Doxycycline Alters the Porcine Renal Proteome and Degradome during Hypothermic Machine Perfusion

Ischemia-reperfusion injury (IRI) is a hallmark for tissue injury in donation after circulatory death (DCD) kidneys. The implementation of hypothermic machine perfusion (HMP) provides a platform for improved preservation of DCD kidneys. Doxycycline administration has shown protective effects during IRI. Therefore, we explored the impact of doxycycline on proteolytic degradation mechanisms and the urinary proteome of perfused kidney grafts. Porcine kidneys underwent 30 min of warm ischemia, 24 h of oxygenated HMP (control/doxycycline) and 240 min of ex vivo reperfusion. A proteomic analysis revealed distinctive clustering profiles between urine samples collected at T15 min and T240 min. High-efficiency undecanal-based N-termini (HUNTER) kidney tissue degradomics revealed significantly more proteolytic activity in the control group at T-10. At T240, significantly more proteolytic activity was observed in the doxycycline group, indicating that doxycycline alters protein degradation during HMP. In conclusion, doxycycline administration during HMP led to significant proteomic and proteolytic differences and protective effects by attenuating urinary NGAL levels. Ultimately, we unraveled metabolic, and complement and coagulation pathways that undergo alterations during machine perfusion and that could be targeted to attenuate IRI induced injury.     

Lactate metabolism in potato tubers deficient in lactate dehydrogenase activity

The aim of this work was to investigate the effect of decreased activity of lactate dehydrogenase (EC 1.1.1.27; LDH) on lactate metabolism in potato tubers. By expressing a cDNA‐encoding potato tuber LDH in the antisense orientation, we generated transgenic potato plants with a preferential decrease in two of the five isozymes of LDH. Surprisingly, transgenic tubers grown under normoxic conditions did not contain less lactate, but rather instead contained approximately two‐fold more lactate than control tubers. This result is explicable if the decreased isozymes of LDH are responsible for the oxidation of lactate to pyruvate in vivo. This was confirmed by measurements of the rate of metabolism of lactate supplied to tuber discs: the rate in transgenic tubers was approximately half that of control tubers. The decrease in LDH activity had no measurable effect on the accumulation of lactate in cold‐stored tubers under anoxia, nor during the subsequent utilization of this lactate upon return to normoxia. In both control and transgenic tubers, the accumulation of lactate during anoxia was not accompanied by an induction of LDH activity or a change in isozyme distribution. In contrast, the metabolism of lactate after a period of anoxia was accompanied by a two‐fold increase in LDH activity and the induction of two isozymes that were distinct from those which had been decreased in the transgenic plants.     

Protective Effects of Pyridoxal Phosphate Against Glucose Deprivation-Induced Damage in Cultured Hippocampal Neurons

Abstract: When hippocampal cultures were deprived of glucose, massive release of lactate dehydrogenase (LDH), an indicator of neuronal death, occurred via NMDA receptor activation. Addition of pyridoxal phosphate (PLP; 1 and 10 µ M ) inhibited this LDH release in a concentration-dependent manner. Prior exposure to PLP evoked more potent inhibitory effects on LDH release compared with those treated at the onset of glucose deprivation. Furthermore, PLP inhibited the reduction of intracellular content of pyruvate induced by glucose deprivation, which was accompanied by the reversal of intracellular ATP depletion. A noteworthy elevation of extracellular glutamate in response to glucose deprivation was completely reversed by addition of PLP. Aminooxyacetic acid, a potent inhibitor of PLP-dependent enzymes, antagonized the effects of PLP on LDH release, pyruvate production, and ATP formation. These results suggest that PLP protects neurons from glucose deprivation-induced damage by enhancing the formation of energy-yielding products and relieving extracellular load of glutamate. The observed phenomena further indicate that PLP might be used prophylactically against neuronal death induced by metabolic disorders.     

Inhibition of lactate glucogneogenesis in rat kidney by dichloroacetate.

1. Sodium dichloroacetate (1mM) inhibited glucose production from L-lactate in kidney-cortex slices from fed, starved or alloxan-diabetic rates. In general gluconeogenesis from other substrates was no inhibited. 2. Sodium dichloracetate inhibited glucose production from L-lactate but no from pyruvate in perfused isolated kidneys from normal or alloxan-diabetic rats. 3. Sodium dichloroacetate is an inhibitor of the pyruvate dehydrogenase kinase reaction and it effected conversion of pyruvate dehydrogenase into its its active (dephosphorylated) form in kidney in vivo. In general, pyruvate dehydrogenase was mainly in the active form in kidneys perfused or incubated with L-lactate and the inhibitory effect of dichloroacetate on glucose production was not dependent on activation of pyruvate dehydrogenase. 4. Balance data from kidney slices showed that dichloroacetate inhibits lactate uptake, glucose and pyruvate production from lactate, but no oxidation of lactate. 5. The mechanism of this effect of dichloroactetate on glucose production from lactate has not been fully defined, but evidence suggests that it may involve a fall in tissue pyruvate concentration and inhibition of pyruvate carboxylation.     

Ceftriaxone ameliorates cyclosporine A-induced oxidative nephrotoxicity in rat

A growing body of evidence now suggested that cyclosporine A (CycA)‐induced nephrotoxicity is a crucial clinical problem and oxidative stress is importantly responsible for its toxicity. Ceftriaxone induced antioxidant effect in brain and neuronal tissues against oxidative damage although its antioxidant potential effect on kidney has not been clarified. The aim of this study was to evaluate whether ceftriaxone protects CycA‐induced oxidative stress kidney injury in rats. Twenty‐four rats were equally divided into four groups. First group was used as control. Ceftriaxone (200 mg/kg) and CycA (15 mg/kg) were administrated to second and third groups for 10 days, respectively. The ceftriaxone and CycA combination was given to rats constituting the fourth group for 10 days. Lipid peroxidation (LP), urea nitrogen and lactate dehydrogenase (LDH) levels were higher in CycA group than in control and ceftriaxone groups although LP, urea nitrogen and LDH levels were lower in ceftriaxone + CycA group than in control and ceftriaxone groups. Glutathione peroxidase and catalase activities were lower in CycA group than in control whereas their activities were increased in control and ceftriaxone groups. Superoxide dismutase activity did not change by the treatments. Ceftriaxone administration recovered also CycA‐induced atrophy, vacuolization and exfoliations of tubular epithelium and glomerular collapse in histopathological evaluation of kidney. In conclusion, we observed that ceftriaxone is beneficial on CycA‐induced oxidative stress in kidney of rats by modulating oxidative and antioxidant system. Copyright © 2011 John Wiley & Sons, Ltd.     

Metabolic engineering of Bacillus subtilis for ethanol production: lactate dehydrogenase plays a key role in fermentative metabolism

Wild-type Bacillus subtilis ferments 20 g/liter glucose in 48 h, producing lactate and butanediol, but not ethanol or acetate. To construct an ethanologenic B. subtilis strain, homologous recombination was used to disrupt the native lactate dehydrogenase (LDH) gene (ldh) by chromosomal insertion of the Zymomonas mobilis pyruvate decarboxylase gene (pdc) and alcohol dehydrogenase II gene (adhB) under the control of the ldh native promoter. The values of the intracellular PDC and ADHII enzymatic activities of the engineered B. subtilis BS35 strain were similar to those found in an ethanologenic Escherichia coli strain. BS35 produced ethanol and butanediol; however, the cell growth and glucose consumption rates were reduced by 70 and 65%, respectively, in comparison to those in the progenitor strain. To eliminate butanediol production, the acetolactate synthase gene (alsS) was inactivated. In the BS36 strain (BS35 delta alsS), ethanol production was enhanced, with a high yield (89% of the theoretical); however, the cell growth and glucose consumption rates remained low. Interestingly, kinetic characterization of LDH from B. subtilis showed that it is able to oxidize NADH and NADPH. The expression of the transhydrogenase encoded by udhA from E. coli allowed a partial recovery of the cell growth rate and an early onset of ethanol production. Beyond pyruvate-to-lactate conversion and NADH oxidation, an additional key physiological role of LDH for glucose consumption under fermentative conditions is suggested. Long-term cultivation showed that 8.9 g/liter of ethanol can be obtained using strain BS37 (BS35 delta alsS udhA+). As far as we know, this is the highest ethanol titer and yield reported with a B. subtilis strain.     

The nucleotide metabolism in lactate perfused hearts under ischaemic and reperfused conditions

It was examined whether lactate influences postischaemic hemodynamic recovery as a function of the duration of ischaemia and whether changes in high-energy phosphate metabolism under ischaemic and reperfused conditions could be held responsible for impairment of cardiac function. To this end, isolated working rat hearts were perfused with either glucose (11 mM), glucose (11 mM) plus lactate (5 mM) or glucose (11 mM) plus pyruvate (5 mM). The extent of ischaemic injury was varied by changing the intervals of ischaemia, i.e. 15, 30 and 45 min. Perfusion by lactate evoked marked depression of functional recovery after 30 min of ischaemia. Perfusion by pyruvate resulted in marked decline of cardiac function after 45 min of ischaemia, while in glucose perfused hearts hemodynamic performance was still recovered to some extent after 45 min of ischaemia. Hence, lactate accelerates postischaemic hemodynamic impairment compared to glucose and pyruvate. The marked decline in functional recovery of the lactate perfused hearts cannot be ascribed to the extent of degradation of high-energy phosphates during ischaemia as compared to glucose and pyruvate perfused hearts. Glycolytic ATP formation (evaluated by the rate of lactate production) can neither be responsible for loss of cardiac function in the lactate perfused hearts. Moreover, failure of reenergization during reperfusion, the amount of nucleosides and oxypurines lost or the level of high-energy phosphates at the end of reperfusion cannot explain lactate-induced impairment. Alternatively, the accumulation of endogenous lactate may have contributed to ischaemic damage in the lactate perfused hearts after 30 min of ischaemia as it was higher in the lactate than in the glucose or pyruvate perfused hearts. It cannot be excluded that possible beneficial effects of the elevated glycolytic ATP formation during 15 to 30 min of ischaemia in the lactate perfused hearts are counterbalanced by the detrimental effects of lactate accumulation.     

Sodium Acetate,Sodium Acid Pyrophosphate,and Citric Acid Impacts on Isolated Peripheral Lymphocyte Viability,Proliferation, and DNA Damage

The present study examined the impacts of sodium acetate (SA), sodium acid pyrophosphate (SAPP), and citric acid (CA) on the viability, proliferation, and DNA damage of isolated lymphocytes in vitro. 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) release assays were adopted to evaluate cell viability, while comet assay was employed to assess the genotoxic effects. The cells were incubated with different levels of SA (50, 100, and 200 mM), SAPP (25, 50, and 100 mM/L), or CA (100, 200, and 300 μg/mL). The lymphocytes treated with the tested food additives showed concentration‐dependent decreases in both cell viability and proliferation. A concentration‐dependent increase in LDH release was also observed. The comet assay results indicated that SA, SAPP, and CA increased DNA damage percentage, tail DNA percentage, tail length, and tail moment in a concentration‐dependent manner. The current results showed that SA, SAPP, and CA are cytotoxic and genotoxic to isolated lymphocytes in vitro.     

Lactate-induced inhibition of glucose catabolism in guinea pig cortical brain slices.

Extracellular lactate concentration rises following ischaemic stroke in both the infarcted area and in the surrounding ischaemic penumbra. We investigated the effect of lactate accumulation on glucose metabolism in cortical slices from guinea pigs initially by varying superfusion medium to tissue volumes. Stable intracellular K+ concentrations indicated that a decrease in media/ tissue volume did not impair viability of the tissue, but 13C NMR demonstrated that lactate accumulation in the superfusion medium reduced glucose oxidation with inhibition of glial metabolism via pyruvate carboxylase. The concentration of lactate which had accumulated when significant inhibition was observed was approximately 0.85 mM. In independent experiments we found that superfusion of brain slices with lactate at this concentration (even using a 'high-volume' of superfusion fluid) decreased oxygen consumption by 40 +/- 3%. K(-)-induced depolarisation partially reversed this effect. These results suggest that even low extracellular lactate concentrations may depress metabolic rates in inactive and poorly perfused brain tissue in vivo through inhibition of glial metabolism of glucose.     

Effect of ethylisopropyl amiloride,a Na+-H+ exchange inhibitor,on cardioprotective effect of ischaemic and angiotensin preconditioning

The present study is designed to investigate the role of Na⁺-H⁺ exchanger in the cardioprotective effect of ischaemic and angiotensin (Ang II) preconditioning. Isolated perfused rat heart was subjected to global ischaemia for 30 min followed by reperfusion for 120 min. Coronary effluent was analysed for LDH and CK release to assess the degree of cardiac injury. Myocardial infarct size was estimated macroscopically using TTC staining. Left ventricular developed pressure (LVDP) and dp/dt were recorded to evaluate myocardial contractility. Four episodes of ischaemic or Ang II preconditioning markedly reduced LDH and CK release in coronary effluent and decreased myocardial infarct size. 5-(N-ethyl-N-isopropyl)amiloride (EIPA), a Na⁺-H⁺ exchange inhibitor, produced no marked effect on ischaemic preconditioning and Ang II preconditioning induced cardioprotection. On the other hand, EIPA administration prior to global ischaemia produced a similar reduction in myocardial injury as was noted with ischaemic preconditioning or Ang II preconditioning. On the basis of these results, it may be concluded that inhibition of Na⁺-H⁺ exchanger protects against ischaemia-reperfusion induced myocardial injury whereas activation of Na⁺-H⁺ exchanger may not mediate the cardioprotective effect of ischaemic and Ang II preconditioning.     

Effect of cooling and warming rate on glycerolized rabbit kidneys

Cooling and warming rates are known to be important determinants of viability for cryopreserved cells, but optimal rates have not previously been determined for any whole organ. In this study, rabbit kidneys, permeated with 2 M glycerol were cooled to -80 degrees C at four rates varying from 1 degrees C/hr to 3.1 degrees C/min and then rewarmed at four rates from 1 degrees C/hr to 4.2 degrees C/min, giving 16 experimental treatments. After gradual deglycerolization at 10 degrees C, each kidney was autografted and observed for 30 min. Assessment was by measurement of vascular resistance, immediate post-thaw lactate dehydrogenase (LDH) release, gross appearance, light- and electron microscopy, and tissue K+/Na+ ratio 30 min after transplantation. The best results were obtained after cooling at 1 degrees C/hr; warming rate had little apparent influence on the criteria used to assess function with the exception of LDH release, which indicated a preferred warming rate around 1 degrees C/min. Histological studies revealed extensive vascular damage, notably to the glomerular capillaries, that was minimized by very slow cooling. Freeze substitution, carried out on samples removed at -80 degrees C, demonstrated extensive ice formation in the interstitial space and, at the faster cooling rates, in the glomerular capillaries. Intracapillary ice formation was reduced in the kidneys cooled at 1 degrees C/hr.     

Proteomic Signature of Mesenchymal Stromal Cell‐Derived Small Extracellular Vesicles

Small extracellular vesicles (EVs) are 50–200 nm vesicles secreted by most cells. They are considered as mediators of intercellular communication, and EVs from specific cell types, in particular mesenchymal stem/stromal cells (MSCs), offer powerful therapeutic potential, and can provide a novel therapeutic strategy. They appear promising and safe (as EVs are non‐self‐replicating), and eventually MSC‐derived EVs (MSC‐EVs) may be developed to standardized, off‐the‐shelf allogeneic regenerative and immunomodulatory therapeutics. Promising pre‐clinical data have been achieved using MSCs from different sources as EV‐producing cells. Similarly, a variety EV isolation and characterization methods have been applied. Interestingly, MSC‐EVs obtained from different sources and prepared with different methods show in vitro and in vivo therapeutic effects, indicating that isolated EVs share a common potential. Here, well‐characterized and controlled, publicly available proteome profiles of MSC‐EVs are compared to identify a common MSC‐EV protein signature that might be coupled to the MSC‐EVs’ common therapeutic potential. This protein signature may be helpful in developing MSC‐EV quality control platforms required to confirm the identity and test for the purity of potential therapeutic MSC‐EVs.     

Can the outcomes of mesenchymal stem cell‐based therapy for myocardial infarction be improved? Providing weapons and armour to cells

Use of mesenchymal stem cell (MSC) transplantation after myocardial infarction (MI) has been found to have infarct‐limiting effects in numerous experimental and clinical studies. However, recent meta‐analyses of randomized clinical trials on MSC‐based MI therapy have highlighted the need for improving its efficacy. There are two principal approaches for increasing therapeutic effect of MSCs: (i) preventing massive MSC death in ischaemic tissue and (ii) increasing production of cardioreparative growth factors and cytokines with transplanted MSCs. In this review, we aim to integrate our current understanding of genetic approaches that are used for modification of MSCs to enable their improved survival, engraftment, integration, proliferation and differentiation in the ischaemic heart. Genetic modification of MSCs resulting in increased secretion of paracrine factors has also been discussed. In addition, data on MSC preconditioning with physical, chemical and pharmacological factors prior to transplantation are summarized. MSC seeding on three‐dimensional polymeric scaffolds facilitates formation of both intercellular connections and contacts between cells and the extracellular matrix, thereby enhancing cell viability and function. Use of genetic and non‐genetic approaches to modify MSC function holds great promise for regenerative therapy of myocardial ischaemic injury.     

Functional GPR37 trafficking protects against toxicity induced by 6‐OHDA,MPP+ or rotenone in a catecholaminergic cell line

G protein‐coupled receptor 37 (GPR37) is suggested to be implicated in the pathogenesis of Parkinson's disease and is accumulating in Lewy bodies within afflicted brain regions. Over‐expressed GPR37 is prone to misfolding and aggregation, causing cell death via endoplasmic reticulum stress. Although the cytotoxicity of misfolded GPR37 is well established, effects of the functional receptor on cell viability are still unknown. An N2a cell line stably expressing green fluorescent protein (GFP)‐tagged human GPR37 was created to study its trafficking and effects on cell viability upon challenge with the toxins 1‐methyl‐4‐phenylpyridinium (MPP+), rotenone and 6‐hydroxydopamine (6‐OHDA). Neuronal‐like differentiation into a tyrosine hydroxylase expressing phenotype, using dibutyryl‐cAMP, induced trafficking of GPR37 to the plasma membrane. 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) cell viability and lactate dehydrogenase (LDH) cell death assays revealed that GPR37 was protective against all three toxins in differentiated cells. In undifferentiated cells, the majority of GPR37 was cytoplasmic and the protective effects were more variable: GPR37 expression protected against rotenone and MPP+ but not against 6‐OHDA in MTT assays, while it protected against 6‐OHDA but not against MPP+ or rotenone in lactate dehydrogenase (LDH) assays. These results suggest that GPR37 functionally trafficked to the plasma membrane protects against toxicity.     

Acetyl‐11‐keto‐β‐boswellic acid ameliorates renal interstitial fibrosis via Klotho/TGF‐β/Smad signalling pathway

Acetyl‐11‐keto‐β‐boswellic acid (AKBA), an active triterpenoid compound from the extract of Boswellia serrate, has been reported previously in our group to alleviate fibrosis in vascular remodelling. This study aimed to elucidate the in vivo and in vitro efficacy and mechanism of AKBA in renal interstitial fibrosis. The experimental renal fibrosis was produced in C57BL/6 mice via unilateral ureteral obstruction (UUO). Hypoxia‐induced HK‐2 cells were used to imitate the pathological process of renal fibrosis in vitro. Results showed that the treatment of AKBA significantly alleviated UUO‐induced impairment of renal function and improved the renal fibrosis by decreasing the expression of TGF‐β1, α‐SMA, collagen I and collagen IV in UUO kidneys. In hypoxia‐induced HK‐2 cells, AKBA displayed remarkable cell protective effects and anti‐fibrotic properties by increasing the cell viability, decreasing the lactate dehydrogenase (LDH) release and inhibiting fibrotic factor expression. Moreover, in obstructed kidneys and HK‐2 cells, AKBA markedly down‐regulated the expression of TGFβ‐RI, TGFβ‐RII, phosphorylated‐Smad2/3 (p‐Smad2/3) and Smad4 in a dose‐dependent fashion while up‐regulated the expression of Klotho and Smad7 in the same manner. In addition, the effects of AKBA on the Klotho/TGF‐β/Smad signalling were reversed by transfecting with siRNA‐Klotho in HK‐2 cells. In conclusion, our findings provide evidence that AKBA can effectively protect kidney against interstitial fibrosis, and this renoprotective effect involves the Klotho/TGF‐β/Smad signalling pathway. Therefore, AKBA could be considered as a promising candidate drug for renal interstitial fibrosis.     

Lactate regulates pyruvate uptake and metabolism in the preimplantation mouse embryo

This study was an investigation of the interaction of lactate on pyruvate and glucose metabolism in the early mouse embryo. Pyruvate uptake and metabolism by mouse embryos were significantly affected by increasing the lactate concentration in the culture medium. In contrast, glucose uptake was not affected by lactate in the culture medium. At the zygote stage, the percentage of pyruvate taken up and oxidized was significantly reduced in the presence of increasing lactate, while at the blastocyst stage, increasing the lactate concentration increased the percentage of pyruvate oxidized. Lactate oxidation was determined to be 3-fold higher (when lactate was present at 20 mM) at the blastocyst stage compared to the zygote. Analysis of the kinetics of lactate dehydrogenase (LDH) determined that while the V(max) of LDH was higher at the zygote stage, the K(m) of LDH was identical for both stages of development, confirming that the LDH isozyme was the same. Furthermore, the activity of LDH isolated from both stages was reduced by 40% in the presence of 20 mM lactate. The observed differences in lactate metabolism between the zygote and blastocyst must therefore be attributed to in situ regulation of LDH. Activity of isolated LDH was found to be affected by nicotinamide adenine dinucleotide(+) (NAD(+)) concentration. In the presence of increasing concentrations of lactate, zygotes exhibited an increase in autofluorescence consistent with a depletion of NAD(+) in the cytosol. No increase was observed for later-stage embryos. Therefore it is proposed that the differences in pyruvate and lactate metabolism at the different stages of development are due to differences in the in situ regulation of LDH by cytosolic redox potential.     

Functional interrelations between isozymes of dehydrogenases in the intact and denervated rabbit muscles]

A correlation is shown to exist between malate dehydrogenase (MDH), lactate dehydrogenase (LDH) and glycerol-3-phosphate dehydrogenase (glycerol-3-PDH activity values, lactate/pyruvate and malate/oxaloacetate coefficients, MDH and LDH isozyme spectra and kinetic properties of LDH isozymes in soluble fractions of cytoplasm from intact rabbit m. soleus (red), m. gastrocnemius (mixed) and m. quadratus lumborum (white). In denervated soleus and gastrocnemius the cytoplasmic MDH/LDH, mitochondrial MDH/LDH, MDH mitochondrial/MDH cytoplasmic activity ratios, concentrations of substrates and isozyme spectra of MDH and LDH tend to equalize. The obtained results indicate the importance of isozyme composition and total activity ratios of the dehydrogenases for regulation of pyruvate and NADH metabolic pathways.     

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.     

Lactate Dehydrogenase Activity is Inhibited by Methylmalonate in vitro

Methylmalonic acidemia (MMAemia) is an inherited metabolic disorder of branched amino acid and odd-chain fatty acid metabolism, involving a defect in the conversion of methylmalonyl-coenzyme A to succinyl-coenzyme A. Systemic and neurological manifestations in this disease are thought to be associated with the accumulation of methylmalonate (MMA) in tissues and biological fluids with consequent impairment of energy metabolism and oxidative stress. In the present work we studied the effect of MMA and two other inhibitors of mitochondrial respiratory chain complex II (malonate and 3-nitropropionate) on the activity of lactate dehydrogenase (LDH) in tissue homogenates from adult rats. MMA potently inhibited LDH-catalyzed conversion of lactate to pyruvate in liver and brain homogenates as well as in a purified bovine heart LDH preparation. LDH was about one order of magnitude less sensitive to inhibition by MMA when catalyzing the conversion of pyruvate to lactate. Kinetic studies on the inhibition of brain LDH indicated that MMA inhibits this enzyme competitively with lactate as a substrate (K _i=3.02±0.59 mM). Malonate and 3-nitropropionate also strongly inhibited LDH-catalyzed conversion of lactate to pyruvate in brain homogenates, while no inhibition was observed by succinate or propionate, when present in concentrations of up to 25 mM. We propose that inhibition of the lactate/pyruvate conversion by MMA contributes to lactate accumulation in blood, metabolic acidemia and inhibition of gluconeogenesis observed in patients with MMAemia. Moreover, the inhibition of LDH in the central nervous system may also impair the lactate shuttle between astrocytes and neurons, compromising neuronal energy metabolism.S. R. Mirandola and E. N. Maciel contributed equally to this work.     

The effects of feed and intracellular pyruvate levels on the redistribution of metabolic fluxes in Escherichia coli

In a previous study, an Escherichia coli strain lacking the key enzymes (acetate kinase and phosphotransacetylase, ACK-PTA) of the major acetate synthesis pathways reduced acetate accumulation. The ackA-pta mutant strain also exhibits an increased lactate synthesis rate. Metabolic flux analysis suggested that the majority of excessive carbon flux was redirected through the lactate formation pathway rather than the ethanol synthesis pathway. This result indicated that lactate dehydrogenase may be competitive at the pyruvate node. However, a 10-fold overexpression of the fermentative lactate dehydrogenase (ldhA) gene in the wild-type parent GJT001 was not able to divert carbon flux from acetate. The carbon flux through pyruvate and all its end products increases at the expense of flux through biosynthesis and succinate. Intracellular pyruvate measurements showed that strains overexpressing lactate dehydrogenase (LDH) depleted the pyruvate pool. This observation along with the observed excretion of pyruvate in the ackA-pta strain indicates the significance of intracellular pyruvate pools. In the current study, we focus on the role of the intracellular pyruvate pool in the redirection of metabolic fluxes at this important node. An increasing level of extracellular pyruvate leads to an increase in the intracellular pyruvate pool. This increase in intracellular pyruvate affects carbon flux distribution at the pyruvate node. Partitioning of the carbon flux to acetate at the expense of ethanol occurs at the acetyl-CoA node while partitioning at the pyruvate node favors lactate formation. The increased competitiveness of the lactate pathway may be due to the allosteric activation of LDH as a result of increased pyruvate levels. The interaction between the reactions catalyzed by the enzymes PFL (pyruvate formate lyase) and LDH was examined.