Presence Of L-lactate Research Articles

Discovery of N-l-Lactoyl-l-Trp as a Bitterness Masker via Structure-Based Virtual Screening and a Sensory Approach.

N-Lactoyl-amino acid derivatives (N-Lac-AAs) are of increasing interest as potential taste-active compounds. The complexity and diversity of N-Lac-AAs pose a significant challenge to the effective discovery of taste-active N-Lac-AAs. Therefore, a structure-based virtual screening was used to identify taste-active N-Lac-AAs. Virtual screening results showed that N-lactoyl-hydrophobic amino acids had a higher affinity for taste receptors, specifically N-l-Lac-l-Trp. And then, N-l-Lac-l-Trp was synthesized in yields of 22.3% by enzymatic synthesis in the presence of l-lactate and l-Trp, and its chemical structure was confirmed by MS/MS and one-dimensional (1D) and two-dimensional (2D) NMR. Sensory evaluation revealed that N-l-Lac-l-Trp had a significant taste-masking effect on quinine, d-salicin, caffeine, and l-Trp, particularly l-Trp and caffeine. N-l-Lac-l-Trp had a better masking effect on the higher concentration of bitter compounds. It reduced the bitterness of caffeine (500 mg/L) and l-Trp (1000 mg/L) by approximately 20 and 26%, respectively. The result of the ligand-receptor interaction and a quantum mechanical analysis showed that N-l-Lac-l-Trp increased the binding affinity to the bitter receptor mainly through hydrogen bonding and lowering the electrostatic potential.

Abstract 4367: Protein kinase D1 induces metabolic switch in pancreatic cancer via modulation of mTORC1

Abstract Objective: Pancreatic cancer (PanCa) is the third most common cause of cancer-related deaths in the US. Protein Kinase D1 (PKD1) is a kinase molecule which is involved in various important cellular signaling processes. While PKD1 has been reported to play a role in PanCa progression, the molecular mechanisms involved have not been adequately studied. Herein, we investigate the underlying mechanisms which enable PKD1 in enhancing glucose metabolism and further contribute to PanCa aggressiveness. Methods: PanCa cells with high PKD1 expressing (Panc-1 and AsPC-1) and low PKD1 (HPAF-II and BXPC-3) were used in the study. Immunohistochemistry and confocal immunofluorescence were performed to analyze the expression of PKD1 in pancreatic cancer/normal tissues and cells, respectively. The effect of PKD1 gain/loss-in function was investigated on glucose uptake and lactate production in PanCa cells using commercially available kits. Western blotting and real-time PCR experiments were performed to analyze the expression of protein and mRNA levels. MTT assay was conducted to access the influence of PKD1 on cell proliferation. Boyden chamber migration assay and Matrigel invasion assays were performed to determine the migratory and invasive abilities of PanCa cells. The gene silencing was performed using specific siRNAs in the study. Results: Our results demonstrate that PKD1 upregulates glucose metabolism in PanCa cells as indicated by enhanced glucose consumption and lactate production. The invasive characteristics of PKD1 expressing cells are augmented in presence of L-Lactate and reduced in presence of 2DG. PKD1 overexpression demonstrated enhanced phosphorylation of mTOR (ser-2448), 4EBP1, s6kinase and AKT, suggesting the role of PKD1 in the activation of mTOR pathway. We further observed that the phosphorylation of mTOR (ser-2448) and ps6kinase was attenuated on silencing PKD1 or in presence of Rapamycin, suggesting the role of PKD1 in activation of mTORC1 complex, both being the main effectors of mTORC1. Additionally, the inhibition in the phosphorylation of mTOR (ser-2448) in presence of kinase dead PKD1 suggests that PKD1 phosphorylates/activates mTORC1 in PanCa. Decrease in glucose uptake and lactate production in presence of PKD1 overexpression was observed on silencing raptor but not rictor in the cells, further suggesting the involvement of mTORC1 in PKD1 induced metabolic reprogramming in PanCa. PKD1 induced enhanced phosphorylation of mTOR resulted in an activation of HIF-1α and Glut-1 proteins, involved in abberant glucose metabolism. Additionally, our results also demonstrate that altered glucose metabolism leads to chemoresistance and silencing of PKD1 expression sensitizes PanCa cells to gemcitabine. Conclusion: Our studies indicate that PKD1 acts as a key regulator of the glucose metabolism via mTOR activation and facilitates proliferation and invasion of PanCa cells. Citation Format: Sonam Kumari, Sheema Khan, Murali Yallapu, Subhash Chauhan, Meena Jaggi. Protein kinase D1 induces metabolic switch in pancreatic cancer via modulation of mTORC1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4367.

L-Lactate-Mediated Neuroprotection against Glutamate-Induced Excitotoxicity Requires ARALAR/AGC1.

Lactate now stands as a metabolite necessary for multiple functions in the brain and is an alternative energy source during excitotoxic brain injury. Here we find that the absence of a functional malate-aspartate NADH shuttle caused by aralar/AGC1 disruption causes a block in lactate utilization by neurons, which prevents the protective role of lactate on excitotoxicity, but not glutamate excitotoxicity itself. Thus, failure to use lactate is detrimental and is possibly responsible for the exacerbated in vivo excitotoxicity in aralar(+/-)mice.

L-Lactate protects neurons against excitotoxicity: implication of an ATP-mediated signaling cascade

Converging experimental data indicate a neuroprotective action of L-Lactate. Using Digital Holographic Microscopy, we observe that transient application of glutamate (100 μM; 2 min) elicits a NMDA-dependent death in 65% of mouse cortical neurons in culture. In the presence of L-Lactate (or Pyruvate), the percentage of neuronal death decreases to 32%. UK5099, a blocker of the Mitochondrial Pyruvate Carrier, fully prevents L-Lactate-mediated neuroprotection. In addition, L-Lactate-induced neuroprotection is not only inhibited by probenicid and carbenoxolone, two blockers of ATP channel pannexins, but also abolished by apyrase, an enzyme degrading ATP, suggesting that ATP produced by the Lactate/Pyruvate pathway is released to act on purinergic receptors in an autocrine/paracrine manner. Finally, pharmacological approaches support the involvement of the P2Y receptors associated to the PI3-kinase pathway, leading to activation of KATP channels. This set of results indicates that L-Lactate acts as a signalling molecule for neuroprotection against excitotoxicity through coordinated cellular pathways involving ATP production, release and activation of a P2Y/KATP cascade.

Enantioselective regulation of lactate racemization by LarR in Lactobacillus plantarum.

Lactobacillus plantarum is a lactic acid bacterium that produces a racemic mixture of l- and d-lactate from sugar fermentation. The interconversion of lactate isomers is performed by a lactate racemase (Lar) that is transcriptionally controlled by the l-/d-lactate ratio and maximally induced in the presence of l-lactate. We previously reported that the Lar activity depends on the expression of two divergently oriented operons: (i) the larABCDE operon encodes the nickel-dependent lactate racemase (LarA), its maturases (LarBCE), and a lactic acid channel (LarD), and (ii) the larR(MN)QO operon encodes a transcriptional regulator (LarR) and a four-component ABC-type nickel transporter [Lar(MN), in which the M and N components are fused, LarQ, and LarO]. LarR is a novel regulator of the Crp-Fnr family (PrfA group). Here, the role of LarR was further characterized in vivo and in vitro. We show that LarR is a positive regulator that is absolutely required for the expression of Lar activity. Using gel retardation experiments, we demonstrate that LarR binds to a 16-bp palindromic sequence (Lar box motif) that is present in the larR-larA intergenic region. Mutations in the Lar box strongly affect LarR binding and completely abolish transcription from the larA promoter (PlarA). Two half-Lar boxes located between the Lar box and the -35 box of PlarA promote LarR multimerization on DNA, and point mutations within one or both half-Lar boxes inhibit PlarA induction by l-lactate. Gel retardation and footprinting experiments indicate that l-lactate has a positive effect on the binding and multimerization of LarR, while d-lactate antagonizes the positive effect of l-lactate. A possible mechanism of LarR regulation by lactate enantiomers is proposed.

Identification of the genes that contribute to lactate utilization in Helicobacter pylori.

Helicobacter pylori are Gram-negative, spiral-shaped microaerophilic bacteria etiologically related to gastric cancer. Lactate utilization has been implicated although no corresponding genes have been identified in the H. pylori genome. Here, we report that gene products of hp0137–0139 (lldEFG), hp0140–0141 (lctP), and hp1222 (dld) contribute to D- and L-lactate utilization in H. pylori. The three-gene unit hp0137–0139 in H. pylori 26695 encodes L-lactate dehydrogenase (LDH) that catalyzes the conversion of lactate to pyruvate in an NAD-dependent manner. Isogenic mutants of these genes were unable to grow on L-lactate-dependent medium. The hp1222 gene product functions as an NAD-independent D-LDH and also contributes to the oxidation of L-lactate; the isogenic mutant of this gene failed to grow on D-lactate-dependent medium. The parallel genes hp0140–0141 encode two nearly identical lactate permeases (LctP) that promote uptake of both D- and L-lactate. Interestingly an alternate route must also exist for lactate transport as the knockout of genes did not completely prevent growth on D- or L-lactate. Gene expression levels of hp0137–0139 and hp1222 were not enhanced by lactate as the carbon source. Expression of hp0140–0141 was slightly suppressed in the presence of L-lactate but not D-lactate. This study identified the genes contributing to the lactate utilization and demonstrated the ability of H. pylori to utilize both D- and L-lactate.

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.

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.

Uptake of α-Ketoglutarate by Citrate Transporter CitP Drives Transamination in Lactococcus lactis

Transamination is the first step in the conversion of amino acids into aroma compounds by lactic acid bacteria (LAB) used in food fermentations. The process is limited by the availability of α-ketoglutarate, which is the best α-keto donor for transaminases in LAB. Here, uptake of α-ketoglutarate by the citrate transporter CitP is reported. Cells of Lactococcus lactis IL1403 expressing CitP showed significant levels of transamination activity in the presence of α-ketoglutarate and one of the amino acids Ile, Leu, Val, Phe, or Met, while the same cells lacking CitP showed transamination activity only after permeabilization of the cell membrane. Moreover, the transamination activity of the cells followed the levels of CitP in a controlled expression system. The involvement of CitP in the uptake of the α-keto donor was further demonstrated by the increased consumption rate in the presence of L-lactate, which drives CitP in the fast exchange mode of transport. Transamination is the only active pathway for the conversion of α-ketoglutarate in IL1403; a stoichiometric conversion to glutamate and the corresponding α-keto acid from the amino acids was observed. The transamination activity by both the cells and the cytoplasmic fraction showed a remarkably flat pH profile over the range from pH 5 to pH 8, especially with the branched-chain amino acids. Further metabolism of the produced α-keto acids into α-hydroxy acids and other flavor compounds required the coupling of transamination to glycolysis. The results suggest a much broader role of the citrate transporter CitP in LAB than citrate uptake in the citrate fermentation pathway alone.

Deletion of pyruvate decarboxylase by a new method for efficient markerless gene deletions in Gluconobacter oxydans

Gluconobacter oxydans, a biotechnologically relevant species which incompletely oxidizes a large variety of carbohydrates, alcohols, and related compounds, contains a gene for pyruvate decarboxylase (PDC). This enzyme is found only in very few species of bacteria where it is normally involved in anaerobic ethanol formation via acetaldehyde. In order to clarify the role of PDC in the strictly oxidative metabolism of acetic acid bacteria, we developed a markerless in-frame deletion system for strain G. oxydans 621H which uses 5-fluorouracil together with a plasmid-encoded uracil phosphoribosyltransferase as counter selection method and used this technique to delete the PDC gene (GOX1081) of G. oxydans 621H. The PDC deletion mutant accumulated large amounts of pyruvate but almost no acetate during growth on D-mannitol, D-fructose or in the presence of L-lactate. This suggested that in G. oxydans acetate formation occurs by decarboxylation of pyruvate and subsequent oxidation of acetaldehyde to acetate. This observation and the efficiency of the markerless deletion system were confirmed by constructing deletion mutants of two acetaldehyde dehydrogenases (GOX1122 and GOX2018) and of the acetyl-CoA-synthetase (GOX0412). Acetate formation during growth of these mutants on mannitol did not differ significantly from the wild-type strain.

Lactate Utilization Is Regulated by the FadR-Type Regulator LldR in Pseudomonas aeruginosa

NAD-independent L-lactate dehydrogenase (l-iLDH) and NAD-independent D-lactate dehydrogenase (D-iLDH) activities are induced coordinately by either enantiomer of lactate in Pseudomonas strains. Inspection of the genomic sequences of different Pseudomonas strains revealed that the lldPDE operon comprises 3 genes, lldP (encoding a lactate permease), lldD (encoding an L-iLDH), and lldE (encoding a D-iLDH). Cotranscription of lldP, lldD, and lldE in Pseudomonas aeruginosa strain XMG starts with the base, C, that is located 138 bp upstream of the lldP ATG start codon. The lldPDE operon is located adjacent to lldR (encoding an FadR-type regulator, LldR). The gel mobility shift assays revealed that the purified His-tagged LldR binds to the upstream region of lldP. An XMG mutant strain that constitutively expresses D-iLDH and L-iLDH was found to contain a mutation in lldR that leads to an Ile23-to-serine substitution in the LldR protein. The mutated protein, LldR(M), lost its DNA-binding activity. A motif with a hyphenated dyad symmetry (TGGTCTTACCA) was identified as essential for the binding of LldR to the upstream region of lldP by using site-directed mutagenesis. L-Lactate and D-lactate interfered with the DNA-binding activity of LldR. Thus, L-iLDH and D-iLDH were expressed when the operon was induced in the presence of L-lactate or D-lactate.

Mechanism of Citrate Metabolism by an Oxaloacetate Decarboxylase-Deficient Mutant of Lactococcus lactis IL1403

Citrate metabolism in resting cells of Lactococcus lactis IL1403(pFL3) results in the formation of two end products from the intermediate pyruvate, acetoin and acetate (A. M. Pudlik and J. S. Lolkema, J. Bacteriol. 193:706-714, 2011). Pyruvate is formed from citrate following uptake by the transporter CitP through the subsequent actions of citrate lyase and oxaloacetate decarboxylase. The present study describes the metabolic response of L. lactis when oxaloacetate accumulates in the cytoplasm. The oxaloacetate decarboxylase-deficient mutant ILCitM(pFL3) showed nearly identical rates of citrate consumption, but the end product profile in the presence of glucose shifted from mainly acetoin to only acetate. In addition, in contrast to the parental strain, the mutant strain did not generate proton motive force. Citrate consumption by the mutant strain was coupled to the excretion of oxaloacetate, with a yield of 80 to 85%. Following citrate consumption, oxaloacetate was slowly taken up by the cells and converted to pyruvate by a cryptic decarboxylase and, subsequently, to acetate. The transport of oxaloacetate is catalyzed by CitP. The parental strain IL1403(pFL3) containing CitP consumed oxaloacetate, while the original strain, IL1403, not containing CitP, did not. Moreover, oxaloacetate consumption was enhanced in the presence of L-lactate, indicating exchange between oxaloacetate and L-lactate catalyzed by CitP. Hence, when oxaloacetate inadvertently accumulates in the cytoplasm, the physiological response of L. lactis is to excrete oxaloacetate in exchange with citrate by an electroneutral mechanism catalyzed by CitP. Subsequently, in a second step, oxaloacetate is taken up by CitP and metabolized to pyruvate and acetate.

Chromate-reducing activity of Hansenula polymorpha recombinant cells over-producing flavocytochrome b2

In spite of the great interest to studies of the biological roles of chromium, as well as the toxic influence of Cr(VI)-species on living organisms, the molecular mechanisms of chromate bioremediation remain vague. A reductive pathway resulting in formation of less toxic Cr(III)-species is suggested to be the most important among possible mechanisms for chromate biodetoxification. The yeast l-lactate:cytochrome c-oxidoreductase (flavocytochrome b 2, FC b 2) has absolute specificity for l-lactate, yet is non-selective with respect to its electron acceptor. These properties allow us to consider the enzyme as a potential candidate for chromate reduction by living cells in the presence of l-lactate. A recombinant strain of thermotolerant, methylotrophic yeast Hansenula polymorpha with sixfold increased FC b 2 enzyme activity (up to 3 μmol min −1 mg −1 protein in cell-free extract) compared to the parental strain was used for approval our suggestion. The recombinant cells, stored in dried state, as well as living yeast cells were tested for chromate-reducing activity in vitro in the presence of l-lactate (as an electron donor for chromate reduction) and different low molecular weight, redox-active mediators facilitating electron transfer from the reduced form of the enzyme to chromate (as a final electron acceptor): dichlorophenolindophenol (DCPIP), Methylene blue, Meldola blue, and Nile blue. It was shown that the highest chromate-reducing activity of the cells was achieved in the presence of DCPIP. The ability of chromate to catch electrons from the reduced flavocytochrome b 2 was confirmed using purified enzyme immobilized on the surface of a platinum electrode. The increasing concentration of Cr(VI) resulted in a decrease of enzyme-mediated current generated on the electrode during l-lactate oxidation. The shift and drop in amplitude of the peak in the cyclic voltammogram are indicative of Cr(VI)-dependent competition between reaction of chromate with reduced FC b 2 and direct electron transfer from the enzyme to the electrode surface. The application of the chromate-reducing ability of FC b 2-over-producing recombinant cells of H. polymorpha toward chromate bioremediation and the construction of cells-based biosensor for chromate monitoring in the environment are discussed.

Effects of L-Lactate and D-Mannitol on γ-Hydroxybutyrate Toxicokinetics and Toxicodynamics in Rats

Overdoses of gamma-hydroxybutyrate (GHB), a drug of abuse, result in coma, respiratory arrest, and death. The objective of this study was to evaluate a potential GHB detoxification strategy by inhibiting the monocarboxylate transporter (MCT)-mediated renal reabsorption of GHB in rats, using the MCT substrate L-lactate. The use of the osmotic diuretic D-mannitol alone or combined with L-lactate was also explored. GHB (208 mg/h/kg) was infused i.v. for 3 h in the absence or presence of L-lactate (60.5, 121, and 302.5 mg h(-1) kg(-1)), D-mannitol (0.5 g/kg), or L-lactate (60.5 mg h(-1) kg(-1)) combined with D-mannitol (0.5 g/kg). GHB in plasma and urine samples was determined along with blood pH, electrolytes, glucose, and L-lactate. Administration of L-lactate, or the combination of L-lactate and D-mannitol, but not D-mannitol alone, significantly increased the renal and total clearances of GHB in rats. Blood pH and electrolyte concentrations exhibited small changes with GHB, GHB/lactate, and GHB/mannitol treatments, although most values remained within their normal range. The concomitant administration of lactated Ringer's solution (28 mM L-lactate) at 300 mul/min with mannitol (0.5 g/kg) resulted in a significant increase in GHB clearance and a decrease in sleep time after an i.v. dose of 1 g/kg. Overall, our results indicated the following: 1) the use of the MCT inhibitor L-lactate can increase the renal and total clearances of GHB, and 2) the combination of lactated Ringer's solution and D-mannitol significantly alters GHB toxicokinetics and toxicodynamics and represents a potential clinical detoxification strategy for the treatment of GHB overdoses.

Regulation of L-lactate utilization by the FadR-type regulator LldR of Corynebacterium glutamicum.

Corynebacterium glutamicum can grow on L-lactate as a sole carbon and energy source. The NCgl2816-lldD operon encoding a putative transporter (NCgl2816) and a quinone-dependent L-lactate dehydrogenase (LldD) is required for L-lactate utilization. DNA affinity chromatography revealed that the FadR-type regulator LldR (encoded by NCgl2814) binds to the upstream region of NCgl2816-lldD. Overexpression of lldR resulted in strongly reduced NCgl2816-lldD mRNA levels and strongly reduced LldD activity, and as a consequence, a severe growth defect was observed in cells grown on L-lactate as the sole carbon and energy source, but not in cells grown on glucose, ribose, or acetate. Deletion of lldR had no effect on growth on these carbon sources but resulted in high NCgl2816-lldD mRNA levels and high LldD activity in the presence and absence of L-lactate. Purified His-tagged LldR bound to a 54-bp fragment of the NCgl2816-lldD promoter, which overlaps with the transcriptional start site determined by random amplification of cDNA ends-PCR and contains a putative operator motif typical of FadR-type regulators, which is -1TNGTNNNACNA10. Mutational analysis revealed that this motif with hyphenated dyad symmetry is essential for binding of LldD to the NCgl2816-lldD promoter. L-Lactate, but not D-lactate, interfered with binding of LldRHis to the NCgl2816-lldD promoter. Thus, during growth on media lacking L-lactate, LldR represses expression of NCgl2816-lldD. In the presence of L-lactate in the growth medium or under conditions leading to intracellular L-lactate accumulation, the L-lactate utilization operon is induced.

Permeabilized cells of flavocytochrome b2 over-producing recombinant yeast Hansenula polymorpha as biological recognition element in amperometric lactate biosensors

A l-lactate-selective microbial biosensor was developed using permeabilized cells of gene-engineered thermotolerant methylotrophic yeast Hansenula polymorpha, over-producing l-lactate:cytochrome c-oxidoreductase (EC 1.1.2.3, flavocytochrome b 2, FC b 2). The construction of FC b 2-producers by over-expression of the gene CYB2 H. polymorpha encoding FC b 2 is described. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in the frame of a plasmid for multicopy integration was transformed to the recipient strain H. polymorpha C-105 ( gcr1 catX) impaired in glucose repression and devoid of catalase activity. The permeabilized cells were either immobilized on the graphite working electrode by physical entrapment of the cell suspension by means of a dialysis membrane or by integration of the cells in an electrochemically generated layer using a cathodic electrodeposition polymer. Phenazine methosulphate was used as a free-diffusing redox mediator. It was assumed that the mediator reacts with mitochondrial FC b 2 after entering the cells in the presence of l-lactate. The biosensor based on recombinant yeast cells exhibited a higher K M app value and hence expanded linear range toward l-lactate as compared to a similar sensor based on the initial cells of H. polymorpha C-105.

Renal Clearance of γ-Hydroxybutyric Acid in Rats: Increasing Renal Elimination as a Detoxification Strategy

Intoxication with gamma-hydroxybutyric acid (GHB) is associated with coma, seizure, and death; treatment of overdoses is symptomatic. The objectives of this investigation were to characterize the renal clearance and total clearance of GHB in rats and to evaluate potential strategies for increasing the elimination of GHB after drug overdoses. GHB was administered by i.v. infusion at low (108 mg/h/kg), medium (128 mg/h/kg), or high (208 mg/h/kg) doses. Crossover studies were performed under steady-state conditions using the medium dose in the absence or presence of l-lactate, pyruvate, d-mannitol, sodium bicarbonate, or normal saline. GHB in plasma and urine samples was assayed using liquid chromatography-tandem mass spectrometry. Infusion of the low, medium, and high doses of GHB produced steady-state plasma concentrations of 0.22 +/- 0.04, 0.43 +/- 0.05, and 0.68 +/- 0.11 mg/ml. The renal clearance of the medium (51.8 +/- 13.0 ml/h/kg) and high (97.1 +/- 43.1 ml/h/kg) doses was significantly higher than that of the low dose (14.9 +/- 5.1 ml/h/kg), whereas the total clearance values were significantly lower than that of the low dose. The renal clearance was significantly increased by the concomitant administration of l-lactate, pyruvate, d-mannitol, or sodium bicarbonate with GHB but was not altered by normal saline. The total and metabolic clearance values were significantly increased by all treatments except normal saline. Overall, our results indicated that the renal clearance of GHB is dose-dependent, involving capacity-limited reabsorption. Monocarboxylate transport inhibitors, osmotic diuresis using d-mannitol, or the administration of sodium bicarbonate can increase the renal and total clearances of GHB. The approaches used in this investigation may offer potential detoxification strategies for the treatment of GHB overdoses.

Kinetics, molecular basis, and differentiation of l-lactate transport in spermatogenic cells

Round spermatid energy metabolism is closely dependent on the presence of L-lactate in the external medium. This L-lactate has been proposed to be supplied by Sertoli cells in the seminiferous tubules. L-Lactate, in conjunction with glucose, modulates intracellular Ca(2+) concentration in round spermatids and pachytene spermatocytes. In spite of this central role of L-lactate in spermatogenic cell physiology, the mechanism of L-lactate transport, as well as possible differentiation during spermatogenesis, has not been studied in these cells. By measuring radioactive L-lactate transport and intracellular pH (pH(i)) changes with pH(i) fluorescent probes, we show that these cells transport L-lactate using monocarboxylate-H(+) transport (MCT) systems. RT-PCR, in situ mRNA hybridization, and immunocyto- and immunohistochemistry data show that pachytene spermatocytes express mainly the MCT1 and MCT4 isoforms of the transporter (intermediate- and low-affinity transporters, respectively), while round spermatids, besides MCT1 and MCT4, also show expression of the MCT2 isoform (high-affinity transporter). These molecular data are consistent with the kinetic data of L-lactate transport in these cells demonstrating at least two transport components for L-lactate. These separate transport components reflect the ability of these cells to switch between the generation of glycolytic L-lactate in the presence of external glucose and the use of L-lactate when this substrate is available in the external environment. The supply of these substrates is regulated by the hormonal control of Sertoli cell glycolytic activity.

The Allosteric Effector l-Lactate Induces a Conformational Change of 2×6-meric Lobster Hemocyanin in the Oxy State as Revealed by Small Angle X-ray Scattering

Hemocyanins are multisubunit respiratory proteins found in many invertebrates. They bind oxygen highly cooperatively. However, not much is known about the structural basis of this behavior. We studied the influence of the physiological allosteric effector l-lactate on the oxygenated quaternary structure of the 2x6-meric hemocyanin from the lobster Homarus americanus employing small angle x-ray scattering (SAXS). The presence of 20 mm l-lactate resulted in different scattering curves compared with those obtained in the absence of l-lactate. The distance distribution functions p(r) indicated a more compact molecule in presence of l-lactate, which is also reflected in a reduction of the radius of gyration by about 0.2 nm (3%). Thus, we show for the first time on a structural basis that a hemocyanin in the oxy state can adopt two different conformations. This is as predicted from the analysis of oxygen binding curves according to the "nesting" model. A comparison of the distance distribution functions p(r) obtained from SAXS with those deduced from electron microscopy revealed large differences. The distance between the two hexamers as deduced from electron microscopy has to be shortened by up to 1.1 nm to agree well with the small angle x-ray curves.

Hepatic uptake of hippurate: a multiple-indicator dilution, perfused rat liver study.

The hepatic transport of hippuric acid (HA), a glycine-conjugated metabolite of benzoic acid that exhibits only modest plasma albumin binding (binding association constant of 2.1 x 10(3) M-1), was studied in the single-pass perfused rat liver (12 ml/min), using the multiple indicator dilution (MID) technique. The venous recovery of [3H]HA on portal venous injection of a MID dose containing a mixture of a set of noneliminated reference indicators and [3H]HA revealed a survival fraction of unity, corroborating the lack of disappearance of bulk HA from plasma. When the outflow recovery was fitted to the barrier-limited model of Goresky et al. (C.A. Goresky, G. G. Bach, and B. E. Nadeau. J. Clin. Invest. 52: 991-1009, 1973), the derived influx (P(in)S) and efflux (P(out)S) permeability-surface area products were found to be dependent on the concentration of HA (1-930 microM); P(in)S and P(out)S were approximately 3.5 times the plasma flow rate at low HA concentration, but decreased with increasing HA concentration. All values, however, greatly exceeded the expected contribution from passive diffusion, because the equilibrium distribution ratio of chloroform to buffer for HA was extremely low (0.0001 at pH 7.4). The tissue equilibrium partition coefficient (P(in)/P(out), or ratio of influx to efflux rate constants, k1/k-1) was less than unity and decreased with concentration. The optimized apparent Michaelis-Menten constant and maximal velocity were 182 +/- 60 microM and 12 +/- 4 nmol.s-1.g-1, respectively, for influx and 390 +/- 190 microM and 29 +/- 13 nmol.s-1.g-1, respectively, for efflux. In the presence of L-lactate (20 mM), however, P(in)S for the uptake of HA (174 +/- 3 microM) was reduced. Benzoic acid (10-873 microM) was also effective in reducing hepatic uptake of HA (5.3 +/- 0.9 microM). These interactions suggest that MCT2, the monocarboxylate transporter that mediates the hepatic uptake of lactate and other monocarboxylic acids, may be involved in HA transport.