L-lactate Uptake Research Articles

Characterization of lactate utilization and its implication on the physiology of Haemophilus influenzae

Haemophilus influenzae is a Gram-negative bacillus and a frequent commensal of the human nasopharynx. Earlier work demonstrated that in H. influenzae type b, l-lactate metabolism is associated with serum resistance and in vivo survival of the organism. To further gain insight into lactate utilization of the non-typeable (NTHi) isolate 2019 and laboratory prototype strain Rd KW20, deletion mutants of the l-lactate dehydrogenase (lctD) and permease (lctP) were generated and characterized. It is shown, that the apparent KM of l-lactate uptake is 20.1μM as determined for strain Rd KW20. Comparison of the COPD isolate NTHi 2019-R with the corresponding lctP knockout strain for survival in human serum revealed no lactate dependent serum resistance. In contrast, we observed a 4-fold attenuation of the mutant strain in a murine model of nasopharyngeal colonization. Characterization of lctP transcriptional control shows that the lactate utilization system in H. influenzae is not an inductor inducible system. Rather negative feedback regulation was observed in the presence of l-lactate and this is dependent on the ArcAB regulatory system. Additionally, for 2019 it was found that lactate may have signaling function leading to increased cell growth in late log phase under conditions where no l-lactate is metabolized. This effect seems to be ArcA independent and was not observed in strain Rd KW20. We conclude that l-lactate is an important carbon-source and may act as host specific signal substrate which fine tunes the globally acting ArcAB regulon and may additionally affect a yet unknown signaling system and thus may contribute to enhanced in vivo survival.

Lactate-mediated glia-neuronal signalling in the mammalian brain

Astrocytes produce and release L-lactate as a potential source of energy for neurons. Here we present evidence that L-lactate, independently of its caloric value, serves as an astrocytic signalling molecule in the locus coeruleus (LC). The LC is the principal source of norepinephrine to the frontal brain and thus one of the most influential modulatory centers of the brain. Optogenetically activated astrocytes release L-lactate, which excites LC neurons and triggers release of norepinephrine. Exogenous L-lactate within the physiologically relevant concentration range mimics these effects. L-lactate effects are concentration-dependent, stereo-selective, independent of L-lactate uptake into neurons and involve a cAMP-mediated step. In vivo injections of L-lactate in the LC evokes arousal similar to the excitatory transmitter, L-glutamate. Our results imply the existence of an unknown receptor for this ‘glio-transmitter’.

Crucial Residue Involved in L-Lactate Recognition by Human Monocarboxylate Transporter 4 (hMCT4)

BackgroundMonocarboxylate transporters (MCTs) transport monocarboxylates such as lactate, pyruvate and ketone bodies. These transporters are very attractive therapeutic targets in cancer. Elucidations of the functions and structures of MCTs is necessary for the development of effective medicine which targeting these proteins. However, in comparison with MCT1, there is little information on location of the function moiety of MCT4 and which constituent amino acids govern the transport function of MCT4. The aim of the present work was to determine the molecular mechanism of L-lactate transport via hMCT4.Experimental approachTransport of L-lactate via hMCT4 was determined by using hMCT4 cRNA-injected Xenopus laevis oocytes. hMCT4 mediated L-lactate uptake in oocytes was measured in the absence and presence of chemical modification agents and 4,4′-diisothiocyanostilbene-2,2′-disulphonate (DIDS). In addition, L-lactate uptake was measured by hMCT4 arginine mutants. Immunohistochemistry studies revealed the localization of hMCT4.ResultsIn hMCT4-expressing oocytes, treatment with phenylglyoxal (PGO), a compound specific for arginine residues, completely abolished the transport activity of hMCT4, although this abolishment was prevented by the presence of L-lactate. On the other hand, chemical modifications except for PGO treatment had no effect on the transport activity of hMCT4. The transporter has six conserved arginine residues, two in the transmembrane-spanning domains (TMDs) and four in the intracellular loops. In hMCT4-R278 mutants, the uptake of L-lactate is void of any transport activity without the alteration of hMCT4 localization.ConclusionsOur results suggest that Arg-278 in TMD8 is a critical residue involved in substrate, L-lactate recognition by hMCT4.

L-Lactate metabolism in HEP G2 cell mitochondria due to the l-lactate dehydrogenase determines the occurrence of the lactate/pyruvate shuttle and the appearance of oxaloacetate, malate and citrate outside mitochondria

As part of an ongoing study of l-lactate metabolism both in normal and in cancer cells, we investigated whether and how l-lactate metabolism occurs in mitochondria of human hepatocellular carcinoma (Hep G2) cells. We found that Hep G2 cell mitochondria (Hep G2-M) possess an l-lactate dehydrogenase (ml-LDH) restricted to the inner mitochondrial compartments as shown by immunological analysis, confocal microscopy and by assaying ml-LDH activity in solubilized mitochondria. Cytosolic and mitochondrial l-LDHs were found to differ from one another in their saturation kinetics. Having shown that l-lactate itself can enter Hep G2 cells, we found that Hep G2-M swell in ammonium l-lactate, but not in ammonium pyruvate solutions, in a manner inhibited by mersalyl, this showing the occurrence of a carrier-mediated l-lactate transport in these mitochondria. Occurrence of the l-lactate/pyruvate shuttle and the appearance outside mitochondria of oxaloacetate, malate and citrate arising from l-lactate uptake and metabolism together with the low oxygen consumption and membrane potential generation are in favor of an anaplerotic role for l-LAC in Hep G2-M.

D‐Lactate Disturbed Mitochondrial Energy Production in Rat Brain and Heart but not Liver

High D-lactate concentrations are associated with neurological and cardiac toxicity in both humans and animals. The neurological symptoms are similar to those of inherited or acquired abnormalities of pyruvate metabolism. We hypothesized that intracellular D-lactate (DLA) inhibits mitochondrial uptake of L-lactate (LLA) and/or pyruvate, thereby decreasing mitochondrial energy production. In our study, respiration rates in rat brain, heart and liver mitochondria using DLA, LLA and pyruvate alone or in combination were measured. In brain (heart), DLA had 53% and 75% (39% and 86%) lower state 3 respiration rates (S3) and 49% and 72% (38% and 66%) lower respiratory control ratios (RCR) compared to LLA and pyruvate, respectively (p<0.05). DLA also reduced S3 and RCR of LLA and pyruvate in brain and heart (p<0.05). DLA dehydrogenase activities were 61% and 51% lower in brain and heart mitochondria compared to liver, respectively, whereas LLA dehydrogenase LDH activities were similar across tissues. In the presence of a LDH inhibitor, LLA reduced S3 and RCR values of pyruvate in all tissues. Respiration was also inhibited by the monocarboxylate transporter inhibitor with DLA, LLA or pyruvate as substrates. In conclusion, DLA was a poor respiratory substrate and inhibited LLA and pyruvate usage in brain and heart. This may explain, in part, the neurological and cardiac toxicity caused by high DLA levels. This work was funded by Natural Sciences and Engineering Research Council of Canada.

Bicarbonate-dependent and bicarbonate-independent mechanisms contribute to nondiffusive uptake of acetate in the ruminal epithelium of sheep

The present study investigated the significance of apical transport proteins for ruminal acetate absorption and their interaction with different anions. In anion competition experiments in the washed reticulorumen, chloride disappearance rate (initial concentration, 28 mM) was inhibited by the presence of a short-chain fatty acid mixture (15 or 30 mM of each acetate, propionate, and butyrate). Disappearance rates of acetate and propionate, but not butyrate (initial concentration, 25 mM each) were diminished by 40 or 80 mM chloride. In isolated ovine ruminal epithelia mounted in Ussing chambers, an increase in chloride concentration from 4.5 to 90 mM led to a decrease of apical acetate uptake at a concentration of 0.5 mM. Mucosal nitrate inhibited acetate uptake most potently whereas sulfate had no effect. Decreasing mucosal pH from 7.4 to 6.1 approximately doubled uptake of acetate both at 0.5 and 10 mM, but this doubling was almost abolished when HCO(3)(-) was absent. The stimulated uptake at mucosal pH 6.1 consisted of a bicarbonate-dependent, nitrate-inhibitable part (K(m) = 54 mM) and a bicarbonate-independent component (K(m) = 12 mM) that was also sensitive to nitrate inhibition. Maximal uptake was three times larger for bicarbonate-dependent vs. bicarbonate-independent uptake. Mucosal addition of 200 microM DIDS, 400 microM p-chloromercuribenzene sulfonic acid, 800 microM p-hydroxymercuribenzoic acid, or 100 microM phloretin had no effects on acetate uptake although the latter two inhibited l-lactate uptake. Our data conclusively show a dominant involvement of proteins in apical acetate uptake. Previously described pH effects on acetate absorption originate mainly from modulation of acetate/bicarbonate exchange. Additionally, there is bicarbonate-independent uptake of acetate anions that is protein coupled but not via monocarboxylate cotransporter.

Inhibition of astrocytic energy metabolism by d-lactate exposure impairs memory

Bead discrimination learning in day-old chicken was inhibited by bilateral injection into the intermediate medial mesopallium (IMM), a homolog of the mammalian brain cortex, of the poorly metabolized enantiomer of l-lactate, d-lactate. The window of vulnerability extended from 10 min before training to 20 min after training. Unilateral injection 10 min before training inhibited only in the left IMM, whereas 10 min after training injection was only inhibitory if made into the right hemisphere. The pre-training administration caused memory loss from the earliest time tested whereas memory was maintained for another 20 min when d-lactate was injected 10 min post-training. The ability of acetate, an astrocyte-specific substrate, injected into the IMM to counteract the inhibitory effect was tested. Following d-lactate injection 10 min before training, rescue of memory immediately after training was achieved by acetate as long as aspartate, an oxaloacetate precursor, was also present. This suggests that pyruvate carboxylation is necessary for net synthesis of glutamate, which is known to occur at this time [Gibbs, M.E., Lloyd, H.G.E., Santa, T., Hertz, L., 2007. Glycogen is a preferred glutamate precursor during learning in 1-day-old chick: biochemical and behavioral evidence. J. Neurosci. Res., 85, 3326–3333]. However, acetate alone rescued memory 20 min post-training (following d-lactate injection 10 min after training), indicating that pyruvate at this time is used for energy production, consistent with memory inhibition by dinitrophenol. These findings suggest that d-lactate acts by inhibiting uptake of l-lactate into astrocytes (an extracellular effect) or metabolism of pyruvate in astrocytic mitochondria (an intracellular effect). An apparent lag phase between the administration of d-lactate and its inhibition of learning favors the latter possibility. Thus, under the present experimental conditions d-lactate acts as an astrocytic metabolic inhibitor rather than as an inhibitor of neuronal l-lactate uptake, as has occasionally been suggested. Analogously, a rare reversible neurological syndrome with memory deficits, d-lactate encephalopathy, may mainly or exclusively be due to astrocytic malfunction.

Transport and metabolism of l-lactate occur in mitochondria from cerebellar granule cells and are modified in cells undergoing low potassium dependent apoptosis

Having confirmed that externally added l-lactate can enter cerebellar granule cells, we investigated whether and how l-lactate is metabolized by mitochondria from these cells under normal or apoptotic conditions.(1)l-lactate enters mitochondria, perhaps via an l-lactate/H+ symporter, and is oxidized in a manner stimulated by ADP. The existence of an l-lactate dehydrogenase, located in the inner mitochondrial compartment, was shown by immunological analysis. Neither the protein level nor the Km and Vmax values changed en route to apoptosis.(2)In both normal and apoptotic cell homogenates, externally added l-lactate caused reduction of the intramitochondrial pyridine cofactors, inhibited by phenylsuccinate. This process mirrored l-lactate uptake by mitochondria and occurred with a hyperbolic dependence on l-lactate concentrations. Pyruvate appeared outside mitochondria as a result of external addition of l-lactate. The rate of the process depended on l-lactate concentration and showed saturation characteristics. This shows the occurrence of an intracellular l-lactate/pyruvate shuttle, whose activity was limited by the putative l-lactate/pyruvate antiporter. Both the carriers were different from the monocarboxylate carrier.(3)l-lactate transport changed en route to apoptosis. Uptake increased in the early phase of apoptosis, but decreased in the late phase with characteristics of a non-competitive like inhibition. In contrast, the putative l-lactate/pyruvate antiport decreased en route to apoptosis with characteristics of a competitive like inhibition in early apoptosis, and a mixed non-competitive like inhibition in late apoptosis.

The Role of Monocarboxylate Transporter 2 and 4 in the Transport of γ-Hydroxybutyric Acid in Mammalian Cells

Monocarboxylate transporter 1 (MCT1) is an important determinant of the renal transport of the drug of abuse, gamma-hydroxybutyric acid (GHB). The objective of this study was to investigate the role of MCT2 and MCT4, present in tissues including intestine, kidney, skeletal muscle, and brain, in the membrane transport of GHB and the MCT substrate l-lactate. mRNA and protein of MCT2 and MCT4 were expressed in MDA-MB231 cells, as detected by reverse transcription-polymerase chain reaction and Western blot analysis; MCT1 and MCT3 were not detected. The uptake of GHB or l-lactate by MDA-MB231 cells was pH-dependent but not sodium-dependent. The concentration-dependent uptake of GHB was best fitted to a single-transporter model with a diffusional clearance component (K(m) of 17.6 +/- 1.5 mM, V(max) of 50.6 +/- 9.0 nmol x mg(-1) min(-1) and diffusional clearance of 0.20 +/- 0.07 microl x mg(-1) min(-1)). On the other hand, the concentration-dependent uptake of l-lactate was best fitted to a two-transporter model (K(m) of 21 +/- 2.5 and 3.0 +/- 1.5 mM, and V(max) of 268 +/- 72 and 62.9 +/- 42.2 nmol x mg(-1)min(-1), respectively). The uptake of GHB and l-lactate was inhibited by MCT inhibitors alpha-cyano-4-hydroxycinnamate (CHC), phloretin, and p-chloromercuribenzoic acid; CHC inhibited GHB and l-lactate uptake with IC(50) values of 1.71 +/- 0.39 and 0.71 +/- 0.11 mM, respectively. Small interfering RNA treatment to silence MCT2 or MCT4 significantly decreased their protein expression and the uptake of l-lactate and GHB; however, the decrease in GHB uptake with MCT2 inhibition was smaller than that for MCT4. This investigation demonstrated that GHB is a substrate for both MCT2 and MCT4; these transporters may be important in the nonlinear disposition of GHB, as well as influencing its tissue distribution.

Monocarboxylate Transporter (MCT) Mediates the Transport of γ-Hydroxybutyrate in Human Kidney HK-2 cells

Previous studies in our laboratory have suggested that GHB may undergo renal reabsorption mediated by monocarboxylic acid transporters (MCT). The objectives of this study were to characterize the renal transport of GHB using HK-2 cells and the role of MCT in the renal transport of GHB. Western blot was used to detect the protein expression of MCT1, 2, and 4. Cellular uptake and directional flux studies were conducted to investigate the transport of GHB and L-lactate. RNA interference assay was used to investigate the involvement of MCT isoforms in the transport of GHB. MCT1, 2 and 4 were present in HK-2 cells. The cellular uptake of L-lactate and GHB exhibited pH- and concentration-dependence (L-lactate: K (m) of 6.5 +/- 1.1 mM and V (max) of 340 +/- 60 nmol mg(-1)min(-1); GHB: K (m) of 2.07 +/- 0.79 mM, V (max) of 27.6 +/- 9.3 nmol mg(-1)min(-1), and a diffusional clearance of 0.54 +/- 0.15 microl mg(-1)min(-1)), but not sodium-dependence. alpha-Cyano-4-hydroxycinnamate (CHC) competitively inhibited the uptake of GHB and L-lactate with inhibition constants (K (i)) of 0.28 +/- 0.1 mM, and 0.19 +/- 0.03 mM, respectively. Using small-interference RNA (siRNA) for MCT1, the protein expression of MCT1 and the uptake of L-lactate and GHB were significantly decreased. The siRNA treatment of MCT2 in HK-2 cells inhibited the uptake of GHB by 17%, and the siRNA treatment of MCT4 demonstrated no inhibition of GHB uptake. GHB exhibited a directional flux across HK-2 monolayer from apical to basal chambers in the presence of a pH gradient of pH 6.0 to pH 7.4. These data suggest that MCT1 represents an important transporter for GHB transport in renal tubule cells, responsible for the reabsorption of GHB in the kidney.

Placental Lactate Transporter Activity and Expression in Intrauterine Growth Restriction

To compare lactate uptake in the microvillous plasma membrane (maternal facing [MVM]) in term and preterm placentas in intrauterine growth restriction (IUGR) and appropriate weight for gestational age (AGA) controls, and in the basal plasma membrane (fetal facing [BM]) at term. In addition, we examine the expression of monocarboxylate transporters (MCT1 and MCT4). We measured [14C] L-lactate uptakes into vesicles prepared from MVM and BM, stimulated by an inwardly directed H+ gradient. MCT expression was examined by Western blotting. In term placentas, mean (+/- SE) [14C] L-lactate uptake into MVM vesicles of the IUGR (n = 6) and AGA (n = 11) groups at initial rate was similar (15.4 +/- 2.3 versus 15.0 +/- 1.1 pmol/mg protein/20 s). In preterm placentas, in IUGR (n = 3) and AGA (n = 3) groups, [14C] l-lactate uptake into MVM was also not significantly different. In BM vesicles from term placentas, [14C] L-lactate uptake was significantly lower in IUGR (n = 5) than in AGA (n = 6) controls (3.6 +/- 0.4 versus 5.6 +/- 0.6 pmol/mg protein/20 s, P <.05). MCT1 and MCT4 were expressed in BM vesicles, but there was no difference in expression between the IUGR and AGA groups. These findings suggest that in IUGR placental lactate transport capacity in the BM is reduced, which may adversely affect placental lactate clearance.

Transport of γ-Hydroxybutyrate in Rat Kidney Membrane Vesicles: Role of Monocarboxylate Transporters

Intoxication with gamma-hydroxybutyrate (GHB) is associated with coma, seizure, and death; treatment of overdoses is symptomatic. Previous studies in our laboratory have demonstrated that L-lactate and pyruvate treatment can increase the renal clearance of GHB and increase its elimination in rats, suggesting that GHB may undergo renal reabsorption mediated by monocarboxylic acid transporters (MCTs). The goals of this study were to characterize the renal transport of GHB in rats and to determine the role of MCT in its renal transport. Brush-border membrane (BBM) and basolateral membrane (BLM) vesicles were isolated from rat kidney cortex, and the uptake of L-lactate and GHB was characterized. L-Lactate and GHB undergo both pH- and sodium-dependent transport in BBM vesicles and pH-dependent transport in BLM vesicles. A simple Michaelis-Menten equation best described the pH-dependent uptake of GHB in BBM (Km, 8.0 +/- 1.8 mM; Vmax, 838 +/- 45 pmol/mg/s) and in BLM vesicles (Km, 10.5 +/- 2.6 mM; Vmax, 806 +/- 253 pmol/mg/s). mRNA of MCT1 and MCT2 was determined in rat kidney cortex using reverse transcriptase-polymerase chain reaction; using Western blot, the protein expression of MCT1 was present mainly in BLM vesicles, with weak expression in BBM vesicles, whereas that of MCT2 was exclusively in BLM vesicles. Studies with rat MCT1 gene-transfected MDA-MB231 cells demonstrated that GHB was a substrate of MCT1. The data suggest that rat MCT1 may represent an important transporter for GHB in renal tubule cells. This investigation provides evidence for the importance of MCTs in the reabsorption of the monocarboxylic acids l-lactate and GHB in the kidney.

Transfer of glycogen‐derived lactate from astrocytes to axons via specific monocarboxylate transporters supports mouse optic nerve activity

It is hypothesized that L-lactate derived from astrocyte glycogen sustains axon excitability in mouse optic nerve (MON). This theory was tested by using a competitive antagonist of L-lactate transport and immunocytochemistry to determine whether transport proteins are appropriately distributed in adult MON. L-lactate sustained the compound action potential (CAP), indicating that exogenous L-lactate was an effective energy substrate. During 60 min of aglycemia, the CAP persisted for 30 min, surviving on a glycogen-derived substrate (probably lactate), before failing. After failing, the CAP could be partially rescued by restoring 10 mM glucose or 20 mM L-lactate. Aglycemia in the presence of 20 mM D-lactate, a metabolically inert but transportable monocarboxylate, resulted in accelerated CAP decline compared with aglycemia alone, suggesting that D-lactate blocked the axonal uptake of glycogen-derived L-lactate, speeding the onset of energy failure and loss of the CAP. The CAP was maintained for up to 2 hr when exposed to 20% of normal bath glucose (i.e., 2 mM). To test whether glycogen-derived L-lactate "supplemented" available glucose (2 mM) in supporting metabolism, L-lactate uptake into axons was reduced by the competitive inhibitor D-lactate. Indeed, in the presence of 20 mM D-lactate, the CAP was lost more rapidly in MONs bathed in 2 mM glucose artificial cerebrospinal fluid. Immunocytochemical staining demonstrated cell-specific expression of monocarboxylate transporter (MCT) subtypes, localizing MCT2 predominantly to axons and MCT1 predominantly to astrocytes, supporting the idea that L-lactate is released from astrocytes and taken up by axons as an energy source for sustaining axon excitability.

Polarized Lactate Transporter Activity and Expression in the Syncytiotrophoblast of the Term Human Placenta

We investigated the polarization of l-lactate transport in human syncytiotrophoblast by measuring uptake of [ 14C] l-lactate by both microvillous (maternal-facing; MVM) and basal (fetal-facing; BM) plasma membranes. [ 14C] l-lactate uptake by MVM and BM was stimulated in the presence of an inwardly directed H +gradient, with a significantly higher uptake in MVM than in BM at initial rate (15.4±2.3 vs 5.6±0.6 pmol/mg protein/20 sec). Stereospecific inhibition was observed in MVM, with a higher affinity for l-lactate compared with d-lactate. In BM, there was no difference in the inhibition by these two stereoisomers. Inhibition of lactate uptake in both MVM and BM by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), an inhibitor of monocarboxylate transporter (MCT) activity, indicated MCT-mediated mechanisms across both membranes. Kinetic modelling supported a two-transporter model as the best fit for both MVM and BM, the K mof the major component being 6.21 m m and 25.01 m m in MVM and BM respectively. Western blotting and immunolocalization examining the distribution of MCT1 and MCT4, showed that MCT expression was polarized, MCT1 being predominantly localized to BM and MCT4 showing greater abundance on MVM. CD147, a chaperone protein for MCT1 and MCT4, was equally expressed by both membranes. These studies demonstrate that the opposing plasma membranes of human syncytiotrophoblast are polarized with respect to both MCT activity and expression.

Intraruminal propionate supplementation modifies hindlimb energy metabolism without changing the splanchnic release of glucose in growing lambs

The influence of propionate supplementation on the splanchnic metabolism of energy-yielding nutrients and the supply of glucose to the hindlimb was investigated in growing lambs. Six rumen-cannulated and multicatheterized lambs (32.2 kg), fed frozen rye grass at 690 kJ metabolizable energy intake/d per kg body weight0.75, were infused intraruminally with a salt solution (control) or with propionate solutions at 0.55 mol/d (P1) or 0.98 mol/d (P2) according to a replicated Latin square design. In the rumen fluid, supplementation decreased the acetate:propionate molar ratio from 2.36:1 to 1.37:1, without modifying the ruminal concentrations of acetate and NH4. As a result, the portal appearance of propionate increased by 51 and 72 % with P1 and P2, respectively, and that of l-lactate doubled. Across the liver, net extraction of propionate increased by 47 and 67 % with P1 and P2, respectively. However, the net hepatic production of glucose remained unchanged, probably as the result of a substantial rise in insulin secretion and its hepatic extraction. Overall, the net splanchnic release of acetate, glucose and butyrate was not modified while that of l-lactate increased. Despite this, the net uptake of acetate, glucose, l-lactate and non-esterified fatty acids by the hindlimb increased. Propionate probably enhanced the storage of energy-yielding nutrients in the hindlimb, despite their unchanged release by the splanchnic tissues and the unmodified insulinaemia. Regulatory mechanisms are not clear.

The Membrane-Bound [formula omitted]- and [formula omitted]-Lactate Dehydrogenase Activities in Mitochondria from Euglena gracilis

The activity of the pyridine nucleotide-independent lactate dehydrogenase (iLDH) was characterized in mitochondria isolated from the protist Euglena gracilis. The dissociation constants for l- and d-lactate were similar, but the Vmax was higher with the d isomer. A ping-pong kinetic mechanism was displayed with 2,4-dichlorophenol-indolphenol (DCPIP), or coenzyme Q1, reacting as the second substrate with the modified, reduced enzyme. Oxamate was a competitive inhibitor against both l- and d-lactate. Oxalate exerted a mixed-type inhibition regarding l- or d-lactate and also against DCPIP. The rate of l-lactate uptake was partially inhibited by mersalyl and lower than the rate of dehydrogenation, which was mersalyl-insensitive. These data suggested that the active site of l-iLDH was orientated toward the intermembrane space. The following observations indicated the existence of two stereo-specific iLDH enzymes in the inner membrane of Euglena mitochondria: a greater affinity of the d-iLDH for both inhibitors, d-iLDH thermo-stability at 70°C and denaturation of l-iLDH, opposite signs in the enthalpy change for the association reaction of the isomers to the enzyme, differential solubilization of both activities with detergents, and different molecular mass.

Determination of transport kinetics of chick MCT3 monocarboxylate transporter from retinal pigment epithelium by expression in genetically modified yeast.

Monocarboxylate transporters (MCTs) comprise a group of highly homologous proteins that reside in the plasma membrane of almost all cells and which mediate the 1:1 electroneutral transport of a proton and a lactate ion. The isoform MCT3 is restricted to the basal membrane of the retinal pigment epithelium where it regulates lactate levels in the neural retina. Kinetic analysis of this transporter poses formidable difficulties due to the presence of multiple lactate transporters and their complex interaction with MCTs in adjacent cells. To circumvent these problems, we expressed both the MCT3 gene and a green fluorescent protein-tagged MCT3 construct in Saccharomyces cerevisiae. Since L-lactate metabolism in yeast depends on the CYB2 gene, we disrupted CYB2 to study the MCT3 transporter activity free from the complications of metabolism. Under these conditions L-lactate uptake varied inversely with pH, greater uptake being associated with lower pH. Whereas the V(max) was invariant, the K(m) increased severalfold as the pH rose from 6 to 8. In addition, MCT3 was highly resistant to a number of "classical" inhibitors of lactate transport. Last, studies with diethyl pyrocarbonate and p-chloromercuribenzenesulfonate set limitations on the locus of potential residues involved in the critical site of lactate translocation.

Net absorption and hepatic metabolism of glucose, L-lactate, and volatile fatty acids by steers fed diets containing sorghum grain processed as dry-rolled or steam-flaked at different densities.

We determined the effect of processing method (dry-rolled [DR] vs steam-flaked [SF]) and degree of processing (flake density; FD) of SF sorghum grain on splanchnic (gut and liver) metabolism of energy-yielding nutrients by growing steers. Diets contained 77% sorghum grain, either DR or SF, with SF at densities of 437, 360, or 283 g/L (SF34, SF28, or SF22). Eight multicatheterized steers (340 kg initial BW) were used in a randomized complete block design. Net output or uptake of glucose, L-lactate, VFA, and beta-hydroxybutyrate (BHBA) were measured across portal-drained viscera (PDV), liver, and splanchnic (PDV plus liver) tissues. Net absorption of glucose across PDV was negative and similar for all treatments (average of -104 g/d). Decreasing FD of SF sorghum grain linearly increased (P < or = .04) net absorption and splanchnic output of L-lactate by 20 and 130%, respectively, and hepatic synthesis (P = .06) and splanchnic output (P = .01) of glucose by 50%. Reducing FD did not alter output or uptake of acetate or n-butyrate by gut and liver tissues, but linearly decreased (P = .06) splanchnic output of BHBA by 40%. Net absorption (P = .18) and splanchnic output (P = .15) of propionate tended to be increased linearly by 50% with decreasing FD. Neither processing method (SF vs DR) nor degree of processing (varying FD) altered hepatic nutrient extraction ratios or estimated net absorption and splanchnic output of energy. Maximal contribution of propionate, L-lactate, and amino acids (alpha-amino N) to gluconeogenesis averaged 49, 11, and 20%, respectively. Feeding steers SF compared to DR diets did not alter net output or uptake of energy-yielding nutrients across splanchnic tissues, except net absorption of acetate tended to be greater (P = .13) for steers fed DR. Increasing degree of grain processing in the present study, by incrementally decreasing FD, tended to linearly increase the net absorption of glucose precursors (propionate and lactate), resulting in linear increases in synthesis and output of glucose by the liver to extrasplanchnic tissues (e.g., muscle).

A Monocarboxylate Transporter MCT1 is Located at the Basolateral Pole of Rat Jejunum

We have functionally expressed and identified a monocarboxylate transporter (MCT1) from rat jejunal enterocyte and we provide evidence for its basolateral localization. Poly(A)+ RNA isolated from rat jejunum was injected into Xenopus laevis oocytes and expression of a proton-lactate symporter was investigated by means of L-[14C]lactate uptake. The existence of an endogenous capacity for L-lactate transport was demonstrated; when, however, oocytes were injected with jejunal mRNA, an expressed L-lactate uptake was seen which differed from the endogenous transporter since it was significantly pH dependent. After sucrose density gradient fractionation, the highest expression of the pH-dependent lactate uptake was detected with the mRNA size fraction of about 2-3 kb in length. The substrate specificity, stereoselectivity and sensitivity to pCMBS (an organomercurial thiol reagent that modifies cysteine residues) of the expressed transport were in good agreement with results previously obtained using isolated jejunal basolateral membranes. Using the reverse transcriptase-polymerase chain reaction, the presence of mRNA coding for the MCT1 isoform was demonstrated in jejunal enterocytes. These data, together with previous results, suggest that MCT1 is a major route for lactate efflux across the basolateral membrane of rat jejunum; this is in contrast to current opinion which restricts the presence of MCT1 to the apical membrane of the whole small intestine.

Identification and characterization of a monocarboxylate transporter (MCT1) in pig and human colon: its potential to transport L-lactate as well as butyrate.

1. Oligonucleotide primers based on the human heart monocarboxylate transporter (MCT1) cDNA sequence were used to isolate a 544 bp cDNA product from human colonic RNA by reverse transcription-polymerase chain reaction (RT-PCR). The sequence of the RT-PCR product was identical to that of human heart MCT1. Northern blot analysis using the RT-PCR product indicated the presence of a single transcript of 3.3 kb in mRNA isolated from both human and pig colonic tissues. Western blot analysis using an antibody to human MCT1 identified a specific protein with an apparent molecular mass of 40 kDa in purified and well-characterized human and pig colonic lumenal membrane vesicles (LMV). 2. Properties of the colonic lumenal membrane L-lactate transporter were studied by the uptake of L-[U-14C]lactate into human and pig colonic LMV. L-Lactate uptake was stimulated in the presence of an outward-directed anion gradient at an extravesicular pH of 5.5. Transport of L-lactate into anion-loaded colonic LMV appeared to be via a proton-activated, anion exchange mechanism. 3. L-Lactate uptake was inhibited by pyruvate, butyrate, propionate and acetate, but not by Cl- and SO4(2-). The uptake of L-lactate was inhibited by phloretin, mercurials and alpha-cyano-4-hydroxycinnamic acid (4-CHC), but not by the stilbene anion exchange inhibitors, 4,4'-diisothiocyanostilbene-2, 2'-disulphonic acid (DIDS) and 4-acetamido-4'-isothiocyanostilbene-2, 2'-disulphonic acid (SITS). 4. The results indicate the presence of a MCT1 protein on the lumenal membrane of the colon that is involved in the transport of L-lactate as well as butyrate across the colonic lumenal membrane. Western blot analysis showed that the abundance of this protein decreases in lumenal membrane fractions isolated from colonic carcinomas compared with that detected in the normal healthy colonic tissue.