Metabolic responses to moderate exercise in lambs with aortopulmonary shunts.

In a previous study we found, after an overnight fast of 18 hours, a lower arterial glucose concentration and a depressed glycogenolysis in lambs with aortopulmonary left-to-right shunts. During exercise, glucose and free fatty acids (FFA) concentrations normally increase. The aim of this study was to investigate whether the shunt lambs could compensate for a depressed glycogenolysis by increasing gluconeogenesis and by increasing levels of blood substrates such as FFA and glycerol during exercise. Therefore, we investigated glucose kinetics, with [U-(13)C]glucose, in five 7-week-old shunt and 7 control lambs of a similar age, at rest and during moderate exercise (treadmill; 50% of VO(2) peak). The glucose production rate and the rate of disappearance of glucose were lower in shunt than in control lambs, both at rest and during exercise. We found no difference in metabolic clearance rate of glucose, glucose recycling, or gluconeogenesis between both groups of lambs. Glycogenolysis was at rest lower in shunt than in control lambs and tended to be lower during exercise. The arterial concentrations of pyruvate, lactate, FFA, and total and free glycerol increased during exercise in both groups of lambs. In conclusion, shunt lambs have lower arterial glucose concentrations than control lambs, both at rest and during moderate exercise. This was due to a lower glucose production rate, in particular a lower glycogenolysis. In addition, the reduced glycogenolysis rate was not offset by an increase in gluconeogenesis nor by an increase in other substrates that can be utilized by working muscles.

Perinatal changes in myocardial metabolism in lambs.

BACKGROUND: Lactate accounts for a third of myocardial oxygen consumption before and in the first 2 weeks after birth. It is unknown how the remainder of myocardial oxygen is consumed. Glucose is thought to be important before birth, whereas long-chain fatty acids (LC-FA) are the prime substrate for the adult. However, the ability of the myocardium of the newborn to use LC-FA has been doubted. METHODS AND RESULTS: We measured the myocardial metabolism of glucose and LC-FA with [U-(13)C]glucose and [1-(13)C]palmitate in chronically instrumented fetal and newborn lambs. In fetal lambs, myocardial oxidation of glucose was high and that of LC-FA was low. Glucose and LC-FA accounted for 48+/-4% and 2+/-2% of myocardial oxygen consumption, respectively. In newborn lambs, oxidation of glucose decreased, whereas oxidation of LC-FA increased. Glucose and LC-FA accounted for 12+/-3% and 83+/-19% of myocardial oxygen consumption. To test whether near-term fetal lambs could use LC-FA, we increased the supply of LC-FA with a fat infusion. In fetal lambs during fat infusion, the oxidation of LC-FA increased 15-fold. Although the oxidation of LC-FA was still lower than in newborn lambs, the contribution to myocardial oxygen consumption (70+/-13%) was the same as in newborn lambs. CONCLUSIONS: These data show that glucose and lactate account for the majority of myocardial oxygen consumption in fetal lambs, whereas in newborn lambs, LC-FA and lactate account for the majority of myocardial oxygen consumption. Moreover, we showed that the fetal myocardium can use LC-FA as an energy substrate.

Lower arterial glucose concentrations in lambs with aortopulmonary shunts after an 18-hour fast.

Spontaneously occurring hypoglycemia has been described in children with severe acute congestive heart failure. Hypoglycemia may be the result of an increase in glucose utilization in tissues, a decrease in glucose production, or a decrease in the dietary intake of nutrients. To determine whether hypoglycemia may also occur in congenital heart disease with volume overloading, we investigated glucose metabolism during and after an 18-hour fast in nine lambs with an aortopulmonary left-to-right shunt and nine control lambs. Plasma levels of hormones involved in the endocrine control of glucose metabolism were determined. The glucose production rate (rate of appearance [Ra]) was studied using [U-13C]glucose. Gluconeogenesis through the Cori cycle was estimated by measuring glucose 13C recycling. The arterial glucose concentration (3,409 +/- 104 v 4,338 +/- 172 micromol/L, P < .001) and Ra of glucose (16.97 +/- 0.89 v 25.49 +/- 4.28 micromol x min(-1) x kg(-1), P < .05) were lower in shunt versus control lambs. There were no differences in hormone levels between control and shunt lambs. Fractional glucose 13C recycling via the Cori cycle (6.9% +/- 2.8% v 7.1% +/- 2.5%) and gluconeogenesis from pyruvate and lactate (1.24 +/- 0.58 v 1.95 +/- 0.67 micromol x min(-1) x kg(-1)) were similar in both groups of lambs. The sum of glycogenolysis and gluconeogenesis from precursors other than pyruvate and lactate was lower in shunt versus control lambs (15.73 +/- 1.07 v 23.54 +/- 4.27 micromol x min(-1) x kg(-1), P < .05). In conclusion, after an 18-hour fast, the arterial glucose concentration is lower in lambs with aortopulmonary shunts. This lower glucose concentration is associated with a decreased glucose production rate. In shunt lambs, glycogenolysis is decreased, while there is no difference in gluconeogenesis or hormonal control.

Cytological evidence that the C-terminus of carnitine palmitoyltransferase I is on the cytosolic face of the mitochondrial outer membrane.

Carnitine palmitoyltransferase I (CPT I) is a key enzyme in the regulation of beta-oxidation. The topology of this enzyme has been difficult to elucidate by biochemical methods. We studied the topology of a fusion protein of muscle-type CPT I (M-CPT I) and green fluorescent protein (GFP) by microscopical means. To validate the use of the fusion protein, designated CPT I-GFP, we checked whether the main characteristics of native CPT I were retained. CPT I-GFP was expressed in HeLa cells after stable transfection. Confocal laser scanning microscopy in living cells revealed an extranuclear punctate distribution of CPT I-GFP, which coincided with a mitochondrial fluorescent marker. Immunogold electron microscopy localized CPT I-GFP almost exclusively to the mitochondrial periphery and showed that the C-terminus of CPT I must be on the cytosolic face of the mitochondrial outer membrane. Western analysis showed a protein that was 6 kDa smaller than predicted, which is consistent with previous results for the native M-CPT I. Mitochondria from CPT I-GFP-expressing cells showed an increased CPT activity that was inhibited by malonyl-CoA and was lost on solubilization with Triton X-100. We conclude that CPT I-GFP adopts the same topology as native CPT I and that its C-terminus is located on the cytosolic face of the mitochondrial outer membrane. The evidence supports a recently proposed model for the domain structure of CPT I based on biochemical evidence.

Myocardial lactate metabolism in fetal and newborn lambs.

BACKGROUND: Around birth, myocardial substrate supply changes from carbohydrates before birth to primarily fatty acids after birth. Parallel to these changes, the myocardium is expected to switch from the use of primarily lactate before birth to fatty acids thereafter. However, myocardial lactate uptake and oxidation around birth has not been measured in vivo. METHODS AND RESULTS: We measured myocardial lactate uptake, oxidation, and release with infusion of [1-13C]lactate and myocardial flux of fatty acids and glucose in chronically instrumented fetal and newborn (1 to 15 days) lambs. Myocardial lactate oxidation was the same in newborn (81.7+/-14.7 micromol. min-1. 100 g-1, n=11) as in fetal lambs (60.7+/-26.7 micromol. min-1. 100 g-1, n=7). Lactate uptake was also the same in newborn as in fetal lambs. Lactate uptake was higher than lactate flux, indicating lactate release simultaneously with uptake. In the newborn lambs, lactate uptake declined with age. Lactate uptake was strongly related to lactate supply, whereas lactate oxidation was not. The supply of fatty acids or glucose did not interfere with lactate uptake, but the flux of fatty acids was inversely related to lactate oxidation. CONCLUSIONS: We show that lactate is an important energy source for the myocardium before birth as well as in the first 2 weeks after birth in lambs. We also show that there is release of lactate by the myocardium simultaneously with uptake of lactate. Furthermore, we show that lactate oxidation may be attenuated by fatty acids but not by glucose, probably at the level of pyruvate dehydrogenase.

Lactate kinetics at rest and during exercise in lambs with aortopulmonary shunts.

In a previous study [G. C. M. Beaufort-Krol, J. Takens, M. C. Molenkamp, G. B. Smid, J. J. Meuzelaar, W. G. Zijlstra, and J. R. G. Kuipers. Am. J. Physiol. 275 (Heart Circ. Physiol. 44): H1503-H1512, 1998], a lower systemic O2 supply was found in lambs with aortopulmonary left-to-right shunts. To determine whether the lower systemic O2 supply results in increased anaerobic metabolism, we used [1-13C]lactate to investigate lactate kinetics in eight 7-wk-old lambs with shunts and eight control lambs, at rest and during moderate exercise [treadmill; 50% of peak O2 consumption (VO2)]. The mean left-to-right shunt fraction in the shunt lambs was 55 +/- 3% of pulmonary blood flow. Arterial lactate concentrations and the rate of appearance (Ra) and disappearance (Rd) of lactate were similar in shunt and control lambs, both at rest (lactate: 1, 201 +/- 76 vs. 1,214 +/- 151 micromol/l; Ra = Rd: 12.97 +/- 1.71 vs. 12.55 +/- 1.25 micromol. min-1. kg-1) and during a similar relative workload. We found a positive correlation between Ra and systemic blood flow, O2 supply, and VO2 in both groups of lambs. In conclusion, shunt lambs have similar lactate kinetics as do control lambs, both at rest and during moderate exercise at a similar fraction of their peak VO2, despite a lower systemic O2 supply.

Increased myocardial lactate oxidation in lambs with aortopulmonary shunts at rest and during exercise.

Free fatty acids are the major fuels for the myocardium, but during a higher load carbohydrates are preferred. Previously, we demonstrated that myocardial net lactate uptake was higher in lambs with aortopulmonary shunts than in control lambs. To determine whether this was caused by an increased lactate uptake and oxidation or by a decreased lactate release, we studied myocardial lactate and glucose metabolism with 13C-labeled substrates in 36 lambs in a fasting, conscious state. The lambs were assigned to two groups: a resting group consisting of 8 shunt and 9 control lambs, and an exercise group (50% of peak O2 consumption) consisting of 9 shunt and 10 control lambs. Myocardial lactate oxidation was higher in shunt than in control lambs (mean +/- SE, rest: 10.33 +/- 2.61 vs. 0. 17 +/- 0.82, exercise: 38.05 +/- 8.87 vs. 16.89 +/- 4.78 micromol. min-1. 100 g-1; P < 0.05). There was no difference in myocardial lactate release between shunt and control lambs. Oxidation of exogenous glucose, which was approximately zero at rest, increased during exercise in shunt and control lambs. The contribution of glucose and lactate to myocardial oxidative metabolism increased during exercise compared with at rest in both shunt and control lambs. We conclude that myocardial lactate oxidation is higher in shunt than in control lambs, both at rest and during exercise, and that the contribution of carbohydrates in myocardial oxidative metabolism in shunt lambs is higher than in control lambs. Thus it appears that this higher contribution of carbohydrates occurs not only in the case of pressure-overloaded hearts but also in myocardial hypertrophy due to volume overloading.

Perinatal changes in myocardial supply and flux of fatty acids, carbohydrates, and ketone bodies in lambs.

No information is available on perinatal changes in myocardial metabolism in vivo. We measured myocardial supply and flux of fatty acids, carbohydrates, and ketone bodies in chronically instrumented fetal, newborn (1-4 days), and juvenile (7 wk) lambs, by measuring aorta-coronary sinus concentration differences and blood flow. In the fetal lambs, myocardial supply and flux of fatty acids were zero. In the newborn lambs, the supply of fatty acids increased tenfold, but there was no flux of fatty acids. Carbohydrates were the major energy source in fetal and newborn lambs, accounting for 89 and 69% of myocardial oxygen consumption, respectively. In the juvenile lambs, the flux of fatty acids was increased threefold. The supply and flux of carbohydrates were decreased (by 31 and 82%, respectively). The supply and flux of ketone bodies gradually increased with age. We show that the myocardium of the lamb in vivo does not switch immediately after birth from carbohydrates to fatty acids. The mechanisms involved in the development of myocardial fatty acid oxidation remain to be elucidated.

Determination of organ substrate oxidation in vivo by measurement of 13CO2 concentration in blood.

Substrate oxidation by various organs in animals as well as in humans is usually studied by experiments in which radioactively labeled substrates are used and the production of 14CO2 is measured. In vivo, substrate oxidation by an organ has, up to now, not been determined by means of stable isotopes. Problems in the determination of the concentration of 13CO2 in blood may have impeded the use of 13C-labeled substrates. For the determination of 13CO2 concentration in blood a direct method for the determination of total CO2 concentration in blood was combined with the determination of the isotope ratio (13C/12C) of CO2 by isotope ratio mass spectrometry. The intra-assay relative standard deviation of the CO2 concentration (mean: 19.26 mmol l-1; n = 7) was 0.8%. The inter-assay relative standard deviation of the CO2 concentration in solutions of a weighed amount of Na2CO3 determined over a 5 year period was 0.64% (mean: 21.99 mmol l-1; n = 22). The intra-assay relative standard deviation of 13C in CO2 was 0.03% (mean 13C/12C: 0.0111557; n = 5). From the 13CO2 concentration in arterial and venous blood, substrate oxidation by various organs can be calculated. As an illustration, the determination of myocardial glucose oxidation in lambs, both at rest and during exercise, is described.

Localization and intron usage analysis of the human CPT1B gene for muscle type carnitine palmitoyltransferase I.

We isolated and sequenced cDNA and genomic DNA fragments of the human CPT1B gene, encoding muscle type camitine palmitoyltransferase I. A recombinant P1 phage containing CPT1B was mapped to chromosome 22qter by fluorescent in situ hybridization. This finding supports the concept that 'liver type' and 'muscle type' isoforms of CPT I are encoded by different loci at separate chromosomal positions. Analysis of CPT1B cDNA sequences revealed the presence of an untranslated 5' exon and differential processing of introns 13 and 19. The alternative splicing of intron 13 causes an in-frame deletion leading to a 10 amino acid residues smaller protein. Using different splice acceptor sites, intron 19 is spliced in the majority of cases, but 4 out of 14 sequenced CPT1B 3' cDNA clones contain part of intron 19 in stead of exon 20. We found that differential polyadenylation is the mechanism behind the existence of these alternative 3' CPT1B mRNA forms.