Andrew Halestrap

We examined the isoform-specific regulation of monocarboxylate transporter (MCT)1 and MCT4 expression by contractile activity in red and white tibialis anterior muscles. After 1 and 3 wk of chronic muscle stimulation (24 h/day), MCT1 protein expression was increased in the red muscles (+78%, P < 0.05). In the white muscles, MCT1 was increased after 1 wk (+191%) and then was decreased after 3 wk. In the red muscle, MCT1 mRNA accumulation was increased only after 3 wk (+21%; P < 0.05). In the white muscle, MCT1 mRNA was increased after 1 wk (+30%; P < 0.05) and 3 wk (+15%; P < 0.05). MCT4 protein was not altered in either the red or white muscles after 1 or 3 wk. MCT4 mRNA was transiently lowered (approximately 15%) in both muscles in the 1st wk, but MCT4 mRNA levels were back to control levels after 3 wk. In conclusion, chronic contractile activity induces the expression of MCT1 but not MCT4. This increase in MCT1 alone was sufficient to increase lactate uptake from the c...

1. Acetyl-CoA carboxylase activity was measured in extracts of rat epididymal fat-pads either on preparation of the extracts (initial activity) or after incubation of the extracts with citrate (total activity). In the presence of glucose or fructose, brief exposure of pads to insulin increased the initial activity of acetyl-CoA carboxylase; no increase occurred in the absence of substrate. Adrenaline in the presence of glucose and insulin decreased the initial activity. None of these treatments led to a substantial change in the total activity of acetyl-CoA carboxylase. A large decrease in the initial activity of acetyl-CoA carboxylase also occurred with fat-pads obtained from rats that had been starved for 36h although the total activity was little changed by this treatment. 2. Conditions of high-speed centrifugation were found which appear to permit the separation of the polymeric and protomeric forms of the enzyme in fat-pad extracts. After the exposure of the fat-pads to insulin...

The general anesthetic propofol has been shown to be cardioprotective. However, its benefits when used in cardioplegia during cardiac surgery have not been demonstrated. In this study, we investigated the effects of propofol on metabolic stress, cardiac function, and injury in a clinically relevant model of normothermic cardioplegic arrest and cardiopulmonary bypass. Twenty anesthetized pigs, randomized to propofol treatment (n = 8) and control (n = 12) groups, were surgically prepared for cardiopulmonary bypass (CPB) and cardioplegic arrest. Doses of warm blood cardioplegia were delivered at 15-min intervals during a 60-min aortic cross-clamped period. Propofol was continuously infused for the duration of CPB and was therefore present in blood cardioplegia. Myocardial biopsies were collected before, at the end of cardioplegic arrest, and 20 mins after the release of the aortic cross-clamp. Hemodynamic parameters were monitored and blood samples collected for cardiac troponin I meas...

1. Langendorff-perfused rat hearts were treated, either alone or in combination, with acetate, adrenaline or supraphysiological [Ca2+] to mimic increased workload. No significant increases in the PPi content of the freeze-clamped tissue were observed. 2. In contrast with liver mitochondria, isolated rat heart mitochondria incubated at a free [Mg2+] of 1.5 mM showed little or no increase in matrix PPi content or volume in the presence of 0.75 microM Ca2+; neither did 5 mM acetate cause heart mitochondrial PPi to increase. 3. At 0.1 mM Mg2+ some increase in matrix PPi content of heart mitochondria was observed, and increases in liver mitochondria were significantly greater. 4. Substantial increases in heart mitochondrial-matrix PPi content were observed at 1.5 mM Mg2+ when [Ca2+] > 1 microM, especially in the presence of acetate, or at 0.75 microM Ca2+ in the presence of bongkrekic acid to inhibit the adenine nucleotide translocase. 5. Total pyrophosphatase activity was similar in ...

We have used the intracellular pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) to characterize the substrate and inhibitor specificity of monocarboxylate transport into isolated rat heart cells. Further evidence was obtained for the presence of two lactate carriers present in heart cells (Wang et al., Biochem. J. 290: 249-258, 1993) both distinct from the recently cloned monocarboxylate transporter isoform 1 (MCT-1) found in many other cell types. Only one isoform was potently inhibited by alpha-cyano-4-hydroxycinnamate [CHC; inhibitor constant (Ki) 190 microM] and the stilbene disulfonates 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (Ki 79 microM) and 4,4'-dinitrostilbene-2,2'-disulfonate (Ki of cis- and trans-isomers 38 and 171 microM, respectively; neither isomer inhibits MCT-1). The second carrier had a Ki of approximately 3 mM for CHC and 0.5-2 mM for the stilbene disulfonates. Thus, unlike in many other tissues,...

We have recently shown that hearts of mice fed high-fat diet exhibit increased vulnerability to ischaemia and reperfusion (I/R) in parallel to changes in catalase protein expression, mitochondrial morphology and intracellular diastolic Ca(2+). To determine whether switching from high-fat back to normal diet alters vulnerability to I/R and to investigate cardiac cellular remodelling in relation to the mechanism(s) underlying I/R injury. Male C57BL/6J mice were fed a high-fat diet for 19-22 weeks; after which a subset of mice was switched back to normal diet for 4-6 weeks. Hearts from mice switched back to normal diet were more resistant to reperfusion injury compared to hearts from mice fed only high-fat diet. This was associated with a significant reversal in catalase expression (western blotting) and recovery of size and density of mitochondria (electron microscopy). In contrast, switching back to normal diet did not alter cardiomyocyte contractility or Ca(2+) transients compared t...

The nucleotide sequence of the rat monocarboxylate transporter MCT2 was determined from brain-derived cDNA. A polyclonal antibody was raised in chickens against the carboxyl terminal end of the deduced amino acid sequence and affinity purified. The MCT2 antibody identified a 46-kDa band on immunoblots and labeled kidney, skeletal muscle, and stomach consistent with the reported cellular expression for this transporter. Light microscopic immunocytochemistry indicated that the MCT2 transporter was abundant in glial limiting membranes, ependymocytes, and neuropil, particularly in the lacunosum molecular layer of hippocampus and the molecular layer of cerebellum. Labeled astrocytes were commonly observed in white matter. The distribution of this transporter differed in several respects from that previously reported for MCT1. MCT2 was abundantly distributed in astrocyte foot processes and was usually not detected in other cells of the cerebrovasculature, including vascular smooth muscle cells, pericytes, and endothelium. In addition, the granular layer of cerebellum, which showed little MCT1 labeling, exhibited MCT2 labeling of cellular processes in the neuropil surrounding the granule and Purkinje cells. The results lend support to the concept that astrocytes play a significant role in cerebral energy metabolism by transporting lactate and other monocarboxylates.

ABSTRACT ROS production during ischaemia-reperfusion induces mitochondrial permeability transition pore opening and so mediates reperfusion injury. Several groups have used real-time fluorescence to determine ROS production by isolated cardiomyocytes during simulated ischaemia-reperfusion but protocols and results vary. Few studies have utilised the more pathophysiologically relevant perfused heart. We have developed an ischaemia-reperfusion protocol that exposes isolated rat cardiac myocytes to conditions closely matching ischaemia-reperfusion in the perfused heart. Temperature and [oxygen] are tightly controlled and continuously monitored while ROS production is measured by fluorescence microscopy using dihydroethidium (DHE) or 2',7'-dichlorofluorescein (DCF). In parallel real-time multiwavelength surface fluorescence and reflectance were used to monitor autofluorescence and ROS production in the perfused rat heart together with measurements of haemodynamic function and infarct size. In isolated myocytes DCF fluorescence increased continuously throughout the experiment, but with pronounced acceleration at the start of ischaemia and at reperfusion. For DHE, nuclear fluorescence increased steadily during ischaemia but dropped to baseline within seconds of reperfusion. In perfused hearts the pattern of fluorescence change was similar; a rapid increase during onset of ischaemia, a larger increase at ischaemic contracture followed by a rapid decrease on reperfusion. To define the nature of the fluorescence changes, emission spectra were determined on the perfused hearts. These confirmed our conclusion that DHE is not a suitable dye to monitor ROS production in situ. DCF appears suitable for use in cardiomyocytes and we are developing suitable loading protocols for perfused heart measurements.

1. The kinetics of transport of pyruvate (Km 0.20 mM), L-lactate (Km 2.2 mM) and D-lactate (Ki 10.2 mM) into rat cardiac myocytes were studied and compared with those for guinea-pig heart cells [Poole, Halestrap, Price and Levi (1989) Biochem. J. 264, 409-418] whose equivalent values were 0.07, 2.3 and 6.6 mM respectively. Maximal rates of transport were about 5-fold higher in the rat heart cells. 2. 4,4'-Dibenzamidostilbene-2,2'-disulphonate (DBDS), a powerful inhibitor of monocarboxylate transport into erythrocytes [Poole & Halestrap (1991) Biochem. J. 275, 307-312], was found to be a potent but apparently partial inhibitor of lactate and pyruvate transport, with an apparent Ki value at 0.5 mM L-lactate of about 16 microM in both species. Maximal inhibition was 50% and 80% in rat and guinea-pig cells respectively. 3. The maximal extent of inhibition and apparent Ki values were dependent on both the substrate transported and its concentration. Maximum inhibition was less and the Ki was greater at higher substrate concentrations. 4. A variety of other stilbene disulphonates were studied which showed different Ki values and maximal extents of inhibition. 5. Phloretin was a significantly less potent inhibitor of transport into both rat (Ki 25 microM) and guinea-pig (Ki 16 microM) heart cells than into rat erythrocytes (Ki 1.4 microM). In the rat but not the guinea-pig heart cells, inhibition appeared partial (maximal inhibition 84%). 6. We demonstrate that our results can be explained by the presence of two monocarboxylate carriers in heart cells, both with Km values for L-lactate of about 2 mM and inhibited by alpha-cyano-4-hydroxycinnamate, but with different affinities for other substrates and inhibitors. One carrier is sensitive to inhibition by stilbene disulphonates and has lower Km values for pyruvate (0.05-0.10 mM) and D-lactate (5 mM), whereas the other has higher Km values for pyruvate (0.30 mM) and D-lactate (25 mM), and is relatively insensitive to stilbene disulphonates. Rat heart cells possess more of the latter carrier and guinea-pig heart cells more of the former. 7. The significance of these results for the study of lactate transport in the perfused heart is discussed.

To investigate the effects of a single session of prolonged cycle exercise [60% peak O2 uptake (VO2 peak) for 5-6 h] on metabolic adaptations in working vastus lateralis muscle, nine untrained males (peak O2 uptake = 47.2 +/- 1.1 ml x kg(-1) x min(-1), means +/- SE) were examined before (Pre) and at 2 (Post-2), 4 (Post-4), and 6 (Post-6) days after the training session. On the basis of 15 min of cycle exercise at 59% VO2 peak, it was found that training reduced (P < 0.05) exercise muscle lactate (mM) at Post-2 (6.65 +/- 0.69), Post-4 (7.74 +/- 0.63), and Post-6 (7.78 +/- 1.2) compared with Pre (10.9 +/- 1.3). No effect of training was observed on exercise ATP, phosphocreatine, and glycogen levels. After the single session of training, plasma volumes were elevated (P < 0.05) at Post-2 (6.7 +/- 1.7%), Post-4 (5.86 +/- 1.9), and Post-6 (5.13 +/- 2.5). The single exercise session also resulted in elevations (P < 0.05) in the monocarboxylate transporters MCT1 and MCT4 throughout the 6 days after exercise. Although epinephrine and norepinephrine both increased with exercise, only norepinephrine was reduced (P < 0.05) with training and only at Post-4. These results indicate that regulation of cellular lactate levels occurs rapidly and independently of other metabolic adaptations. It is proposed that increases in MCT and plasma volume are at least partly involved in the lower muscle lactate content observed after the training session by increasing lactate membrane transport and removal, respectively.

The pH-sensitive fluorescent indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) was used to measure lactate transport in single cardiac myocytes. Addition of lactate externally caused a rapid fall of intracellular pH (pHi), which was largely inhibited by 5 mM alpha-cyano-4-hydroxycinnamate (CHC), a specific inhibitor of the lactate carrier. Stilbene disulfonates such as 4,4'-dibenzamidostilbene-2,2'-disulfonate (DBDS) only partially inhibited the response, with inhibition being greater in guinea pig than rat myocytes. The data are consistent with two isoforms of the lactate carrier, one sensitive and one insensitive to DBDS, coexisting within a single myocyte and both having a stoichiometry of 1 lactate:1 proton. The initial rate of pHi fall was used to determine carrier kinetics. Rat myocytes had a Michaelis constant (Km) for external L-lactate of 2.74 mM and a Km for external pyruvate of 0.2 mM. Guinea pig cells had a Km for external L-lactate of 2.2 mM. Kinetics of lactate efflux were also evaluated using the rate of pHi recovery on removing external lactate. The Km and maximal rate values for efflux were both threefold higher than for influx and were related to each other and the transmembrane pH gradient as predicted by the Haldane relationship. It is suggested that under hypoxic conditions, the carrier may be the rate-limiting factor for lactate extrusion.

Rat epididymal fat-pads were incubated for 30min with glucose (2mg/ml) in the presence or absence of insulin. A twofold or greater increase in acetyl-CoA carboxylase activity was observed in extracts from insulin-treated tissue provided that assays were performed rapidly after extraction. This effect of insulin was evident whether or not extracts were prepared with albumin, and was not noticeably diminished by the presence of citrate or albumin or both in the assay. Incubation of extracts before assay led to activation of acetyl-CoA carboxylase and a marked diminution in the insulin effect. The enzyme in extracts was very sensitive to reversible inhibition by palmitoyl-CoA even in the presence of albumin (10mg/ml); inhibition persisted on dilution of enzyme and inhibitor. It is suggested that the observed activation of acetyl-CoA carboxylase by insulin may reflect changes in enzyme activity in the fat-cell resulting from the reduction of long-chain fatty-acyl-CoA that occurs in the presence of insulin. Activation of the enzyme with loss of the insulin effect on incubation of the extracts may be due to the slow dissociation of long-chain fatty acyl-CoA from the enzyme.

1. Effects of alpha-cyano-4-hydroxycinnamate and alpha-cyanocinnamate on a number of enzymes involved in pyruvate metabolism have been investigated. Little or no inhibition was observed of any enzyme at concentrations that inhibit completely mitochondrial pyruvate transport. At much higher concentrations (1 mM) some inhibition of pyruvate carboxylase was apparent. 2. Alpha-Cyano-4-hydroxycinnamate (1-100 muM) specifically inhibited pyruvate oxidation by mitochondria isolated from rat heart, brain, kidney and from blowfly flight muscle; oxidation of other substrates in the presence or absence of ADP was not affected. Similar concentrations of the compound also inhibited the carboxylation of pyruvate by rat liver mitochondria and the activation by pyruvate of pyruvate dehydrogenase in fat-cell mitochondria. These findings imply that pyruvate dehydrogenase, pyruvate dehydrogenase kinase and pyruvate carboxylase are exposed to mitochondrial matrix concentrations of pyruvate rather than to cytoplasmic concentrations. 3. Studies with whole-cell preparations incubated in vitro indicate that alpha-cyano-4-hydroxycinnamate or alpha-cyanocinnamate (at concentrations below 200 muM) can be used to specifically inhibit mitochondrial pyruvate transport within cells and thus alter the metabolic emphasis of the preparation. In epididymal fat-pads, fatty acid synthesis from glucose and fructose, but not from acetate, was markedly inhibited. No changes in tissue ATP concentrations were observed. The effects on fatty acid synthesis were reversible. In kidney-cortex slices, gluconeogenesis from pyruvate and lactate but not from succinate was inhibited. In the rat heart perfused with medium containing glucose and insulin, addition of alpha-cyanocinnamate (200 muM) greatly increased the output and tissue concentrations of lactate plus pyruvate but decreased the lactate/pyruvate ratio. 4. The inhibition by cyanocinnamate derivatives of pyruvate transport across the cell membrane of human erythrocytes requires much higher concentrations of the derivatives than the inhibition of transport across the mitochondrial membrane. Alpha-Cyano-4-hydroxycinnamate appears to enter erythrocytes on the cell-membrane pyruvate carrier. Entry is not observed in the presence of albumin, which may explain the small effects when these compounds are injected into whole animals.

alpha-Cyano-4-hydroxycinnamate greatly inhibits the transport of pyruvate but not that of acetate or butyrate in liver mitochondria and erythrocytes. In the latter, lactate uptake is also inhibited. It is concluded that a specific carrier is involved in membrane transport of pyruvate and that the plasma-membrane carrier may also be involved in lactate transport.

Reperfusion following a period of ischemia can salvage the myocardium only if the ischemic episode has not exceeded a certain time limit; beyond this point damage becomes irreversible. A key feature of the transition from reversible to irreversible injury is mitochondrial dysfunction which may involve the opening of a non-specific pore in the mitochondrial inner membrane. Pore opening can be induced in vitro by exposure of isolated mitochondria to high [Ca2+] and Pi. Such pore formation is sensitized by adenine nucleotide depletion and oxidative stress and can be blocked by the immunosuppressant cyclosporin A. Here we show that in isolated perfused rat hearts subjected to 30 min ischemia and 15 min reperfusion, 0.2 microM cyclosporin A restored the ATP/ADP ratio and AMP content (decreased and increased respectively during ischemia) to pre-ischemic values. In separate experiments functional recovery was assessed by monitoring the restoration of left ventricular developed pressure (LVP) during reperfusion after 30, 40 or 45 min ischemia. LVP was substantially improved in the presence of 0.2 microM cyclosporin A but did not return to pre-ischemic levels. The cyclosporin analogues G and H were less effective than cyclosporin A in protecting the heart during reperfusion. This is consistent with their reduced ability to protect isolated mitochondria from damage caused by Ca2+ overload. Surprisingly, reperfusion of hearts with 1 microM cyclosporin A reversed the protective effect seen at 0.2 microM.