Quantifying reductive carboxylation flux of glutamine to lipid in a brown adipocyte cell line

We previously reported that glutamine was a major source of carbon for de novo fatty acid synthesis in a brown adipocyte cell line. The pathway for fatty acid synthesis from glutamine may follow either of two distinct pathways after it enters the citric acid cycle. The glutaminolysis pathway follows the citric acid cycle, whereas the reductive carboxylation pathway travels in reverse of the citric acid cycle from alpha-ketoglutarate to citrate. To quantify fluxes in these pathways we incubated brown adipocyte cells in [U-(13)C]glutamine or [5-(13)C]glutamine and analyzed the mass isotopomer distribution of key metabolites using models that fit the isotopomer distribution to fluxes. We also investigated inhibitors of NADP-dependent isocitrate dehydrogenase and mitochondrial citrate export. The results indicated that one third of glutamine entering the citric acid cycle travels to citrate via reductive carboxylation while the remainder is oxidized through succinate. The reductive carboxylation flux accounted for 90% of all flux of glutamine to lipid. The inhibitor studies were compatible with reductive carboxylation flux through mitochondrial isocitrate dehydrogenase. Total cell citrate and alpha-ketoglutarate were near isotopic equilibrium as expected if rapid cycling exists between these compounds involving the mitochondrial membrane NAD/NADP transhydrogenase. Taken together, these studies demonstrate a new role for glutamine as a lipogenic precursor and propose an alternative to the glutaminolysis pathway where flux of glutamine to lipogenic acetyl-CoA occurs via reductive carboxylation. These findings were enabled by a new modeling tool and software implementation (Metran) for global flux estimation.     

Cardioselective dominant-negative thyroid hormone receptor (Delta337T) modulates myocardial metabolism and contractile efficiency

Dominant-negative thyroid hormone receptors (TRs) show elevated expression relative to ligand-binding TRs during cardiac hypertrophy. We tested the hypothesis that overexpression of a dominant-negative TR alters cardiac metabolism and contractile efficiency (CE). We used mice expressing the cardioselective dominant-negative TRbeta(1) mutation Delta337T. Isolated working Delta337T hearts and nontransgenic control (Con) hearts were perfused with (13)C-labeled free fatty acids (FFA), acetoacetate (ACAC), lactate, and glucose at physiological concentrations for 30 min. (13)C NMR spectroscopy and isotopomer analyses were used to determine substrate flux and fractional contributions (Fc) of acetyl-CoA to the citric acid cycle (CAC). Delta337T hearts exhibited rate depression but higher developed pressure and CE, defined as work per oxygen consumption (MVo(2)). Unlabeled substrate Fc from endogenous sources was higher in Delta337T, but ACAC Fc was lower. Fluxes through CAC, lactate, ACAC, and FFA were reduced in Delta337T. CE and Fc differences were reversed by pacing Delta337T to Con rates, accompanied by an increase in FFA Fc. Delta337T hearts lacked the ability to increase MVo(2). Decreases in protein expression for glucose transporter-4 and hexokinase-2 and increases in pyruvate dehydrogenase kinase-2 and -4 suggest that these hearts are unable to increase carbohydrate oxidation in response to stress. These data show that Delta337T alters the metabolic phenotype in murine heart by reducing substrate flux for multiple pathways. Some of these changes are heart rate dependent, indicating that the substrate shift may represent an accommodation to altered contractile protein kinetics, which can be disrupted by pacing stress.     

Decreased cardiac mitochondrial NADP+-isocitrate dehydrogenase activity and expression: a marker of oxidative stress in hypertrophy development

Mitochondrial dysfunction subsequent to increased oxidative stress and alterations in energy metabolism is considered to play a role in the development of cardiac hypertrophy and its progression to failure, although the sequence of events remains to be elucidated. This study aimed at characterizing the impact of hypertrophy development on the activity and expression of mitochondrial NADP+-isocitrate dehydrogenase (mNADP+-ICDH), a metabolic enzyme that controls redox and energy status. We expanded on our previous finding of its inactivation through posttranslational modification by the lipid peroxidation product 4-hydroxynonenal (HNE) in 7-wk-old spontaneously hypertensive rat (SHR) hearts before hypertrophy development (Benderdour et al. J Biol Chem 278: 45154-45159, 2003). In this study, we used 7-, 15-, and 30-wk-old SHR and Sprague-Dawley (SD) rats with abdominal aortic coarctation. Compared with age-matched control Wistar-Kyoto (WKY) rats, SHR hearts showed a significant 25% decrease of mNADP+-ICDH activity, which preceded in time 1) the decline in its protein and mRNA expression levels (between 10% and 35%) and 2) the increase in hypertrophy markers. The chronic and persistent loss of mNADP+-ICDH activity in SHR was associated with enhanced tissue accumulation of HNE-mNADP+-ICDH and total HNE-protein adducts at all ages and contrasted with the profile of changes in the activity of other mitochondrial enzymes involved in antioxidant or energy metabolism. Two-way ANOVA of the data also revealed a significant effect of age on most parameters measured in SHR and WKY hearts. The mNADP+-ICDH activity, protein, and mRNA expression were reduced between 25% and 35% in coarctated SD rats and were normalized by treatment of SHR or coarctated SD rats with renin-angiotensin system inhibitors, which prevented or attenuated hypertrophy. Altogether, our data show that cardiac mNADP+-ICDH activity and expression are differentially and sequentially affected in hypertrophy development and, to a lesser extent, with aging. Decreased cardiac mNADP+-ICDH activity, which is attributed at least in part to HNE adduct formation, appears to be a relevant early and persistent marker of mitochondrial oxidative stress-related alterations in hypertrophy development. Potentially, this could also contribute to the aetiology of cardiomyopathy.     

Differential modulation of citrate synthesis and release by fatty acids in perfused working rat hearts

The objective of this study was to test the effect of increasing fatty acid concentrations on substrate fluxes through pathways leading to citrate synthesis and release in the heart. This was accomplished using semirecirculating work-performing rat hearts perfused with substrate mixtures mimicking the in situ milieu (5.5 mM glucose, 8 nM insulin, 1 mM lactate, 0.2 mM pyruvate, and 0.4 mM oleate-albumin) and 13C methods. Raising the fatty acid concentration from 0.4 to 1 mM with long-chain oleate or medium-chain octanoate resulted in a lowering ( approximately 20%) of cardiac output and efficiency with unaltered O2 consumption. At the metabolic level, beyond the expected effects of high fatty acid levels on the contribution of pyruvate decarboxylation (reduced >3-fold) and beta-oxidation (enhanced approximately 3-fold) to citrate synthesis, there was also a 2.4-fold lowering of anaplerotic pyruvate carboxylation. Despite the dual inhibitory effect of high fatty acids on pyruvate decarboxylation and carboxylation, tissue citrate levels were twofold higher, but citrate release rates remained unchanged at 11-14 nmol/min, representing <0.5% of citric acid cycle flux. A similar trend was observed for most metabolic parameters after oleate or octanoate addition. Together, these results emphasize a differential modulation of anaplerotic pyruvate carboxylation and citrate release in the heart by fatty acids. We interpret the lack of effects of high fatty acid concentrations on citrate release rates as suggesting that, under physiological conditions, this process is maximal, probably limited by the activity of its mitochondrial or plasma membrane transporter. Limited citrate release at high fatty acid concentrations may have important consequences for the heart's fuel metabolism and function.     

Partitioning of pyruvate between oxidation and anaplerosis in swine hearts

The goal of this study was to measure flux through pyruvate carboxylation and decarboxylation in the heart in vivo. These rates were measured in the anterior wall of normal anesthetized swine hearts by infusing [U-(13)C(3)]lactate and/or [U-(13)C(3)] pyruvate into the left anterior descending (LAD) coronary artery. After 1 h, the tissue was freeze-clamped and analyzed by gas chromatography-mass spectrometry for the mass isotopomer distribution of citrate and its oxaloacetate moiety. LAD blood pyruvate and lactate enrichments and concentrations were constant after 15 min of infusion. Under near-normal physiological concentrations of lactate and pyruvate, pyruvate carboxylation and decarboxylation accounted for 4.7 +/- 0.3 and 41.5 +/- 2.0% of citrate formation, respectively. Similar relative fluxes were found when arterial pyruvate was raised from 0.2 to 1.1 mM. Addition of 1 mM octanoate to 1 mM pyruvate inhibited pyruvate decarboxylation by 93% without affecting carboxylation. The absence of M1 and M2 pyruvate demonstrated net irreversible pyruvate carboxylation. Under our experimental conditions we found that pyruvate carboxylation in the in vivo heart accounts for at least 3-6% of the citric acid cycle flux despite considerable variation in the flux through pyruvate decarboxylation.     

Repression of reserve lipid turnover in Cunninghamella echinulata and Mortierella isabellina cultivated in multiple-limited media

AIMS: To study patterns of reserve lipid biosynthesis and turnover (degradation) in two oleaginous Zygomycetes, namely Cunninghamella echinulata and Mortierella isabellina under various growth conditions. Fatty acid composition of the reserve lipid of both strains was also studied in all growth steps. METHODS AND RESULTS: Cunninghamella echinulata and Mortierella isabellina were grown in carbon-excess batch cultures. In the investigated strains, accumulation of reserve lipid occurred only when the activity of both NAD(+)-isocitrate dehydrogenase (ICDH) and NADP(+)-ICDH were not detectable in the cell-free extract. Specifically, in C. echinulata, NAD(+)-ICDH activity was detected even after depletion of ammonium nitrogen in the medium, resulting in a delay of the initiation of lipid accumulation period. On the contrary, in M. isabellina, lipid accumulation occurred simultaneously with ammonium nitrogen exhaustion in the growth medium, as the activity of both NAD(+)- and NADP(+)-ICDH were not detectable after nitrogen depletion. In C. echinulata reserve lipid was not degraded after glucose had been exhausted. Supplementations of the medium with Fe(3+), yeast extract or Mg(2+) induced, however, reserve lipid breakdown and formation of lipid-free material. In M. isabellina after glucose exhaustion, notable lipid degradation occurred, accompanied by a significant lipid-free material biosynthesis. Nevertheless, in multiple-limited media, in which Mg(2+) or yeast extract, besides carbon and nitrogen, were limiting nutrients, reserve lipid breakdown was repressed. In both strains, the quantity of gamma-linolenic acid (GLA) in the reserve lipids [varying between 9 and 16% (w/w) in C. echinulata and 1.5-4.5% (w/w) in M. isabellina] was proportional to lipid-free biomass. CONCLUSIONS: Lipid accumulation period in Zygomycetes is initiated by the attenuation of ICDH activity in the mycelium while the regulation of ICDH from ammonium nitrogen is strain specific. While a single nitrogen limitation was enough to induce lipid accumulation, however, multiple limitations were needed in order to repress lipid turnover in oleaginous Zygomycetes. As for GLA, its biosynthesis in the mycelium seemed proportional to lipid-free biomass synthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: Several nutrients are indispensable for functioning the mechanisms involved in the mobilization of reserve lipid in oleaginous moulds. Therefore, reserve lipid turnover in oleaginous moulds could be repressed in multiple-limited media.     

Effects of increased heart work on glycolysis and adenine nucleotides in the perfused heart of normal and diabetic rats

1. In the isolated perfused rat heart, the contractile activity and the oxygen uptake were varied by altering the aortic perfusion pressure, or by the atrial perfusion technique (;working heart'). 2. The maximum increase in the contractile activity brought about an eightfold increase in the oxygen uptake. The rate of glycolytic flux rose, while tissue contents of hexose monophosphates, citrate, ATP and creatine phosphate decreased, and contents of ADP and AMP rose. 3. The changes in tissue contents of adenine nucleotides during increased heart work were time-dependent. The ATP content fell temporarily (30s and 2min) after the start of left-atrial perfusion; at 5 and 10min values were normal; and at 30 and 60min values were decreased. ADP and AMP values were increased in the first 15min, but were at control values 30 or 60min after the onset of increased heart work. 4. During increased heart work changes in the tissue contents of adenine nucleotide and of citrate appeared to play a role in altered regulation of glycolysis at the level of phosphofructokinase activity. 5. In recirculation experiments increased heart work for 30min was associated with increased entry of [(14)C]glucose (11.1mm) and glycogen into glycolysis and a comparable increase in formation of products of glycolysis (lactate, pyruvate and (14)CO(2)). There was no major accumulation of intermediates. Glycogen was not a major fuel for respiration. 6. Increased glycolytic flux in Langendorff perfused and working hearts was obtained by the addition of insulin to the perfusion medium. The concomitant increases in the tissue values of hexose phosphates and of citrate contrasted with the decreased values of hexose monophosphates and of citrate during increased glycolytic flux obtained by increased heart work. 7. Decreased glycolytic flux in Langendorff perfused hearts was obtained by using acute alloxan-diabetic and chronic streptozotocin-diabetic rats; in the latter condition there were decreased tissue contents of hexose phosphates and of citrate. There were similar findings when working hearts from streptozotocin-diabetic rats with insulin added to the medium were compared with normal hearts. 8. The effects of insulin addition or of the chronic diabetic state could be explained in terms of an action of insulin on glucose transport. Increased heart work also acted at this site, but in addition there was evidence for altered regulation of glycolysis mediated by changes in tissue contents of adenine nucleotides or of citrate.     

Metabolic phenotyping of the diseased rat heart using 13C-substrates and ex vivo perfusion in the working mode

The objective of the present study was to compare energy substrate fluxes through metabolic pathways leading to mitochondrial citrate synthesis and release in normal and diseased rat hearts using ¹³C-substrates and mass isotopomer analysis by gas chromatography-mass spectrometry (GCMS). This study was prompted by our previous finding of a modulated citrate release by perfused rat hearts and by the possibility that a dysregulated myocardial citrate release represents a specific chronic alteration of energy metabolism in cardiac patients. The 15-week-old spontaneously hypertensive rat (SHR) was chosen as our animal model of disease and the Wistar-Kyoto (WKY) rat as its matched control. Ex vivo work-performing hearts were perfused with a semi-recirculating buffer containing physiological concentrations of unlabeled (glucose) and ¹³C-labeled ([U-¹³C₃](lactate + pyruvate) and/or [1-¹³C]oleate) substrates. In parallel to the continuous monitoring of indices of the heart's functional and physiological status, the following metabolic parameters were documented: (i) citrate release rates and citric acid cycle intermediate tissue levels, (ii) the contribution of fatty acids as well as pyruvate decarboxylation and carboxylation to citrate synthesis, and (iii) lactate and pyruvate uptake and efflux rates. Working hearts from both rat species showed a similar percent contribution of carbohydrates for citrate synthesis through decarboxylation (70%) and carboxylation (10%). SHR hearts showed the following metabolic alterations: a higher citrate release rate, which was associated with a parallel increase in its tissue level, a lower contribution of oleate -oxidation to citrate synthesis, and an accelerated efflux rate of unlabeled lactate from glycolysis. These metabolic changes were not explained by differences in myocardial oxygen consumption, cardiac performance or efficiency, nor correlated with indices of tissue necrosis or ischemia. This study demonstrates how the alliance between ex vivo semi-recirculating working perfused rat hearts with ¹³C-substrates and mass isotopomer analysis by GCMS, can provide an unprecedented insight into the metabolic phenotype of normal and diseased rat hearts. The clinical relevance of metabolic alterations herein documented in the SHR heart is suggested by its resemblance to those reported in cardiac patients. Taken altogether, our results raise the possibility that the increased citrate release of diseased hearts results from an imbalance between citrate synthesis and utilization rates, which becomes more apparent under conditions of substrate abundance.     

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

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

Acute hibernation decreases myocardial pyruvate carboxylation and citrate release

In the well-perfused heart, pyruvate carboxylation accounts for 3-6% of the citric acid cycle (CAC) flux, and CAC carbon is lost via citrate release. We investigated the effects of an acute reduction in coronary flow on these processes and on the tissue content of CAC intermediates. Measurements were made in an open-chest anesthetized swine model. Left anterior descending coronary artery blood flow was controlled by a extracorporeal perfusion circuit, and flow was decreased by 40% for 80 min to induce myocardial hibernation (n = 8). An intracoronary infusion of [U-(13)C(3)]lactate and [U-(13)C(3)]pyruvate was given to measure the entry of pyruvate into the CAC through pyruvate carboxylation from the (13)C-labeled isotopomers of CAC intermediates. Compared with normal coronary flow, myocardial hibernation resulted in parallel decreases of 65% and 79% in pyruvate carboxylation and net citrate release by the myocardium, respectively, and maintenance of the CAC intermediate content. Elevation of the arterial pyruvate concentration by 1 mM had no effect. Thus a 40% decrease in coronary blood flow resulted in a concomitant decrease in pyruvate carboxylation and citrate release as well as maintenance of the CAC intermediates.     

Metabolic adaptations during lactogenesis. Fatty acid synthesis in rabbit mammary tissue during pregnancy and lactation

1. Mammary tissue was obtained from rabbits at various stages of pregnancy and lactation and used for tissue-slice incubations (to measure the rate of fatty acid synthesis and CO(2) production) and to determine relevant enzymic activities. A biphasic adaptation in fatty acid synthetic capacity during lactogenesis was noted. 2. The first lactogenic response occurred between day 15 and 24 of pregnancy. Over this period fatty acid synthesis (from acetate) increased 14-fold and the proportions of fatty acids synthesized changed to those characteristic of milk fat (77-86% as C(8:0)+C(10:0) acids). 3. The second lactogenic response occurred post partum as indicated by increased rates of fatty acid synthesis and CO(2) production (from acetate and glucose) and increased enzymic activities. 4. Major increases in enzymic activities between mid-pregnancy and lactation were noted for ATP citrate lyase (EC 4.1.3.8), acetyl-CoA synthetase (EC 6.2.1.1), acetyl-CoA carboxylase (EC 6.4.1.2), fatty acid synthetase, glucose 6-phosphate dehydrogenase (EC 1.1.1.49), and 6-phosphogluconate dehydrogenase (EC 1.1.1.44). Smaller increases in activity occurred with glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) and NADP(+)-isocitrate dehydrogenase (EC 1.1.1.42) and the activity of NADP(+)-malate dehydrogenase (EC 1.1.1.40) was negligible at all periods tested. 5. During pregnancy and lactation there was a close temporal relationship between fatty acid synthetic capacity and the activities of ATP citrate lyase (r=0.94) and acetyl-CoA carboxylase (r=0.90).     

Isotopomer analysis of myocardial substrate metabolism: a systems biology approach

The increasing accessibility of mass isotopomer data via GC-MS and NMR technology has necessitated the use of a systematic and reliable method to take advantage of such data for flux analysis. Here we applied a nonlinear, optimization-based method to study substrate metabolism in cardiomyocytes using (13)C data from perfused mouse hearts. The myocardial metabolic network used in this study accounts for 257 reactions and 240 metabolites, which are further compartmentalized into extracellular space, cytosol, and mitochondrial matrix. Analysis of the perfused mouse heart showed that the steady-state ATP production rate was 16.6 +/- 2.3 micromol/min . gww, with 30% of the ATP coming from glycolysis. Of the four substrates available in the perfusate (glucose, pyruvate, lactate, and oleate), exogenous glucose forms the majority of cytosolic pyruvate. Pyruvate decaboxylation is significantly higher than carboxylation, suggesting that anaplerosis is low in the perfused heart. Exchange fluxes were predicted to be high for reversible enzymes in the citric acid cycle (CAC), but low in the glycolytic pathway. Pseudoketogenesis amounted to approximately 50% of the net ketone body uptake. Sensitivity analysis showed that the estimated flux distributions were relatively insensitive to experimental errors. The application of isotopomer data drastically improved the estimation of reaction fluxes compared to results computed with respect to reaction stoichiometry alone. Further study of 12 commonly used (13)C glucose mixtures showed that the mixtures of 20% [U-(13)C(6)] glucose, 80% [3 (13)C] glucose and 20% [U-(13)C(6)] glucose, 80% [4 (13)C] were best for resolving fluxes in the current network.     

Measurements of fatty acid and carbohydrate metabolism in te isolated working rat heart

The isolated working rat heart is a useful experimental model which allows contractile function to be measured in hearts perfused at physiologically relevant workloads. To maintain these high workloads the heart is required to generate a tremendous amount of energy. In vivo this energy is derived primarily from the oxidation of fatty acids. In many experimental situations it is desirable to perfuse the isolated working heart in the presence of physiologically relevant concentrations of fatty acids. This is particularly important when studying energy metabolism in the heart, or in determining how fatty acids alter the outcome of myocardial ischemic injury [1, 2]. The other major source of energy for the heart is derived from the oxidation of carbohydrates (glucose and lactate), with a smaller amount of ATP also being derived from glycolysis. Two byproducts of both fatty acid and carbohydrate metabolism are H2O and CO2. By labeling the glucose, lactate, or fatty acids in the perfusate with 3H or 14C the experimenter can quantitatively collect either 3H2O or 14CO2 produced by the heart. By using radioisotopes that are labeled at specific hydrogen or carbon molecules on the various energy substrates, and by knowing the specific activity of the radiolabeled substrate used, it is possible to determine the actual rate of flux through these individual pathways. This paper will describe the experimental protocols for directly measuring fatty acid and carbohydrate metabolism in isolated working rat hearts.     

Effects of pent-4-enoate on cellular redox state, glycolysis and fatty acid oxidation in isolated perfused rat heart

The metabolic effects of pent-4-enoate were studied in beating and potassium-arrested perfused rat hearts. The addition of 0.8mm-pent-4-enoate to the fluid used to perfuse a potassium-arrested heart resulted in a 70% increase in the O(2) consumption and a 66% decrease in the glycolytic flux as measured in terms of the de-tritiation of [3-(3)H]glucose, although the proportion of the O(2) consumption attributable to glucose oxidation decreased from an initial 30% to 10%. The pent-4-enoate-induced increase in O(2) consumption was only 15% in the beating heart. In the potassium-arrested heart, pent-4-enoate stimulated palmitate oxidation by more than 100% when measured in terms of the production of (14)CO(2) from [1-(14)C]palmitate, but in the beating heart palmitate oxidation was inhibited. Perfusion of the heart with pent-4-enoate had no effect on the proportion of pyruvate dehydrogenase found in the active form, in spite of large changes in the CoASH and acetyl-CoA concentrations and changes in their concentration ratios. The effects of pent-4-enoate on the cellular redox state were dependent on the ATP consumption of the heart. In the beating heart, pent-4-enoate caused a rapid mitochondrial NAD(+) reduction that subsequently faded out, so that the final state was more oxidized than the initial state. The arrested heart, however, remained in a more reduced state than initially, even after the partial re-oxidation that followed the initial rapid NAD(+) reduction. The ability of pent-4-enoate to increase or decrease fatty acid oxidation can be explained on the basis of the differential effects of pent-4-enoate on the concentration of citric acid-cycle intermediates under conditions of high or low ATP consumption of the myocardial cell. The proportion of the fatty acids in the fuel consumed by the heart is probably primarily determined by the regulatory mechanisms of glycolysis. When pent-4-enoate causes an increase in the citric acid-cycle intermediates, feedback inhibition of glycolysis results in an increase in the oxidation of fatty acids.     

Thyroid Hormone Reverses Aging-Induced Myocardial Fatty Acid Oxidation Defects and Improves the Response to Acutely Increased Afterload

Background

Subclinical hypothyroidism occurs during aging in humans and mice and may contribute to the development of heart failure. Aging also impairs myocardial fatty acid oxidation, causing increased reliance on flux through pyruvate dehydrogenase (PDH) to maintain function. We hypothesize that the metabolic changes in aged hearts make them less tolerant to acutely increased work and that thyroid hormone supplementation reverses these defects.

Methods

Studies were performed on young (Young, 4–6 months) and aged (Old, 22–24 months) C57/BL6 mice at standard (50 mmHg) and high afterload (80 mmHg). Another aged group received thyroid hormone for 3 weeks (Old-TH, high afterload only). Function was measured in isolated working hearts along with substrate fractional contributions (Fc) to the citric acid cycle (CAC) using perfusate with ¹³C labeled lactate, pyruvate, glucose and unlabeled palmitate and insulin.

Results

Old mice maintained cardiac function under standard workload conditions, despite a marked decrease in unlabeled (presumably palmitate) Fc and relatively similar individual carbohydrate contributions. However, old mice exhibited reduced palmitate oxidation with diastolic dysfunction exemplified by lower -dP/dT. Thyroid hormone abrogated the functional and substrate flux abnormalities in aged mice.

Conclusion

The aged heart shows diminished ability to increase cardiac work due to substrate limitations, primarily impaired fatty acid oxidation. The heart accommodates slightly by increasing efficiency through oxidation of carbohydrate substrates. Thyroid hormone supplementation in aged mice significantly improves cardiac function potentially through restoration of fatty acid oxidation.     

Regulation of NAD- and NADP-dependent isocitrate dehydrogenases by reduction levels of pyridine nucleotides in mitochondria and cytosol of pea leaves

Regulation of NAD- and NADP-dependent isocitrate dehydrogenases (NAD-ICDH, EC 1.1.1.41, and NADP-ICDH, EC 1.1.1.42) by the level of reduced and oxidized pyridine nucleotides has been investigated in pea (Pisum sativum L.) leaves. The affinities of mitochondrial and cytosolic ICDH enzymes to substrates and inhibitors were determined on partially purified preparations in forward and reverse directions. From the kinetic data, it follows that NADP(+)- and NAD(+)-dependent isocitrate dehydrogenases in mitochondria represent a system strongly responding to the intramitochondrial NADPH and NADH levels. The NADPH, NADP(+), NADH and NAD(+) concentrations were determined by subcellular fractionation of pea leaf protoplasts using membrane filtration in mitochondria and cytosol in darkness and in the light under saturating and limiting CO(2) conditions. The cytosolic NADPH/NADP ratio was about 1 and almost constant both in darkness and in the light. In mitochondria, the NADPH/NADP ratio was low in darkness (0.2) and increased in the light, reaching 3 in limiting CO(2) conditions compared to 1 in saturating CO(2). At high reduction levels of NADP and NAD observed at limiting CO(2) in the light, i.e. when photorespiratory glycine is the main mitochondrial substrate, isocitrate oxidation in mitochondria will be suppressed and citrate will be transported to the cytosol ('citrate valve'), where the cytosolic NADP-ICDH supplies 2-oxoglutarate for the photorespiratory ammonia refixation.     

Triiodothyronine increases myocardial function and pyruvate entry into the citric acid cycle after reperfusion in a model of infant cardiopulmonary bypass

Triiodothyronine (T3) supplementation improves clinical outcomes in infants after cardiac surgery using cardiopulmonary bypass by unknown mechanisms. We utilized a translational model of infant cardiopulmonary bypass to test the hypothesis that T3 modulates pyruvate entry into the citric acid cycle (CAC), thereby providing the energy support for improved cardiac function after ischemia-reperfusion (I/R). Neonatal piglets received intracoronary [2-(13)Carbon((13)C)]pyruvate for 40 min (8 mM) during control aerobic conditions (control) or immediately after reperfusion (I/R) from global hypothermic ischemia. A third group (I/R-Tr) received T3 (1.2 μg/kg) during reperfusion. We assessed absolute CAC intermediate levels and flux parameters into the CAC through oxidative pyruvate decarboxylation (PDC) and anaplerotic carboxylation (PC) using [2-(13)C]pyruvate and isotopomer analysis by gas and liquid chromatography-mass spectrometry and (13)C-nuclear magnetic resonance spectroscopy. When compared with I/R, T3 (group I/R-Tr) increased cardiac power and oxygen consumption after I/R while elevating flux of both PDC and PC (～4-fold). Although neither I/R nor I/R-Tr modified absolute CAC levels, T3 inhibited I/R-induced reductions in their molar percent enrichment. Furthermore, (13)C-labeling of CAC intermediates suggests that T3 may decrease entry of unlabeled carbons at the level of oxaloacetate through anaplerosis or exchange reaction with asparate. T3 markedly enhances PC and PDC fluxes, thereby providing potential substrate for elevated cardiac function after reperfusion. This T3-induced increase in pyruvate fluxes occurs with preservation of the CAC intermediate pool. Our labeling data raise the possibility that T3 reduces reliance on amino acids for anaplerosis after reperfusion.     

Citrate release by perfused rat hearts: a window on mitochondrial cataplerosis

Cytosolic citrate is proposed to play a crucial role in substrate fuel selection in the heart. However, little is known about factors regulating the transfer of citrate from the mitochondria, where it is synthesized, to the cytosol. Further to our observation that rat hearts perfused under normoxia release citrate whose (13)C labeling pattern reflects that of mitochondrial citrate (B. Comte, G. Vincent, B. Bouchard, and C. Des Rosiers. J. Biol. Chem. 272: 26117-26124, 1997), we report here data indicating that this citrate release is a specific process reflecting the mitochondrial efflux of citrate, a process referred to as cataplerosis. Indeed, measured rates of citrate release, which vary between 2 and 21 nmol/min, are modulated by the nature and concentration of exogenous substrates feeding acetyl-CoA (fatty acid) and oxaloacetate (lactate plus pyruvate) for the mitochondrial citrate synthase reaction. Such release rates that represent at most 2% of the citric acid cycle flux are in agreement with the activity of the mitochondrial tricarboxylate transporter whose participation is also substantiated by 1) parallel variations in citrate release rates and tissue levels of citrate plus malate, the antiporter, and 2) a lowering of the citrate release rate by 1,2, 3-benzenetricarboxylic acid, a specific inhibitor of the transporter. Taken together, the results from the present study indicate that citrate cataplerosis is modulated by substrate supply, in agreement with the role of cytosolic citrate in fuel partitioning, and occurs, at least in part, through the mitochondrial tricarboxylate transporter.