Energy Substrate Preference Research Articles

Regulation of Cardiac Energy Metabolism in Newborn

Energy in the form of ATP is supplied from the oxidation of fatty acids and glucose in the adult heart in most species. In the fetal heart, carbohydrates, primarily glucose and lactate, are the preferred sources for ATP production. As the newborn matures the contribution of fatty acid oxidation to overall energy production increases and becomes the dominant substrate for the adult heart. The mechanisms responsible for this switch in energy substrate preference in the heart are complicated to identify due to slight differences between species and differences in techniques that are utilized. Nevertheless, our current knowledge suggests that the switch in energy substrate preference occurs due to a combination of events. During pregnancy, the fetus receives a constant supply of nutrients that is rich carbohydrates and poor in fatty acids in many species. Immediately after birth, the newborn is fed with milk that is high in fat and low in carbohydrates. The hormonal environment is also different between the fetal and the newborn. Moreover, direct subcellular changes occur in the newborn period that play a major role in the adaptation of the newborn heart to extrauterin life. The newborn period is unique and provides a very useful model to examine not only the metabolic changes, but also the effects of hormonal changes on the heart. A better understanding of developmental physiology and metabolism is also very important to approach certain disorders in energy substrate metabolism.

Decrease in fatty acid oxidation increases tolerance of the ageing heart to ischaemic injury

Ageing is associated with reduced tolerance to myocardial infarction. Alterations in mitochondrial oxidative metabolism are important determinants in the contractile recovery of the adult heart after ischaemia. However, the profile of substrate metabolism during reperfusion in the ageing heart is unknown. The aim of this study was to investigate energy substrate preference and functional recovery following low flow ischaemia in the ageing heart. Hearts from 6 and 24 mo male Wistar rats were perfused in an isovolumic mode with buffer containing 3% albumin and a physiological mixture of substrates including 0.3mM [U-13C] palmitate, 1mM [3-13C] lactate and 5mM glucose. Cardiac work was increased by dobutamine infusion for 20 min (2.8±0.1 μg/min/g heart weight) followed by 30 min low flow ischaemia and 30 min reperfusion. Ischaemia caused similar reductions in left ventricular developed pressure and cardiac work in adult and senescent hearts. On reperfusion, 24 mo hearts demonstrated improved functional recovery relative to 6-mo hearts. This was associated with a significant decline in palmitate oxidation and increase in lactate oxidation. Thus, the ageing heart has increased tolerance to severe ischaemia, which may be attributed to the reduction in fatty acid oxidation. JS is funded by a PhD scholarship from the University of Hull and Academic Dept of Cardiology.

Malonyl coenzyme a decarboxylase inhibition protects the ischemic heart by inhibiting fatty acid oxidation and stimulating glucose oxidation.

Abnormally high rates of fatty acid oxidation and low rates of glucose oxidation are important contributors to the severity of ischemic heart disease. Malonyl coenzyme A (CoA) regulates fatty acid oxidation by inhibiting mitochondrial uptake of fatty acids. Malonyl CoA decarboxylase (MCD) is involved in the decarboxylation of malonyl CoA to acetyl CoA. Therefore, inhibition of MCD may decrease fatty acid oxidation and protect the ischemic heart, secondary to increasing malonyl CoA levels. Ex vivo working rat hearts aerobically perfused in the presence of newly developed MCD inhibitors showed an increase in malonyl CoA levels, which was accompanied by both a significant decrease in fatty acid oxidation rates and an increase in glucose oxidation rates compared with controls. Using a model of demand-induced ischemia in pigs, MCD inhibition significantly increased glucose oxidation rates and reduced lactate production compared with vehicle-treated hearts, which was accompanied by a significant increase in cardiac work compared with controls. In a more severe rat heart global ischemia/reperfusion model, glucose oxidation was significantly increased and cardiac function was significantly improved during reperfusion in hearts treated with the MCD inhibitor compared with controls. Together, our data show that MCD inhibitors, which increase myocardial malonyl CoA levels, decrease fatty acid oxidation and accelerate glucose oxidation in both ex vivo rat hearts and in vivo pig hearts. This switch in energy substrate preference improves cardiac function during and after ischemia, suggesting that pharmacological inhibition of MCD may be a novel approach to treating ischemic heart disease.

Effects of amino acid supplementation on left ventricular remodeling in patients with chronic heart failure with decreased systolic function and diabetes mellitus: rationale and design of a magnetic resonance imaging study

A considerable number of patients with reduced systolic function caused by primary or ischemic cardiomyopathy have viable and noncontractile myocardium. This may be related to numerous and perhaps overlapping factors, such as chronic ischemia (stunning/hibernation), neurohormonal abnormalities, oxidative stress, metabolic imbalances, and/or nutritional depletion. Changes in myocardial substrate utilization have adverse effects on the metabolism of the viable but noncontractile myocardium. Shifting the energy substrate preference away from fatty acids and replenishing the tricarboxylic acid cycle components via amino acids rather than via fatty acids would increase adenosine triphosphate production, with positive effects on cellular metabolism. A proposed study design is described and will be piloted through the Effects of Diatrofen on Myocardial Function in Patients with Chronic Heart Failure trial (D-CHF), an evaluation of an oral amino acid supplementation treatment in outpatients with heart failure.

Effects of amino acid supplementation on left ventricular remodeling in patients with chronic heart failure with decreased systolic function and diabetes mellitus: rationale and design of a magnetic resonance imaging study

A considerable number of patients with reduced systolic function caused by primary or ischemic cardiomyopathy have viable and noncontractile myocardium. This may be related to numerous and perhaps overlapping factors, such as chronic ischemia (stunning/hibernation), neurohormonal abnormalities, oxidative stress, metabolic imbalances, and/or nutritional depletion. Changes in myocardial substrate utilization have adverse effects on the metabolism of the viable but noncontractile myocardium. Shifting the energy substrate preference away from fatty acids and replenishing the tricarboxylic acid cycle components via amino acids rather than via fatty acids would increase adenosine triphosphate production, with positive effects on cellular metabolism. A proposed study design is described and will be piloted through the Effects of Diatrofen on Myocardial Function in Patients with Chronic Heart Failure trial (D-CHF), an evaluation of an oral amino acid supplementation treatment in outpatients with heart failure.

Short- and long-term beneficial effects of trimetazidine in patients with diabetes and ischemic cardiomyopathy

Background Trimetazidine (TMZ) has been shown to partially inhibit free fatty acid oxidation by shifting substrate utilization from fatty acid to glucose. The aim of this study was to assess the effects of TMZ in patients with diabetes and ischemic cardiomyopathy. Methods Sixteen patients with diabetes and ischemic hypokinetic cardiomyopathy (all males) on conventional therapy were randomized to receive either placebo or TMZ (20 mg 3 times per day), each arm lasting 15 days, and then again to receive either placebo or TMZ for 2 additional 6-month periods, according to a double-blind, crossover design. At the end of each period, all patients underwent exercise testing, 2-dimensional echocardiography, and hyperinsulinemic/euglycemic clamp. Among the others, New York Heart Association class, ejection fraction, exercise time, fasting blood glucose, end-clamp M value (index of total body glucose disposal) and endothelin-1 levels were evaluated. Results Both in the short and long term (completed by 13 patients), on TMZ compared to placebo, ejection fraction (47 ± 7 vs 41 ± 9 and 45 ± 8 vs 36 ± 8%, P < .001 for both) and M value (4.0 ± 1.8 vs 3.3 ± 1.6, P = .003, and 3.5 ± 1.5 vs 2.7 ± 1.6 mg/kg body weight/min, P < .01) increased, while fasting blood glucose (121 ± 30 vs 136 ± 40, P = .02 and 125 ± 36 vs 140 ± 43, P = .19) and endothelin-1 (8.8 ± 3.8 vs 10.9 ± 3.8, P < .001 and 6.2 ± 2.4 vs 9.2 ± 4.3 pg/mL, P = .03) decreased. In the short term, 10 patients decreased 1 class on the NYHA scale during treatment with TMZ ( P = .019 vs placebo). Eight patients decreased 1 NYHA class while on long-term TMZ treatment, while on placebo 1 patient increased 1 NYHA class and none improved ( P = .018 vs placebo). Conclusions In a short series of patients with diabetes and ischemic cardiomyopathy, TMZ improved left ventricular function, symptoms, glucose metabolism, and endothelial function. Shifting energy substrate preference away from fatty acid metabolism and toward glucose metabolism by TMZ appears an effective adjunctive treatment in patients with diabetes with postischemic cardiomyopathy.

Pharmacologic Rationale for Trimetazidine in the Treatment of Ischemic Heart Disease

The classic approach to treating coronary heart disease involves interventions aimed at either increasing the oxygen supply to the heart muscle or decreasing the oxygen demand of the muscle. A new approach to treating coronary heart disease involves improving the efficiency of oxygen utilization by the tissue. Inhibiting fatty acid oxidation and stimulating glucose oxidation can significantly improve cardiac efficiency (cardiac work/oxygen consumed). This metabolic approach is not only beneficial as monotherapy in the treatment of angina pectoris, but is also beneficial in angina patients when used in combination with conventional therapy. Trimetazidine is a widely used metabolic agent that directly optimizes energy metabolism in the heart without causing any direct negative hemodynamic, inotropic or chronotropic effects. Trimetazidine acts by directly inhibiting fatty acid oxidation, resulting in an increase in glucose oxidation in the heart. The actions of trimetazidine occur as a result of inhibition of long-chain 3-ketoacyl CoA thiolase (3-KAT), an important enzyme involved in mitochondrial fatty acid oxidation. Because of this action, trimetazidine has been classified as a 3-KAT inhibitor, and is the first metabolic agent used clinically for treating coronary heart disease. Inhibition of fatty acid oxidation and stimulation of glucose oxidation with trimetazidine result in a decrease in acidosis in the ischemic muscle, thereby decreasing the requirement of energy for non-contractile purposes (i. e. increasing cardiac efficiency). This increase in glucose oxidation and decreased production of acidosis in the myocardium probably account for the cardioprotective effects of trimetazidine, which have been observed in many experimental and clinical studies. Both during and after ischemia, trimetazidine causes a significant improvement in cardiac efficiency, allowing the heart to perform a greater amount of work at a given level of oxygen. This therapeutic approach not only lessens the severity and symptoms of an angina attack in patients with coronary artery disease, but also decreases the incidence of angina attacks. As a result, using trimetazidine to optimize energy substrate preference is a novel and effective approach to treating ischemic heart disease.

Transcriptional activation of energy metabolic switches in the developing and hypertrophied heart.

1. The present review focuses on the gene regulatory mechanisms involved in the control of cardiac mitochondrial energy production in the developing heart and following the onset of pathological cardiac hypertrophy. Particular emphasis has been given to the mitochondrial fatty acid oxidation (FAO) pathway and its control by members of the nuclear receptor transcription factor superfamily. 2. During perinatal cardiac development, the heart undergoes a switch in energy substrate preference from glucose in the fetal period to fatty acids following birth. This energy metabolic switch is paralleled by changes in the expression of the enzymes and protein involved in the respective pathways. 3. The postnatal activation of the mitochondrial energy production pathway involves the induced expression of nuclear genes encoding FAO enzymes, as well as other proteins important in mitochondrial energy transduction/production pathways. Recent evidence indicates that this postnatal gene regulatory effect involves the actions of the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha) and its coactivator the PPARgamma coactivator 1 (PGC-1). 4. The PGC-1 not only activates PPARalpha to induce FAO pathway enzymes in the postnatal heart, but it also plays a pivotal role in the control of cardiac mitochondrial number and function. Thus, PGC-1 plays a master regulatory role in the high-capacity mitochondrial energy production system in the adult mammalian heart. 5. During the development of pathological forms of cardiac hypertrophy, such as that due to pressure overload, the myocardial energy substrate preference shifts back towards the fetal pattern, with a corresponding reduction in the expression of FAO enzyme genes. This metabolic shift is due to the deactivation of the PPARalpha/PGC-1 complex. 6. The deactivation of PPARalpha and PGC-1 during the development of cardiac hypertrophy involves regulation at several levels, including a reduction in the expression of these genes, as well as post-translational effects due to the mitogen-activated protein kinase pathway. Future studies aim at defining whether this transcriptional 'switch' and its effects on myocardial metabolism are adaptive or maladaptive in the hypertrophied heart.

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.

Fatty Acid Utilization in the Hypertrophied and Failing Heart: Molecular Regulatory Mechanisms

During the development of cardiac hypertrophy and in the failing heart, the chief myocardial energy source switches from fatty acid β-oxidation to glycolysis: a reversion to the fetal energy substrate preference pattern. This review describes recent molecular studies aimed at delineating the gene regulatory pathway involved in the energy metabolic switch in the hypertrophied heart and the potential role of the attendant metabolic consequences in the pathogenesis of heart failure. Studies have been performed with the ‘spontaneous hypertensive and heart failure’ rat strain and with human cardiomyopathic tissue. These studies have demonstrated that expression of the gene that encodes medium-chain acyl-coenzyme A dehydrogenase (MCAD), a key fatty acid β-oxidation enzyme, is down-regulated during the progression from cardiac hypertrophy to ventricular dysfunction. A series of studies performed in mice transgenic for the human MCAD gene promoter have identified a transcriptional regulatory pathway involved in the repression of MCAD gene expression in the hypertrophied mouse heart. Two categories of transcription factors, nuclear hormone receptors and Sp factors, bind MCAD gene promoter regulatory elements in response to pressure overload to reactivate a fetal metabolic gene program. Studies are under way to manipulate this transcriptional regulatory pathway in mice using genetic engineering strategies to determine whether this energy metabolic derangement plays a primary role in the development of cardiac hypertrophy and heart failure.

Fatty acid utilization in the hypertrophied and failing heart: molecular regulatory mechanisms.

During the development of cardiac hypertrophy and in the failing heart, the chief myocardial energy source switches from fatty acid beta-oxidation to glycolysis: a reversion to the fetal energy substrate preference pattern. This review describes recent molecular studies aimed at delineating the gene regulatory pathway involved in the energy metabolic switch in the hypertrophied heart and the potential role of the attendant metabolic consequences in the pathogenesis of heart failure. Studies have been performed with the 'spontaneous hypertensive and heart failure' rat strain and with human cardiomyopathic tissue. These studies have demonstrated that expression of the gene that encodes medium-chain acyl-coenzyme A dehydrogenase (MCAD), a key fatty acid beta-oxidation enzyme, is down-regulated during the progression from cardiac hypertrophy to ventricular dysfunction. A series of studies performed in mice transgenic for the human MCAD gene promoter have identified a transcriptional regulatory pathway involved in the repression of MCAD gene expression in the hypertrophied mouse heart. Two categories of transcription factors, nuclear hormone receptors and Sp factors, bind MCAD gene promoter regulatory elements in response to pressure overload to reactivate a fetal metabolic gene program. Studies are under way to manipulate this transcriptional regulatory pathway in mice using genetic engineering strategies to determine whether this energy metabolic derangement plays a primary role in the development of cardiac hypertrophy and heart failure.

Role of developmental factors in the switch from pyruvate to glucose as the major exogenous energy substrate in the preimplantation mouse embryo.

Preimplantation mouse embryos, cultured in vitro and those freshly flushed from the reproductive tract, exhibit a switch in energy substrate preference, from pyruvate during the early preimplantation stages, to glucose at the blastocyst stage. Although the biochemical basis of this phenomenon is quite well characterized, its timing and possible association with developmental factors have not been considered. We have therefore examined the role of five developmental factors in determining the timing of the switch, namely: (1) embryo age (in hours post hCG); (2) developmental stage; (3) cytokinesis; (4) cell number; and (5) activation of the embryonic genome. One-cell embryos, which develop more slowly than 2-cell embryos in vitro, were used to investigate the role of embryo age and developmental stage. Cytochalasin D, which inhibits cytokinesis and delays the timing of compaction and cavitation, was used to investigate the role of cell division and developmental stage. Finally, transcription of the embryonic genome was examined with the inhibitor, alpha-amanitin. Pyruvate and glucose consumption by single embryos were measured using a noninvasive ultramicrofluorometric technique. The results showed that the timing of the switch in energy substrate preference is precisely regulated in the mouse preimplantation embryo. Activation of the embryonic genome is a prerequisite for the switch and its timing is closely associated with developmental stage, specifically compaction and/or cavitation. Cell number, cytokinesis and embryo age appeared to be unrelated to the timing of the switch. These conclusions may well be extrapolated to other species, since an increase in net glucose uptake, if not always at the expense of pyruvate, is a feature of preimplantation embryo metabolism in all mammals studied.

Acetyl-CoA carboxylase control of fatty acid oxidation in hearts from hibernating Richardson's ground squirrels

Although mammalian hibernators rely on stored body fat as a source of energy, direct measurement of energy substrate preference in heart tissue during hibernation, as well as potential mechanisms controlling fatty acid oxidation has not been examined. In order to determine whether an increase in fatty acid utilization occurs during hibernation, glucose and palmitate oxidation were measured in isolated working hearts from hibernating and non-hibernating Richardson's ground Squirrels. Hearts were perfused at either 37° or 5°C with perfusate containing 11 mM [U– 14C]glucose and 1.2 mM [9,10- 3H]palmitate, which allowed for direct measurement of both glucose oxidation ( 14CO 2 production) and fatty acid oxidation ( 3H 2O production). The contribution of fatty acid oxidation as a source of citric acid cycle acetyl-CoA was significantly greater in hearts from hibernating animals, compared to hearts from non-hibernating animals. Since acetyl-CoA carboxylase (ACC) regulates cardiac fatty acid oxidation (producing malonyl-CoA, a potent inhibitor of mitochondrial fatty acid uptake), we measured the activity and expression of ACC in these hearts. ACC activity was significantly decreased in hibernating ground squirrels, regardless of whether ACC was assayed at 37° or 5°C. This decrease in activity could not be explained by a change in the activity of 5′AMP-activated protein kinase, which can phosphorylate and inhibit ACC. Rather, the expression of the 280 kDa isoform of ACC (which predominates in cardiac muscle) was decreased in hearts from hibernating squirrel hearts. This suggests that a down regulation of ACC expression occurs as an adaptation for the increased utilization of fatty acid in hearts of hibernating ground squirrels.

Maturation of fatty acid and carbohydrate metabolism in the newborn heart.

During fetal life, myocardial ATP is derived predominantly from glycolysis and lactate oxidation. Following birth, a rapid maturational increase in fatty acid oxidation occurs along with a decline in glycolytic and lactate oxidative rates, thus changing the major source of myocardial ATP production. This shift in energy substrate preference occurs in response to changes in the circulating substrate content of newborn plasma with the onset of suckling, and is also due to alterations in circulating levels of hormones, such as insulin and glucagon. Important changes in subcellular regulatory mechanisms of both fatty acid and carbohydrate metabolism in the heart also characterize this response. This review deals with recent advances in the understanding of these subcellular mechanisms which regulate this important shift in myocardial energy metabolism, with particular emphasis on the molecular events occurring in the heart during the transition from fetal to newborn life.

Alterations in Fatty Acid Oxidation During Reperfusion of the Heart After Myocardial Ischemia

Energy substrate preference of the heart both during and after ischemia is an important determinant of the degree of functional recovery postischemia. For instance, high rates of fatty acid oxidation after ischemia can decrease cardiac function and efficiency during reperfusion. These high rates of fatty acid oxidation can be explained by a decrease in malonyl coenzyme-A (CoA) levels, a potent inhibitor of mitochondrial fatty acid uptake. This review discusses the biochemical changes in the heart after ischemia that are responsible for these high rates of fatty acid oxidation. In particular, activation of 5′-AMP-activated protein kinase and inhibition of acetyl CoA carboxylase appear to contribute to this decrease in malonyl CoA. As a result, we propose that inhibition of 5′-AMP-activated protein kinase and/or stimulation of acetyl CoA carboxylase may be a pharmacologic approach to inhibiting myocardial fatty acid oxidation during reperfusion. Decreasing fatty acid oxidation is accompanied by a parallel increase in glucose oxidation that results in an improvement in both cardiac function and efficiency in the reperfused ischemic heart.

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.

Activation of a novel metabolic gene regulatory pathway by chronic stimulation of skeletal muscle.

To determine whether expression of a nuclear gene encoding a mitochondrial fatty acid oxidation enzyme is regulated in parallel with skeletal muscle fibre-type-specific energy substrate preference, expression of the gene encoding medium-chain acyl-CoA dehydrogenase (MCAD) was delineated in canine latissimus dorsi muscle subjected to chronic motor nerve stimulation. In predominantly fast-twitch canine latissimus dorsi muscle, MCAD mRNA levels were regulated by chronic stimulation in a biphasic pattern. During the 1st wk of stimulation, steady-state MCAD mRNA levels decreased to 50% of unstimulated levels. MCAD mRNA levels began to increase during the 3rd wk of stimulation to reach a level 3.0-fold higher than levels in unstimulated contralateral control muscle by day 70. Immunodetectable MCAD mRNA levels throughout the stimulation period. The temporal pattern and magnitude of MCAD mRNA accumulation in response to muscle stimulation was distinct from that of mRNAs encoding other enzymes known to be regulated by this stimulus, including glyceraldehyde phosphate dehydrogenase, citrate synthase, and sarcoplasmic reticulum Ca-ATPase, but paralleled the protein levels of the peroxisome proliferator-activated receptor (PPAR), an orphan member of the nuclear hormone receptor superfamily known to regulate genes encoding fatty acid oxidation enzymes in liver. The skeletal muscle expression pattern of PPAR was also similar to that of MCAD in unstimulated rat skeletal muscles with distinct fiber-type compositions. These results demonstrate that a nuclear gene encoding a mitochondrial beta-oxidation enzyme is dynamically regulated in a pattern that parallels skeletal muscle fiber-type-specific energy substrate utilization and implicate an orphan nuclear receptor transcription factor as a candidate transducer of this response.

Calcium regulation of glycolysis, glucose oxidation, and fatty acid oxidation in the aerobic and ischemic heart.

Although Ca2+ is an important regulator of energy metabolism, the effects of increasing extracellular [Ca2+] on energy substrate preference are not clear. We determined the relationship between [Ca2+], fatty acids, and ischemia on rates of glycolysis, glucose oxidation, and palmitate oxidation in isolated working rat hearts. Hearts were perfused with Krebs-Henseleit buffer containing 11 mM glucose, 100 microU/mL insulin, and either 1.25 or 2.5 mM Ca2+, in the presence or absence of 1.2 mM palmitate. Rates of glycolysis and glucose oxidation or palmitate oxidation were measured in the hearts using [5-3H,14C(U)]glucose or [1-14C]palmitate, respectively. In the absence of fatty acids, glycolysis and glucose oxidation rates were similar, regardless of whether [Ca2+ was 1.25 or 2.5 mM. Addition of 1.2 mM palmitate to the perfusate of hearts perfused with 1.25 mM Ca2+ significantly decreased rates of both glycolysis (from 4623 +/- 438 to 1378 +/- 238 nmol.min-1.g-1 dry weight) and glucose oxidation (from 1392 +/- 219 to 114 +/- 22 nmol.min-1.g-1 dry weight). When [Ca2+] was increased from 1.25 to 2.5 mM in hearts perfused with 1.2 mM palmitate, glycolysis and glucose oxidation increased by 164 and 271%, respectively, with no change in palmitate oxidation rates. Increasing [Ca2+] from 1.25 to 2.5 mM increased the contribution of glucose to ATP production from 9.3 to 18.7%. When hearts were subjected to low-flow ischemia (by reducing coronary flow to 0.5 mL.min-1) oxidative metabolism was essentially abolished. Under these conditions, glycolytic rates were not dependent on either [Ca2+] or the presence or absence of fatty acids. These results demonstrate that perfusate [Ca2+] is an important determinant of myocardial glucose metabolism in aerobic hearts, and that glycolysis and glucose oxidation are more responsive to changes in [Ca2+] than is fatty acid oxidation.

Golden hamster embryonic genome activation occurs at the two-cell stage: correlation with major developmental changes.

The earliest time of onset of embryonic genome activation in golden hamsters was investigated. The inhibition of transcription by alpha-amanitin (11 micrograms/ml) in cultured embryos resulted in a total arrest of development of early 2-cell embryos (26 hr post-egg activation); under similar conditions, immediate cleavage divisions of 1-, late 2-, 4-, and 8-cell embryos were not affected. Electrophoretic analysis of [35S]methionine-labeled embryonic proteins showed that alpha-amanitin treatment apparently inhibited transcription-dependent protein synthesis in early 2-cell and, to some extent, in late 2-cell when compared to 4-cell embryos. Analysis of total RNA synthesis, using [alpha 32P]-UTP or [32P]-orthophosphate, showed that there was a high proportion of radioactivity associated with the macromolecular fraction (RNA) at the early and late 2-cell stages and at the 4-cell stage compared to that at the 1-cell stage. These results indicate that the de novo synthesis of RNA, encoded by the embryonic genome, occurs at the 2-cell stage and that the second and subsequent cleavage divisions of hamster preimplantation embryos are dependent on new transcriptional activity. This initial activity of the embryonic genome in hamsters is coincident with several characteristic features of in vitro development such as a block to development, synthesis of major proteins, change in energy substrate preference, phosphate-inhibition of development and a requirement for amino acids.

Glucose and palmitate oxidation in isolated working rat hearts reperfused after a period of transient global ischemia.

Alterations in energy substrate utilization during reperfusion of ischemic hearts can influence the functional recovery of the myocardium. Energy substrate preference by the reperfused myocardium, however, has received limited attention. Therefore, we measured oxidation rates of glucose and palmitate during reperfusion of ischemic hearts. Isolated working rat hearts were perfused with 1.2 mM palmitate and 11 mM [14C]glucose, 1.2 mM [14C]palmitate and 11 mM glucose, or 11 mM [14C]glucose alone, at an 11.5 mm Hg preload and 80 mm Hg afterload. Hearts were subjected to 60-minute aerobic perfusion or 25-minute global ischemia followed by 60-minute aerobic reperfusion. Steady-state oxidative rates of glucose or palmitate were determined by measuring 14CO2 production. In hearts perfused with glucose alone, oxidative rates during reperfusion were not significantly different than nonischemic hearts (1,008 +/- 335 vs. 1,372 +/- 117 nmol [14C]glucose oxidized/min/g dry wt, respectively). In the presence of palmitate, glucose oxidation was markedly reduced in reperfused and nonischemic hearts (81 +/- 11 and 101 +/- 15 nmol [14C]glucose oxidized/min/g dry wt, respectively). Palmitate oxidation rates were not significantly different in reperfused compared with nonischemic hearts (369 +/- 55 and 455 +/- 50 nmol [14C]palmitate oxidized/min/g dry wt, respectively). [14C]Palmitate was incorporated into myocardial triglycerides to a greater extent in reperfused ischemic hearts than in nonischemic hearts (26.0 and 13.8 mumol/g dry wt, respectively). Under the perfusion conditions used, palmitate provided over 90% of the ATP produced from exogenous substrates. Addition of the carnitine palmitoyltransferase I inhibitor, ethyl 2-[6-(4-chlorophenoxy)hexyl]oxirane-2-carboxylate (Etomoxir, 10(-6) M), during reperfusion stimulated glucose oxidation and improved mechanical recovery of ischemic hearts.(ABSTRACT TRUNCATED AT 250 WORDS)