Excess exogenous pyruvate inhibits lactate dehydrogenase activity in live cells in an MCT1-dependent manner.

Cellular pyruvate is an essential metabolite at the crossroads of glycolysis and oxidative phosphorylation, capable of supporting fermentative glycolysis by reduction to lactate mediated by lactate dehydrogenase (LDH) among other functions. Several inherited diseases of mitochondrial metabolism impact extracellular (plasma) pyruvate concentrations, and [1-13C]pyruvate infusion is used in isotope-labeled metabolic tracing studies, including hyperpolarized magnetic resonance spectroscopic imaging. However, how these extracellular pyruvate sources impact intracellular metabolism is not clear. Herein, we examined the effects of excess exogenous pyruvate on intracellular LDH activity, extracellular acidification rates (ECARs) as a measure of lactate production, and hyperpolarized [1-13C]pyruvate-to-[1-13C]lactate conversion rates across a panel of tumor and normal cells. Combined LDH activity and LDHB/LDHA expression analysis intimated various heterotetrameric isoforms comprising LDHA and LDHB in tumor cells, not only canonical LDHA. Millimolar concentrations of exogenous pyruvate induced substrate inhibition of LDH activity in both enzymatic assays ex vivo and in live cells, abrogated glycolytic ECAR, and inhibited hyperpolarized [1-13C]pyruvate-to-[1-13C]lactate conversion rates in cellulo. Of importance, the extent of exogenous pyruvate-induced inhibition of LDH and glycolytic ECAR in live cells was highly dependent on pyruvate influx, functionally mediated by monocarboxylate transporter-1 localized to the plasma membrane. These data provided evidence that highly concentrated bolus injections of pyruvate in vivo may transiently inhibit LDH activity in a tissue type- and monocarboxylate transporter-1-dependent manner. Maintaining plasma pyruvate at submillimolar concentrations could potentially minimize transient metabolic perturbations, improve pyruvate therapy, and enhance quantification of metabolic studies, including hyperpolarized [1-13C]pyruvate magnetic resonance spectroscopic imaging and stable isotope tracer experiments.

Enhanced pyruvate production in Candida glabrata by overexpressing the CgAMD1 gene to improve acid tolerance.

OBJECTIVES: To enhance acid tolerance of Candida glabrata for pyruvate production by engineering AMP metabolism. RESULTS: The physiological function of AMP deaminase in AMP metabolism from C. glabrata was investigated by deleting or overexpresseing the corresponding gene, CgAMD1. At pH 4, CgAMD1 overexpression resulted in 59 and 51% increases in biomass and cell viability compared to those of wild type strain, respectively. In addition, the intracellular ATP level of strain Cgamd1Δ/CgAMD1 was down-regulated by 22%, which led to a 94% increase in pyruvate production. Further, various strengths of CgAMD1 expression cassettes were designed, thus resulting in a 59% increase in pyruvate production at pH 4. Strain Cgamd1Δ/CgAMD1 (H) was grown in a 30 l batch bioreactor at pH 4, and pyruvate reached 46.1 g/l. CONCLUSION: CgAMD1 overexpression plays an active role in improving acid tolerance and pyruvate fermentation performance of C. glabrata at pH 4.

Role of GlnR in acid-mediated repression of genes encoding proteins involved in glutamine and glutamate metabolism in Streptococcus mutans.

The acid tolerance response (ATR) is one of the major virulence traits of Streptococcus mutans. In this study, the role of GlnR in acid-mediated gene repression that affects the adaptive ATR in S. mutans was investigated. Using a whole-genome microarray and in silico analyses, we demonstrated that GlnR and the GlnR box (ATGTNAN(7)TNACAT) were involved in the transcriptional repression of clusters of genes encoding proteins involved in glutamine and glutamate metabolism under acidic challenge. Reverse transcription-PCR (RT-PCR) analysis revealed that the coordinated regulation of the GlnR regulon occurred 5 min after acid treatment and that prolonged acid exposure (30 min) resulted in further reduction in expression. A lower level but consistent reduction in response to acidic pH was also observed in chemostat-grown cells, confirming the negative regulation of GlnR. The repression by GlnR through the GlnR box in response to acidic pH was further confirmed in the citBZC operon, containing genes encoding the first three enzymes in the glutamine/glutamate biosynthesis pathway. The survival rate of the GlnR-deficient mutant at pH 2.8 was more than 10-fold lower than that in the wild-type strain 45 min after acid treatment, suggesting that the GlnR regulon participates in S. mutans ATR. It is hypothesized that downregulation of the synthesis of the amino acid precursors in response to acid challenge would promote citrate metabolism to pyruvate, with the consumption of H(+) and potential ATP synthesis. Such regulation will ensure an optimal acid adaption in S. mutans.

Effects of copper, iron and fluoride co-crystallized with sugar on caries development and acid formation in deslivated rats.

The purpose was to explore the effects of combinations of copper, iron and fluoride (Cu, Fe and F) incorporated in sucrose by co-crystallization on caries development in the deslivated rat model and to examine acid formation by bacteria in the rat mouth. Ninety-six Sprague-Dawley rats were infected with Streptococcus sobrinus 6715 and desalivated when aged 26 days. Eight groups were placed in a König-Höfer programmed feeder and received 17 meals daily at hourly intervals, and essential nutrition (NCP No. 2) by gavage twice daily for 21 days. The groups received (1) plain sucrose, (2) F (8 parts/10(6)) co-crystallized with sucrose, (3) Fe (88 parts/10(6)) sucrose, (4) Cu (75 parts/10(6)) sucrose, (5) Cu + F sucrose, (6) Cu + L Fe sucrose, (7) F + Fe sucrose, and (8) Cu + Fe + F sucrose. At death the jaws were removed and sonicated in 0.9% saline solution for microbial assessment. In addition, organic acid assays were performed for each animal. Keyes smooth-surface and sulcal caries scores were lowest in the Cu + Fe + F sucrose group, but not statistically significantly different from those of the other Cu groups. The numbers of Strep. sobrinus found in the groups that received Cu, Cu + Fe, Cu + F, F + Fe and Cu + Fe + F sugar were lower than in the control group. Lactic acid was found in lower concentrations in Fe, Cu, Cu + F, Cu + Fe and F + Fe groups than in the other groups. It appears that combinations of Cu; Fe and F co-crystallized with sugar may have an additive effect in reducing the cariogenic potential of sugar by affecting lactic acid formation and reducing bacterial colonization.

Intracellular pH responses of hybridoma and yeast to substrate addition and acid challenge.

The pHi responses of hybridoma and yeast cells to substrate and external acid additions were measured using the fluorescent pHi indicator, 9-aminoacridine. The pHi change, following CCCP addition, indicated by 9AA, compared very well with that indicated by BCECF. No change in pHi was observed following glucose or glutamine additions to hybridoma cells under glucose- and glutamine-absent conditions. Also, no change in pHi was observed when glucose was added in the presence of low glutamine and when glutamine was added in the presence of low glucose. However, in the presence of amiloride, the pHi of hybridoma cells decreased following glucose addition. Intracellular pH responses of hybridoma and yeast cells to decreases in external pH, effected by acid addition, were dependent on the cellular energy state and acid type. Cells controlled their pHi more tightly under energy-poor conditions compared to energy-rich conditions, and sulfuric acid (strong acid) caused larger changes in pHi compared to pyruvic acid (weak acid). In response to acid addition under energy-rich conditions, the magnitudes of pHi increases in hybridoma were smaller in the presence of amiloride compared to that in the absence of amiloride. Further, pHi responses to a decrease in external pH were slower at submetabolic temperatures.