiologic levels of carnitine (50 microM) and insulin (75 mU/l [0.5 nM]) on the following metabolic processes: (1) glycolysis (release of 3H2O from 5-3H-glucose); (2) oxidation of glucose and pyruvate (production of 14CO2 from U-14C-glucose, 1-14C-glucose, 3,4-14C-glucose, 1-14C-pyruvate, and 2-14C-pyruvate); and (3) oxidation of palmitate (release of 3H2O from 9,10-3H-palmitate). We found that addition of carnitine (50 microM) to a perfusate containing both glucose (10 mM) and palmitate (0.5 mM) stimulated glycolytic flux by 20%, nearly doubled the rate of glucose oxidation, and inhibited palmitate oxidation by 20%. These actions of carnitine were uniformly similar to those of insulin. When carnitine and insulin were administered together, their effects on the oxidation of glucose and palmitate, but not on glycolysis, were additive. When pyruvate (1 mM) was substituted for glucose, neither carnitine nor insulin influenced the rate of oxidation of pyruvate or palmitate. In combination, however, carnitine and insulin sharply suppressed pyruvate oxidation (75%) and doubled the rate of palmitate oxidation. None of the responses to carnitine or insulin was affected by varying the isotopic labeling of glucose or pyruvate. The results show that carnitine, at normal blood levels, exerts insulin-like effects on myocardial fuel utilization. They also suggest that plasma carnitine in vivo may interact with insulin both additively and permissively on the metabolism of carbohydrates and fatty acids.

Insulin stimulation of glucose uptake fails to decrease palmitate oxidation in muscle if AMPK is activated.

Fatty acid oxidation in muscle has been reported to be diminished when insulin and glucose levels are elevated. This study was designed to determine whether activation of AMP-activated protein kinase (AMPK) will prevent inhibitory effects of insulin and glucose on the rate of fatty acid oxidation. Rat hindlimbs were perfused with medium containing 0, 0.3, or 60 nM insulin with or without 2 mM 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR). Glucose uptake was stimulated four- to fivefold by inclusion of insulin in the medium. Insulin attenuated the increase in AMPK caused by AICAR both in perfused hindlimbs and in isolated epitrochlearis muscles. The activation constant for citrate activation of acetyl-CoA carboxylase (ACC) was significantly increased in response to AICAR, and the increase was slightly attenuated if insulin was present in the perfusion medium. Insulin stimulated an increase in malonyl-CoA content of the muscles in the absence of AICAR. Malonyl-CoA was decreased to approximately the same value in AICAR-perfused muscle, regardless of insulin concentration. Muscle glucose 6-phosphate and citrate were significantly increased in response to AICAR and insulin. The rate of palmitate oxidation tended to decrease in response to insulin and in the absence of AICAR. AICAR increased palmitate oxidation to approximately the same level regardless of the insulin concentration or the rate of glucose uptake into the muscle. The rate of palmitate oxidation showed a curvilinear relationship as a function of muscle malonyl-CoA content, with half-maximal inhibition at approximately 0.6 nmol/g. We conclude that AMPK activation can prevent high rates of glucose uptake and glycolytic flux from inhibiting palmitate oxidation in predominantly fast-twitch muscle under these conditions.

Deactivation of peroxisome proliferator-activated receptor-alpha during cardiac hypertrophic growth.

We sought to delineate the molecular regulatory events involved in the energy substrate preference switch from fatty acids to glucose during cardiac hypertrophic growth. alpha(1)-adrenergic agonist-induced hypertrophy of cardiac myocytes in culture resulted in a significant decrease in palmitate oxidation rates and a reduction in the expression of the gene encoding muscle carnitine palmitoyltransferase I (M-CPT I), an enzyme involved in mitochondrial fatty acid uptake. Cardiac myocyte transfection studies demonstrated that M-CPT I promoter activity is repressed during cardiac myocyte hypertrophic growth, an effect that mapped to a peroxisome proliferator-activated receptor-alpha (PPARalpha) response element. Ventricular pressure overload studies in mice, together with PPARalpha overexpression studies in cardiac myocytes, demonstrated that, during hypertrophic growth, cardiac PPARalpha gene expression falls and its activity is altered at the posttranscriptional level via the extracellular signal-regulated kinase mitogen-activated protein kinase pathway. Hypertrophied myocytes exhibited reduced capacity for cellular lipid homeostasis, as evidenced by intracellular fat accumulation in response to oleate loading. These results indicate that during cardiac hypertrophic growth, PPARalpha is deactivated at several levels, leading to diminished capacity for myocardial lipid and energy homeostasis.

Dietary coconut oil affects more lipoprotein lipase activity than the mitochondria oxidative capacities in muscles of preruminant calves

The presence of coconut oil in a milk replacer stimulates the growth rate of calves, suggesting a better oxidation of fatty acid in muscles. Because dietary fatty acid composition influences carnitine palmitoyltransferase I (CPT I) activity in rat muscles, this study was designed to examine the effects of a milk replacer containing either tallow (TA) or coconut oil (CO) on fatty acid utilization and oxidation and on the characteristics of intermyofibrillar (IM) and subsarcolemmal (SS) mitochondria in the heart and skeletal muscles of preruminant calves. Feeding CO did not affect palmitate oxidation rate by whole homogenates, but induced higher palmitate oxidation by IM mitochondria (+37%, P < 0.05). CPT I activity did not significantly differ between the two groups of calves. Heart and longissimus thoracis muscle of calves fed CO had higher lipoprotein lipase activity (+27% and 58%, respectively; P < 0.05) but showed no differences in fatty acid binding protein content or activity of oxidative enzymes. Whatever the muscle and the diet, IM mitochondria had higher respiration rates and enzyme activities than those of SS mitochondria ( P < 0.05). Furthermore, CPT I activity of the heart was 28-fold less sensitive to malonyl-coenzyme A inhibition in IM mitochondria than in SS mitochondria. In conclusion, dietary CO marginally affected the activity of the two mitochondrial populations and the oxidative activity of muscles in the preruminant calf. In addition, this study showed that differences between IM and SS mitochondria in the heart and muscles were higher in calves than in other species studied so far.

The antianginal drug trimetazidine shifts cardiac energy metabolism from fatty acid oxidation to glucose oxidation by inhibiting mitochondrial long-chain 3-ketoacyl coenzyme A thiolase.

Trimetazidine is a clinically effective antianginal agent that has no negative inotropic or vasodilator properties. Although it is thought to have direct cytoprotective actions on the myocardium, the mechanism(s) by which this occurs is as yet undefined. In this study, we determined what effects trimetazidine has on both fatty acid and glucose metabolism in isolated working rat hearts and on the activities of various enzymes involved in fatty acid oxidation. Hearts were perfused with Krebs-Henseleit solution containing 100 microU/mL insulin, 3% albumin, 5 mmol/L glucose, and fatty acids of different chain lengths. Both glucose and fatty acids were appropriately radiolabeled with either (3)H or (14)C for measurement of glycolysis, glucose oxidation, and fatty acid oxidation. Trimetazidine had no effect on myocardial oxygen consumption or cardiac work under any aerobic perfusion condition used. In hearts perfused with 5 mmol/L glucose and 0.4 mmol/L palmitate, trimetazidine decreased the rate of palmitate oxidation from 488+/-24 to 408+/-15 nmol x g dry weight(-1) x minute(-1) (P<0.05), whereas it increased rates of glucose oxidation from 1889+/-119 to 2378+/-166 nmol x g dry weight(-1) x minute(-1) (P<0.05). In hearts subjected to low-flow ischemia, trimetazidine resulted in a 210% increase in glucose oxidation rates. In both aerobic and ischemic hearts, glycolytic rates were unaltered by trimetazidine. The effects of trimetazidine on glucose oxidation were accompanied by a 37% increase in the active form of pyruvate dehydrogenase, the rate-limiting enzyme for glucose oxidation. No effect of trimetazidine was observed on glycolysis, glucose oxidation, fatty acid oxidation, or active pyruvate dehydrogenase when palmitate was substituted with 0.8 mmol/L octanoate or 1.6 mmol/L butyrate, suggesting that trimetazidine directly inhibits long-chain fatty acid oxidation. This reduction in fatty acid oxidation was accompanied by a significant decrease in the activity of the long-chain isoform of the last enzyme involved in fatty acid beta-oxidation, 3-ketoacyl coenzyme A (CoA) thiolase activity (IC(50) of 75 nmol/L). In contrast, concentrations of trimetazidine in excess of 10 and 100 micromol/L were needed to inhibit the medium- and short-chain forms of 3-ketoacyl CoA thiolase, respectively. Previous studies have shown that inhibition of fatty acid oxidation and stimulation of glucose oxidation can protect the ischemic heart. Therefore, our data suggest that the antianginal effects of trimetazidine may occur because of an inhibition of long-chain 3-ketoacyl CoA thiolase activity, which results in a reduction in fatty acid oxidation and a stimulation of glucose oxidation.

The coactivator PGC-1 cooperates with peroxisome proliferator-activated receptor alpha in transcriptional control of nuclear genes encoding mitochondrial fatty acid oxidation enzymes.

Peroxisome proliferator-activated receptor alpha (PPARalpha) plays a key role in the transcriptional control of genes encoding mitochondrial fatty acid beta-oxidation (FAO) enzymes. In this study we sought to determine whether the recently identified PPAR gamma coactivator 1 (PGC-1) is capable of coactivating PPARalpha in the transcriptional control of genes encoding FAO enzymes. Mammalian cell cotransfection experiments demonstrated that PGC-1 enhanced PPARalpha-mediated transcriptional activation of reporter plasmids containing PPARalpha target elements. PGC-1 also enhanced the transactivation activity of a PPARalpha-Gal4 DNA binding domain fusion protein. Retroviral vector-mediated expression studies performed in 3T3-L1 cells demonstrated that PPARalpha and PGC-1 cooperatively induced the expression of PPARalpha target genes and increased cellular palmitate oxidation rates. Glutathione S-transferase "pulldown" studies revealed that in contrast to the previously reported ligand-independent interaction with PPARgamma, PGC-1 binds PPARalpha in a ligand-influenced manner. Protein-protein interaction studies and mammalian cell hybrid experiments demonstrated that the PGC-1-PPARalpha interaction involves an LXXLL domain in PGC-1 and the PPARalpha AF2 region, consistent with the observed ligand influence. Last, the PGC-1 transactivation domain was mapped to within the NH(2)-terminal 120 amino acids of the PGC-1 molecule, a region distinct from the PPARalpha interacting domains. These results identify PGC-1 as a coactivator of PPARalpha in the transcriptional control of mitochondrial FAO capacity, define separable PPARalpha interaction and transactivation domains within the PGC-1 molecule, and demonstrate that certain features of the PPARalpha-PGC-1 interaction are distinct from that of PPARgamma-PGC-1.

The mechanism of inhibition of β-oxidation by aspirin metabolites in skin fibroblasts from Reye’s syndrome patients and controls

The effects of aspirin metabolites on β-oxidation were studied in skin fibroblasts from eight typical Reye’s syndrome (RS) patients and controls. RS patients’ cells did not differ from controls in rates of palmitate oxidation or in the three component activities of the mitochondrial trifunctional enzyme (MTE), indicating no inherited β-oxidation defect. Aspirin metabolites salicylate, hydroxyhippurate and gentisate, but not aspirin, directly inhibited palmitate oxidation in control and RS cells. RS cells were significantly more sensitive to inhibition than controls at 0.5 to 5 mM salicylate. Inhibition was concentration-dependent and reversible. Inhibition did not occur in fibroblasts lacking activity of the long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) activity of MTE. Salicylate was therefore inhibiting β-oxidation at this step. Hydroxyhippurate and salicylate reversibly inhibited HAD activities in extracts of control and RS cells. Studies with pure short-chain HAD and LCHAD (MTE) showed hydroxyhippurate and salicylate were competitive inhibitors of the former but mixed (not competitive) inhibitors of the latter. Both compounds inhibited the combined, three-step, MTE reaction measured in the physiological direction. We conclude that (1) salicylate and hydroxyhippurate decrease β-oxidation in intact cells by reversible inhibition of LCHAD activity of the MTE, and (2) β-oxidation in RS cells is inherently more sensitive to inhibition by low concentrations of salicylate than controls.

Hyperthyroidism facilitates cardiac fatty acid oxidation through altered regulation of cardiac carnitine palmitoyltransferase: studies in vivo and with cardiac myocytes.

The aim was to establish whether increased cardiac fatty acid oxidation in hyperthyroidism is due to direct alterations in cardiac metabolism which favour fatty acid oxidation and/or whether normal regulatory links between changes in glucose supply and fatty acid oxidation are dysfunctional. Euthyroid rats were sampled in the absorptive state or after 48 h starvation. Rats were rendered hyperthyroid by injection of tri-iodothyronine (1000 microg/kg body wt. per day; 3 days). We evaluated the regulatory significance of direct effects of hyperthyroidism by measuring rates of palmitate oxidation in the absence or presence of glucose using cardiac myocytes. The results were examined in relation to the activity/regulatory characteristics of cardiac carnitine palmitoyltransferase (CPT) estimated by measuring rates of [3H]palmitoylcarnitine formation from [3H]carnitine and palmitoyl-CoA by isolated mitochondria. To define the involvement of other hormones, we examined whether hyperthyroidism altered basal or agonist-stimulated cardiac cAMP concentrations in cardiac myocytes and whether the effects of hyperthyroidism could be reversed by 24 h exposure to insulin infused subcutaneously (2 i. u. per day; Alzet osmotic pumps). Rates of 14C-palmitate oxidation (to 14CO2) by cardiac myocytes were significantly increased (1.6 fold; P< 0.05) by hyperthyroidism, whereas the percentage suppression of palmitate oxidation by glucose was greatly diminished. Cardiac CPT activities in mitochondria from hyperthyroid rats were 2-fold higher and the susceptibility of cardiac CPT activity to inhibition by malonyl-CoA was decreased. These effects were not mimicked by 48 h starvation. The decreased susceptibility of cardiac CPT activities to malonyl-CoA inhibition in hyperthyroid rats was normalised by 24 h exposure to elevated insulin concentration. Acute insulin addition did not influence the response to glucose in cardiac myocytes from euthyroid or hyperthyroid rats and basal and agonist-stimulated cAMP concentrations were unaffected by hyperthyroidism in vivo. The data provide insight into possible mechanisms by which hyperthyroidism facilitates fatty acid oxidation by the myocardium, identifying changes in cardiac CPT activity and malonyl-CoA sensitivity that would be predicted to render cardiac fatty acid oxidation less sensitive to external factors influencing malonyl-CoA content, and thereby to favour fatty acid oxidation. The increased CPT activity observed in response to hyperthyroidism may be a consequence of an impaired action of insulin but occurs through a cAMP-independent mechanism.

Influence of malonyl-CoA and palmitate concentration on rate of palmitate oxidation in rat muscle

5-Aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside (AICAR) is taken up by perfused skeletal muscle and phosphorylated to form 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuraosyl-5'-monopho sph ate (analog of 5'-AMP) with consequent activation of AMP-activated protein kinase, phosphorylation of acetyl-CoA carboxylase, decrease in malonyl-CoA, and increase in fatty acid oxidation. This study was designed to determine the effect of increasing levels of palmitate on the rate of fatty acid oxidation. Malonyl-CoA concentration was manipulated with AICAR at different palmitate concentrations. Rat hindlimbs were perfused with Krebs-Henseleit bicarbonate containing 4% bovine serum albumin, washed bovine red cells, 200 microU/ml insulin, 10 mM glucose, and different concentrations of palmitate (0. 1-1.0 mM) without or with AICAR (2.0 mM). Perfusion with medium containing AICAR was found to activate AMP-activated protein kinase in skeletal muscle, inactivate acetyl-CoA carboxylase, and decrease malonyl-CoA at all concentrations of palmitate. The rate of palmitate oxidation increased as a function of palmitate concentration in both the presence and absence of AICAR but was always higher in the presence of AICAR. These results provide additional evidence that malonyl-CoA is an important regulator of the rate of fatty acid oxidation at palmitate concentrations in the physiological range.

Muscle contraction increases palmitate esterification and oxidation and triacylglycerol oxidation.

We examined the oxidation and esterification of palmitate and the hydrolysis and oxidation of intramuscular lipids in isolated soleus muscles at rest and during tetanic contractions (2-40 tetani/min). Muscles were pulsed with [14C]palmitate to prelabel all intramuscular lipid pools. Muscles remained at rest or were then stimulated to contract at 2, 8, 20, or 40 tetani/min (30 min) in the presence of [3H]palmitate. Palmitate oxidation was increased 412% at 2 tetani/min (P < 0.05) and 880% at 8 tetani/min (P < 0.05). During contraction there was an absolute increase in esterification of palmitate to triacylglycerol in proportion with the increasing rate of palmitate oxidation. Intramuscular lipid oxidation provided approximately 77% of the total muscle energy compared with approximately 3% provided by exogenous palmitate under all conditions, with carbohydrate sources (glycogen and glucose) providing approximately 20% of the total energy. Thus, during muscle contraction, the oxidation rates of both exogenous and intramuscular lipids are increased in proportion to each other, while concomitantly palmitate is esterified in proportion to its oxidation.

Contribution of mitochondria and peroxisomes to palmitate oxidation in rat and bovine tissues

Total and peroxisomal palmitate oxidation capacities and mitochondrial enzyme activities were compared in tissues from growing rats, preruminant calves and 15-month-old bulls. Total palmitate oxidation rates were 1.9–5.2-fold higher in rat than in bovine tissues and 1.7-fold higher in the heart and muscles from calves than from growing bulls. The peroxisomal contribution to palmitate oxidation was similar between rats and bovines (i.e. calves and bulls) in liver (35–51%), heart (26%) but not in muscles (14±3% in rats vs 33±4.5% in bovines, P<0.05). Mitochondrial enzyme activities were 1.8–4.8-fold higher in rat than in bovine tissues but the citrate synthase to cytochrome- c oxidase ratio was the highest in the liver (17–38), intermediate in the heart and muscles from calves and rats (6–10) and the lowest in heart and muscles from bulls (2–3, P<0.05). In all tissues and animal groups, palmitate oxidation rates were similar per unit cytochrome- c oxidase activity, but not always per unit citrate synthase activity. Therefore, differences in mitochondrial contents (as between rats and bovines) or in mitochondrial characteristics (as between liver and muscles) relate to the differences in palmitate oxidation capacity.

Competition Between Lactate and Fatty Acids as Sources of ATP in the Isolated Working Rat Heart

Fatty acid oxidation is generally considered the major source of energy in the heart, although lactate oxidation can be a major contributor to ATP production, depending on the concentration and availability of other competing substrates. In this study, isolated working rat hearts were used to directly determine the relationship between lactate and fatty acid oxidation to overall ATP production from exogenous sources. A range of lactate from 0.5 to 8.0 mmlactate was added to hearts perfused with buffer containing 5.5 mmglucose, and either 0.4 or 1.2 mmpalmitate over a 100 min period. Rates of glycolysis, glucose oxidation, lactate oxidation, and palmitate oxidation were determined. In the presence of 0.5 mmlactate and 0.4 mmpalmitate, lactate oxidation provided 17% of the ATP production and palmitate oxidation provided 68%, with the remainder coming from glucose oxidation and glycolysis. In the presence of 0.4 mmpalmitate, an increase in lactate from 0.5 to 8.0 mmincreased the steady state rates of lactate oxidation from 1239±236 to 5247±940 nmol/min/g dry weight, respectively. The contribution of lactate oxidation to total ATP production increased to 37%, with palmitate oxidation now contributing only 52% of the total ATP produced. At 8.0 mmlactate and 1.2 mmpalmitate, lactate oxidation contributed 13% of the total ATP production, while palmitate oxidation contributed 81%. This data demonstrates that under near physiological conditions of lactate (0.5 mm) and fatty acids (0.4 mm), the preferred energy substrate of the heart remains to be fatty acids, and that only at high levels of lactate, such as can be observed during exercise or severe stress, does lactate oxidation become a significant source of ATP production.

Molecular mechanisms underlying the long-term impact of dietary fat to increase cardiac pyruvate dehydrogenase kinase: regulation by insulin, cyclic AMP and pyruvate.

Previous studies have demonstrated that pyruvate dehydrogenase kinase (PDHK) activity in extracts of rat cardiac mitochondria is increased @two-fold by providing a high-fat diet for 28 days. The present study sought to establish the factor(s) that might underlie the response of cardiac PDHK to the provision of a high-fat diet. ELISA assays of PDHKII, conducted over a range of PDHK activities, demonstrated that the increase in cardiac PDHK activity was not due to an increase in mitochondrial immunoreactive PDHKII concentration. The pyruvate concentration giving 50% active PDHC (PDHa) in mitochondria incubated with respiratory substrates was unaffected by high-fat feeding, demonstrating a dissociation between increased PDHK activity and altered sensitivity of PDHK to suppression by pyruvate. In cardiac myocytes cultured (25 h) with n-octanoate (1 mm) plus dibutyryl cAMP (50 microM), insulin at 12.5 microU/ml, 25 microU/ml and 75 microU/ml, suppressed PDHK activities in cells prepared from control rats, but insulin at concentrations <100 microU/ml failed to suppress PDHK activities in cardiac myocytes prepared from high-fat-fed rats. In vivo, cardiac insulin sensitivity (assessed by euglycaemic hyperinsulinaemic clamp in combination with 2-[3H] deoxyglucose administration) was suppressed after high-fat feeding. A sustained (24 h) two- to four-fold elevation in plasma insulin concentration (achieved by insulin infusion via osmotic pumps) did not affect PDHK activity in hearts of control rats. In contrast, PDHK activity in hearts of high-fat-fed rats was suppressed to values not significantly different from (insulin-infused) control rats. Basal and agonist-stimulated cAMP concentrations were unaffected by high-fat-feeding or insulin. Furthermore, rates of palmitate oxidation (to CO2) in cardiac myocytes (in the absence or presence of insulin or adrenergic agonists) were not statistically significantly affected by high-fat-feeding. The results indicate that an impaired action of insulin to suppress PDHK participates in the mechanism by which increased PDHK activity is achieved in response to high-fat feeding, but insulin does not act through decreasing cAMP concentrations or suppressing fatty acid oxidation.

Control of oxidative metabolism in volume-overloaded rat hearts: effect of propionyl-L-carnitine.

The objective of the present work was the assessment of metabolic events responsible for the improvement of hemodynamic function of volume-overloaded hearts from rats receiving propionyl-L-carnitine. A severe cardiac hypertrophy was induced in 2-mo-old rats by surgical opening of an aortocaval communication. Three months later, during in vitro perfusions with 1.2 mM palmitate, 11 mM glucose, and 10 IU/l insulin, the mechanical performance and overall energy turnover (myocardial O2 consumption) of hypertrophied rat hearts were significantly decreased under conditions of moderate and high workloads. These changes in cardiac energetics paralleled the decrease in total tissue carnitine content and alterations in exogenous palmitate oxidation. The oxidative utilization of glucose was also slightly depressed in volume-overloaded hearts while steady-state glycolysis rates increased, especially in hearts subjected to high mechanical loads. This slowing of metabolic pathways involved in acetyl-CoA generation resulted in decreased NADH availability and in an apparent substrate limitation of oxidative phosphorylation suggested by a failure of cytosolic unbound ADP to drive respiration. Long-term administration of propionyl-L-carnitine normalized the degree of reduction of mitochondrial pyridine nucleotides and improved the kinetics of mitochondrial ATP production in volume-overloaded hearts. The resulting acceleration of energy turnover was essentially related to improved oxidative utilization of glucose, but steady-state palmitate oxidation rates also increased in severely hypertrophied hearts. This concomitant acceleration of glucose and palmitate oxidation may be related to the particular experimental conditions (high exogenous palmitate concentrations, elevated workloads) used in this study. We assume that the increase in intracellular carnitine, together with a stimulation of acetyl-CoA demands related to high workloads, creates conditions that are compatible with the simultaneous relief of pyruvate dehydrogenase and carnitine palmitoyltransferase I. The resulting increase in the rate of steady-state ATP production improves, in turn, the mechanical activity of volume-overloaded hearts.

Physiological role of D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein.

The second and third reactions of the peroxisomal beta-oxidation spiral are thought to be catalyzed by enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase bifunctional protein (L-bifunctional protein). Recently, we found the presence of D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein (D-bifunctional protein) in mammalian peroxisomes. Therefore, we studied the physiological role of the D-bifunctional protein. The contents of the L- and D-bifunctional proteins were about 0.01 and 0.5 microg/mg protein, respectively, in cultured human skin fibroblasts. The activity of conversion of hexadecenoyl-CoA to 3-ketopalmitoyl-CoA by the D-bifunctional protein was estimated to be about 0.5 milliunit/mg of fibroblast protein. This value was about 100-fold that of the L-bifunctional protein in the fibroblasts. From comparison of the activities of the bifunctional proteins with the rate of palmitate oxidation and the activities of acyl-CoA oxidase and 3-ketoacyl-CoA thiolase, it is proposed that the D-bifunctional protein plays a major role in the peroxisomal oxidation of palmitate in the fibroblasts. The contents of both the L- and D-bifunctional proteins in liver were about 2.5 microg/mg protein. Therefore, it is suggested that the D-bifunctional protein also plays a significant role in human liver peroxisomal fatty acid oxidation. Actions of the bifunctional proteins on enoyl forms of other acyl-CoA derivatives were examined. The D-bifunctional protein but not the L-bifunctional protein reacted with 2-methylhexadecenoyl-CoA and 3alpha,7alpha,12alpha-trihydroxy-5beta-cholest-2 4-enoyl-CoA. We propose that, among the reactions of the distinct group of carboxylates oxidized specifically in peroxisomes, oxidation of 2-methyl-branched fatty acids and side-chain shortening of cholesterol for bile acid formation are catalyzed by the D-bifunctional protein, but not the L-bifunctional protein.

Cardiac efficiency is improved after ischemia by altering both the source and fate of protons.

Cardiac efficiency is decreased in hearts after severe ischemia. We determined whether reducing the production of H+ from glucose metabolism or inhibiting the clearance of H+ via Na(+)-H+ exchange could increase cardiac efficiency during reperfusion. This was achieved using dichloroacetate (DCA) to stimulate glucose oxidation and 5-(N,N-dimethyl)-amiloride (DMA) to inhibit Na(+)-H+ exchange, respectively. Isolated working rat hearts were subjected to 30 minutes of global ischemia and 60 minutes of reperfusion. Glycolysis and oxidation rates of glucose, lactate, and palmitate were measured. Recovery of cardiac work, O2 consumption (MVO2), and rates of acetyl-coenzyme A and ATP production during reperfusion were determined. After ischemia, cardiac work recovered to 35 +/- 5% of preischemic values in control hearts (n = 23), although MVO2, tricarboxylic acid (TCA) cycle activity, and ATP production from glycolysis and oxidative metabolism rapidly recovered to preischemic levels. This decrease in cardiac efficiency was accompanied by a substantial production of H+ from glucose metabolism DCA caused a 2.2-fold increase in glucose oxidation, a 46 +/- 17% decrease in H+ production, a 1.6-fold increase in cardiac efficiency, and a 2.0-fold increase in cardiac work during reperfusion (n = 17). Inhibition of Na(+)-H+ exchange with DMA did not alter TCA cycle activity and ATP production rates but did result in a 1.8-fold increase in cardiac efficiency and a 1.7-fold increase in cardiac work (n = 12). These data show that cardiac efficiency and the contractile function after ischemia can be improved by either reducing the rate of H+ production from glucose metabolism during reperfusion or inhibiting the clearance of H+ via Na(+)-H+ exchange. Our data suggest that an increased requirement for ATP to restore ischemia-reperfusion-induced alterations in ion homeostasis contributes to the decrease in cardiac efficiency and contractile function after ischemia.

Recovery of glycolysis and oxidative metabolism during postischemic reperfusion of hypertrophied rat hearts.

We investigated the source and extent of recovery of ATP production during postischemic reperfusion of isolated working hearts from abdominal aortic-banded rats. Rates of glycolysis, glucose oxidation, lactate oxidation, and palmitate oxidation were measured in hypertrophied and control hearts [perfused with (in mM) 11 glucose, 0.5 lactate, and 1.2 palmitate] during and after 30 min of no-flow ischemia. In the initial aerobic period glycolytic rates were 1.87-fold higher in hypertrophied hearts compared with control hearts (P < 0.05), with rates of carbohydrate and palmitate oxidation being similar. During reperfusion, hypertrophied hearts recovered 40% of preischemic function compared with 71% in control hearts. Rates of glycolysis during reperfusion of hypertrophied hearts remained accelerated compared with control hearts (2.01-fold higher, P < 0.05), whereas oxidative metabolism returned to preischemic values in both groups. The efficiency of converting ATP production into mechanical work decreased to 29% of preischemic values in hypertrophied hearts during the postischemic reperfusion compared with a decrease to only 59% of preischemic values in control hearts. This suggests that the recovery of glycolysis and oxidative metabolism in the hypertrophied heart during postischemic reperfusion is not impaired, but rather the efficiency of converting ATP produced into mechanical function decreases.