Increased Lactate Formation Research Articles

Dimethyl Fumarate Mediates Sustained Vascular Smooth Muscle Cell Remodeling in a Mouse Model of Cerebral Aneurysm.

Cerebral aneurysms (CA) are a type of vascular disease that causes significant morbidity and mortality with rupture. Dysfunction of the vascular smooth muscle cells (VSMCs) from circle of Willis (CoW) vessels mediates CA formation, as they are the major cell type of the arterial wall and play a role in maintaining vessel integrity. Dimethyl fumarate (DMF), a first-line oral treatment for relapsing-remitting multiple sclerosis, has been shown to inhibit VSMC proliferation and reduce CA formation in a mouse model. Potential unwanted side effects of DMF on VSMC function have not been investigated yet. The present study characterizes the impact of DMF on VSMC using single-cell RNA-sequencing (scRNA-seq) in CoW vessels following CA induction and further explores its role in mitochondrial function using in vitro VSMC cultures. Two weeks of DMF treatment following CA induction impaired the transcription of the glutathione redox system and downregulated mitochondrial respiration genes in VSMCs. In vitro, DMF treatment increased lactate formation and enhanced the mitochondrial production of reactive oxygen species (ROS). These effects rendered VSMCs vulnerable to oxidative stress and led to mitochondrial dysfunction and enhancement of apoptosis. Taken together, our data support the concept that the DMF-mediated antiproliferative effect on VSMCs is linked to disturbed antioxidative functions resulting in altered mitochondrial metabolism. This negative impact of DMF treatment on VSMCs may be linked to preexisting alterations of cerebrovascular function due to renal hypertension. Therefore, before severe adverse effects emerge, it would be clinically relevant to develop indices or biomarkers linked to this disturbed antioxidative function to monitor patients undergoing DMF treatment.

AMPK activation is sufficient to increase skeletal muscle glucose uptake and glycogen synthesis but is not required for contraction-mediated increases in glucose metabolism

The AMP-activated protein kinase (AMPK) is a cellular sensor of energetics and when activated in skeletal muscle during contraction can impart changes in skeletal muscle metabolism. Therapeutics that selectively activate AMPK have been developed to lower glucose levels through increased glucose disposal rates as an approach to abrogate the hyperglycemic state of diabetes; however, the metabolic fate of glucose following AMPK activation remains unclear. We have used a combination of in vivo evaluation of glucose homeostasis and ex vivo skeletal muscle incubation to systematically evaluate metabolism following pharmacological activation of AMPK with PF-739, comparing this with AMPK activation through sustained intermittent electrical stimulation of contraction. These methods to activate AMPK result in increased glucose uptake but divergent metabolism of glucose: pharmacological activation results in increased glycogen accumulation while contraction-induced glucose uptake results in increased lactate formation and glucose oxidation. These results provide additional evidence to support a role for AMPK in control of skeletal muscle metabolism and additional insight into the potential for AMPK stimulation with small molecule direct activators.

An experimental and in silico analysis of Lacticaseibacillus paracasei isolated from whey shows an association between lactate production and amino acid catabolism.

The production of lactic acid from agroindustry waste products, such as whey, heavily relies on microorganisms within the genusLactobacillus. In this work, a genome-scale metabolic model was implemented from Vinay-Lara (iLca334_548), improved adding some enzymatic reactions and used to analyse metabolic fluxes ofLacticaseibacillus paracasei, which is aLactobacillusstrain isolated from whey used in the large-scale production of lactic acid. Overall, the highest rate of lactic acid productivity was 2.9 g l-1h-1, which equates to a dilution rate of 0.125 h-1, when continuous culture conditions were established. Restrictions on lactic acid production caused by exchange reactions, complex culture medium and intracellular metabolite concentrations were considered and included in the model. In total, theiLca334_548 model consisted of 1046 reactions and 959 metabolites, and flow balance analysis better predicted lactate flux than biomass. The distribution of fluxes exhibited an increase in lactate formation as biomass decreased. This finding is supported by the reactions carried out by glyceraldehyde 3-phosphate dehydrogenase, pyruvate formate lyase and ribose-5-phosphate isomerase, corroborating the modelled phenotype with experimental data. In conclusion, there is potential for the improvement of lactate production in a complex media by amino acid catabolism, especially when lactate is derived from pyruvate.

Sulforaphane exposure impairs contractility and mitochondrial function in three-dimensional engineered heart tissue

Sulforaphane (SFN) is a phytochemical compound extracted from cruciferous plants, like broccoli or cauliflower. Its isothiocyanate group renders SFN reactive, thus allowing post-translational modification of cellular proteins to regulate their function with the potential for biological and therapeutic actions. SFN and stabilized variants recently received regulatory approval for clinical studies in humans for the treatment of neurological disorders and cancer. Potential unwanted side effects of SFN on heart function have not been investigated yet. The present study characterizes the impact of SFN on cardiomyocyte contractile function in cardiac preparations from neonatal rat, adult mouse and human induced-pluripotent stem cell-derived cardiomyocytes. This revealed a SFN-mediated negative inotropic effect, when administered either acutely or chronically, with an impairment of the Frank-Starling response to stretch activation. A direct effect of SFN on myofilament function was excluded in chemically permeabilized mouse trabeculae. However, SFN pretreatment increased lactate formation and enhanced the mitochondrial production of reactive oxygen species accompanied by a significant reduction in the mitochondrial membrane potential. Transmission electron microscopy revealed disturbed sarcomeric organization and inflated mitochondria with whorled membrane shape in response to SFN exposure. Interestingly, administration of the alternative energy source l-glutamine to the medium that bypasses the uptake route of pyruvate into the mitochondrial tricarboxylic acid cycle improved force development in SFN-treated EHTs, suggesting indeed mitochondrial dysfunction as a contributor of SFN-mediated contractile dysfunction. Taken together, the data from the present study suggest that SFN might impact negatively on cardiac contractility in patients with cardiovascular co-morbidities undergoing SFN supplementation therapy. Therefore, cardiac function should be monitored regularly to avoid the onset of cardiotoxic side effects.

A retrospective analysis of CO2 derived goal-directed perfusion variables under normothermic conditions: do we need to re-evaluate threshold values?

This study investigated if current predictive values for increased lactate formation: VCO2i > 60 ml min-1 m-2, respiratory quotient (RQ) > 0.90, and DO2/VCO2 < 5.0, are valid under normothermic conditions. CO2 derived parameters were analyzed in 91 patients undergoing normothermic CABG and related to increase of blood lactate concentrations during bypass. In this study population, 85 patients (93%) had a median VCO2i above 60 ml min-1 m-2 and 53 patients (58%) had a DO2/VCO2 ⩽ 5.0. Eighteen patients (20%) had a median RQ ⩾ 0.90, but RQ remained with a maximum value of 0.94 below the biological threshold of 1.0. Increase of lactate concentrations remained without clinical significance and showed weak correlations with VCO2i (rs = 0.277, p = 0.008) and RQ (rs = 0.346, p = 0.001).The cohort was separated for the different CO2 variables by their median value to compare increase in lactate concentration. Patients with a high VCO2i (⩾70 ml min-1 m-2) and a high RQ (⩾0.82) showed significant higher increase in lactate concentration compared to patients with VCO2i < 70 ml min-1 m-2 (p = 0.004), and a RQ < 0.82 (p = 0.012). Groups separated by a median DO2/VCO2 of 4.8 did not show a difference in increase of lactate concentration in blood. In summary, specific CO2 derived threshold values for the prediction of lactate formation, which have been reported in other studies, cannot be confirmed with our findings. However, a CO2 production ⩾70 ml min-1 m-2 and a RQ ⩾ 0.82 in this study population were correlated with increased lactate formation. Because CO2 production during bypass depends on patient temperature, a different cutoff value, that may take into account the influence of demographic variables, should be determined during normothermic CPB.

Regulation of pyruvate dehydrogenase complex related to lactate switch in CHO cells.

The metabolism of Chinese hamster ovary (CHO) cell lines is typically characterized by high rates of aerobic glycolysis with increased lactate formation, known as the ”Warburg” effect. Although this metabolic state can switch to lactate consumption, the involved regulations of the central metabolism have only been partially studied so far. An important reaction transferring the lactate precursor, pyruvate, into the tricarboxylic acid cycle is the decarboxylation reaction catalyzed by the pyruvate dehydrogenase enzyme complex (PDC). Among other mechanisms, PDC is mainly regulated by phosphorylation–dephosphorylation at the three sites Ser232, Ser293, and Ser300. In this work, the PDC phosphorylation in antibody‐producing CHO DP‐12 cell culture is investigated during the lactate switch. Batch cultivations were carried out with frequent sampling (every 6 h) during the transition from lactate formation to lactate uptake, and the PDC phosphorylation levels were quantified using a novel indirect flow cytometry protocol. Contrary to the expected activation of PDC (i.e., reduced PDC phosphorylation) during lactate consumption, Ser293 and Ser300 phosphorylation levels were 33% higher compared to the phase of glucose excess. At the same time, the relative phosphorylation level of Ser232 increased steadily throughout the cultivation (66% increase overall). The intracellular pyruvate was found to accumulate only during the period of high lactate production, while acetyl‐CoA showed nearly no accumulation. These results indicate a deactivation of PDC and reduced oxidative metabolism during lactate switch even though the cells undergo a metabolic transition to lactate‐based cell growth and metabolism. Overall, this study provides a unique view on the regulation of PDC during the lactate switch, which contributes to an improved understanding of PDC and its interaction with the bioprocess.

Astaxanthin inhibits the reduction of glycolysis during the activation of hepatic stellate cells

AimsHepatic stellate cells (HSCs) play an essential role in the development of liver fibrosis by producing extracellular matrix proteins, growth factors, and pro-inflammatory and pro-fibrogenic cytokines once activated. We previously demonstrated that astaxanthin (ASTX), a xanthophyll carotenoid, attenuates HSC activation. The objective of this study was to investigate whether there is a difference in glycolysis between quiescent and activated HSCs and the effect of ASTX on glycolysis during HSC activation. Materials and methodsMouse primary HSCs were activated for 7 days in the presence or absence of 25 μM of ASTX. Quiescent HSCs (qHSCs), 1 day after isolation, and activated HSCs (aHSCs) treated with/without ASTX were plated in a Seahorse XF24 cell culture microplate for Glycolysis Stress tests. Key findingsaHSCs had significantly lower glycolysis, but higher glycolytic capacity, maximum capacity of glycolysis, and non-glycolytic acidification than qHSCs. Importantly, ASTX markedly increased glycolysis during HSC activation with a concomitant increase in lactate formation and secretion. Compared with qHSCs, aHSCs had significantly lower expression of glucose transporter 1, the major glucose transporter in HSCs, and its transcription factor hypoxia-inducible factor 1α, which was markedly increased by ASTX in aHSCs. SignificanceOur data suggest that ASTX may prevent the activation of HSCs by altering glycolysis and the expression of genes involved in the pathways.

Molecular basis for increased lactate formation in the Müller glial cells of retina

Müller glial cells are highly metabolic active cells that compensate for the high energy demand of retinal neurons. It has been believed that glucose provides the energy needs by the complete oxidation within Müller cells. However, numerous studies indicated that glial cells convert the majority of glucose to lactate, which may serve as an energy source for neurons. It is still not well understood why within glia, glucose is not completely oxidized under aerobic glycolysis conditions. The aspartate glutamate carrier (AGC) is a major component of the malate-aspartate shuttle (MAS) responsible for transporting the reducing equivalent of glycolysis to the mitochondria for the complete oxidation of glucose. Here, we report the absence of AGC within Müller glial cells which impairs the ability to oxidize glucose. We investigated the expression and localization of AGC and its isoforms (aralar and citrin) in the intact rat retina. We also analyzed the expression and regulation of AGC and its metabolic activity within cultured Müller cells (TR-MUL). The results suggest that AGC and its isoforms seem to be neuronal, with no or low expression within Müller cells of the intact retina. The study of cultured Müller cells suggests a very low expression of AGC and a decreased metabolic activity of the carrier especially under cell differentiation conditions due to low serum and hydrocortisone treatments. Thus, these data give a molecular explanation of increased levels of lactate formation due to a lack of AGC in the retina by Müller glial cells.

Abstract 371: Utilizing metabolic pathways to improve diagnosis and risk stratification of prostate cancer

Abstract Prostate cancer has well-characterized changes that take place metabolically in the citric acid cycle (CAC) such as increased lactate formation, reversal of physiologic buildup of citrate and breakdown into downstream CAC products. Metabolic MR spectroscopy utilizing 13C-labeled pyruvate now allows for clinical characterization of metabolic pathways in tissue. However, the traditionally utilized substrate of [1-13C]pyruvate has limitations because as the pyruvate is broken down, the labeled carbon does not enter the CAC and instead is lost as CO2. We propose that [2-13C]pyruvate, is better to characterize changes in the CAC. Specifically, [2-13]C-pyruvate can be converted to 13C-labeled acetyl CoA and subsequently metabolized in the CAC as 13C-labeled intermediate compounds (such as glutamate which rapidly pools alpha-ketogluterate). Alternatively in altered bioenergetic states, [2-13C]pyruvate is reduced to 13C-labeled lactate (Warburg effect) or transaminated to 13C-labeled alanine. By exposing prostate cancer cells to [2-13C]pyruvate and determining the relative labeling of lactate, glutamate, and alanine, we can elucidate which metabolic pathways (CAC, Warburg, transamination) are utilized in prostate cancer metabolism. The aim of this study was to determine how the metabolism of [2-13C]pyruvate via the CAC, reduction to lactate, and transamination differed in high and low malignancy prostate cancer cells.Methods: Tumorigenic prostate cells LNCaP and PC3 were exposed a medium containing [2-13C]pyruvate for 4 hours. PCA extraction was then performed to obtain the metabolites. 13C labeled compounds formed intracellularly and those released into the medium were analyzed using NMR chemical shifts with focus on peaks associated with pyruvate, lactate, alanine, glutamate, and other compounds related to the CAC. Dioxane was used as an internal standard for NMR characterization. Results: The labeled carbon from the [2-13C]pyruvate can be identified in the downstream metabolites of the citric acid cycle, after reduction to lactate, and/or transamination by determining labeled metabolites by 13C-NMR analysis. This allows for the ability to calculate the relative metabolism of pyruvate via the three pathways; transamination (74%), lactate formation (16%), and CAC (10%). Conclusions: [2-13C]pyruvate can be successfully utilized as a tool to explore the alterations in pathways of metabolism, including the citric acid cycle, in prostate cancer cells. Citation Format: Aymen Alqazzaz, Dexue Fu, Arman Karimi, Gustavo Ferreira, Mary McKenna, Mohummad M. Siddiqui. Utilizing metabolic pathways to improve diagnosis and risk stratification of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 371.

Protein and Glucose Absorption and Gastrointestinal Hormone Secretion Differ between Roux-en-Y Gastric Bypass and Sleeve Gastrectomy

Sleeve gastrectomy (SG) and Roux-en-Y gastric bypass (RYGB) induce comparable weight loss and improvements in glycemic control despite marked differences in gastrointestinal rearrangements. We hypothesized that absorption rates of oral glucose and protein derived amino acids (AA) were accelerated after both procedures resulting in comparable gut hormone responses. 12 SG and 12 RYGB operated and 12 control (C) subjects carefully matched on BMI, age, sex and post-operative weight loss received primed-continuous infusions of stable isotopes of glucose, glycerol, phenylalanine (phe), tyrosine and urea combined with a 6-hour mixed meal test (400 kcal, 50E% carb, 35E% fat, 15E% protein) containing a glucose isotope and intrinsically phe labelled casein. Peak rate of appearance (Ra) of oral glucose was higher after RYGB and SG compared with C (RYGB 36±1 µmol/kg FFM/min, SG 27±1, C 22±1; p&amp;lt;0.for all comparisons) and peak Ra of phe originating from meal protein was higher after RYGB but similar in SG and C (RYGB 0.33±0.04, SG 0.17±0.02, C 0.13 ±0.01 p&amp;lt;0.01 for RYGB vs. both SG and C). Oral recovery of ingested glucose and phe as well as net protein synthesis were clearly enhanced within the first postprandial hour after RYGB, whereas total net protein balance and oral recovery did not differ between groups. Urea turnover was equal between groups. The rapid absorption of glucose and phe was associated with larger but more transient excursions of plasma glucose and AAs followed by increased lactate formation and higher secretion of GLP-1, insulin and PYY, exclusively after RYGB. Ghrelin concentration was lower after SG. Postprandial glucose and protein metabolism and gastro-entero-pancreatic hormone secretion clearly differ after RYGB and SG, with accelerated absorption of glucose and casein after RYGB, whereas casein uptake after SG resembled controls. Hence different mechanisms may underlie the improved glycemic control and weight loss after RYGB and SG. Disclosure M.S. Svane: None. K.N. Bojsen-Moller: None. C. Martinussen: None. C. Dirksen: None. S. Reitelseder: None. L. Holm: None. V.B. Kristiansen: None. G. van Hall: None. J.J. Holst: Advisory Panel; Self; Novo Nordisk A/S. Board Member; Self; Zealand Pharma A/S. Speaker's Bureau; Self; Merck Sharp &amp; Dohme Corp., AstraZeneca. Research Support; Self; Danish Diabetes Academy, Novo Nordisk Foundation. Other Relationship; Self; Antag Therapeutics. Other Relationship; Spouse/Partner; Antag Therapeutics. S. Madsbad: Advisory Panel; Self; AstraZeneca. Speaker's Bureau; Self; AstraZeneca. Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc.. Advisory Panel; Self; Boehringer Ingelheim Pharmaceuticals, Inc.. Speaker's Bureau; Self; Eli Lilly and Company, Eli Lilly and Company. Advisory Panel; Self; Merck Sharp &amp; Dohme Corp., Sanofi-Aventis, Novo Nordisk A/S. Speaker's Bureau; Self; Novo Nordisk A/S. Research Support; Self; Novo Nordisk A/S.

To cleave or not to cleave: role of ADAM17 in cell proliferation in PKD.

To cleave or not to cleave: role of ADAM17 in cell proliferation in PKD.

ADAM17 promotes proliferation of collecting duct kidney epithelial cells through ERK activation and increased glycolysis in polycystic kidney disease.

Polycystic kidney disease (PKD) is a common genetic disorder leading to cyst formation in the kidneys and other organs that ultimately results in kidney failure and death. Currently, there is no therapy for slowing down or stopping the progression of PKD. In this study, we identified the disintegrin metalloenzyme 17 (ADAM17) as a key regulator of cell proliferation in kidney tissues of conditional knockout Ift88(-/-) mice and collecting duct epithelial cells from Ift88°(rpk) mice, animal models of autosomal recessive polycystic kidney disease (ARPKD). Using Western blotting, an enzyme activity assay, and a growth factor-shedding assay in the presence or absence of the specific ADAM17 inhibitor TMI-005, we show that increased expression and activation of ADAM17 in the cystic kidney and in collecting duct epithelial cells originating from the Ift88°(rpk) mice (designated as PKD cells) lead to constitutive shedding of several growth factors, including heparin-binding EGF-like growth factor (HB-EGF), amphiregulin, and transforming growth factor-α (TGF-α). Increased growth factor shedding induces activation of the EGFR/MAPK/ERK pathway and maintains higher cell proliferation rate in PKD cells compared with control cells. PKD cells also displayed increased lactate formation and extracellular acidification indicative of aerobic glycolysis (Warburg effect), which was blocked by ADAM17 inhibition. We propose that ADAM17 is a key promoter of cellular proliferation in PKD cells by activating the EGFR/ERK axis and a proproliferative glycolytic phenotype.

Effect of electron mediators on current generation and fermentation in a microbial fuel cell

Effects of select electron mediators [9,10-anthraquinone-2,6-disulfonic acid disodium salt (AQDS), safranine O, resazurin, methylene blue, and humic acids] on metabolic end-products and current production from cellulose digestion by Clostridium cellulolyticum in microbial fuel cells (MFCs) were studied using capillary electrophoresis and traditional electrochemical techniques. Addition of the mediator resazurin greatly enhanced current production but did not appear to alter the examined fermentation end-products compared to MFCs with no mediator. Assays for lactate, acetate, and ethanol indicate that the presence of safranine O, methylene blue, and humic acids alters metabolite production in the MFC: safranine O decreased the examined metabolites, methylene blue increased lactate formation, and humic acids increased the examined metabolites. Mediator standard redox potentials (E (0)) reported in the literature do not coincide with redox potentials in MFCs due presumably to the electrolytic complexity of media that supports bacterial survival and growth. Current production in MFCs: (1) can be effected by the mediator redox potential while in the media, which may be significantly shifted from E (0), and (2) depended on the ability of the mediator to access the bacterial electron source, which may be cytoplasmic. In addition, some electron mediators had significant effects on metabolic end-products and therefore the metabolism of the organism itself.

Effect of nitric oxide synthase (NOS) inhibition on macro- and microcirculation in a model of rat endotoxic shock

Treatment of hemodynamic instability in septic shock often demands the administration of vasopressor agents, although these may have deleterious effects on microcirculatory homeostasis. Inhibition of nitric oxide synthase (NOS) has been suggested as an alternative therapeutic approach, as NO formation may be excessively increased in sepsis. To compare the effects of epinephrine titration, non-selective NOS inhibition by L-NMMA and selective inhibition of inducible NOS (iNOS) by 1400W on hemodynamics and on the regulation of microcirculation in a rat model of endotoxic shock, we intravenously injected endotoxin (LPS) or saline to male Wistar rats and after 2 hours randomized LPS treated rats into four different groups that received either saline, norepinephrine, L-NMMA or 1400W (n = 6 per group). Three hours after LPS administration, rats presented with severe systemic arterial hypotension (64 +/- 3 vs. 115 +/- 4 mmHg, p < 0.001), unresponsiveness to volume treatment, lactate acidosis and a marked increase in plasmatic nitrite and nitrate levels (15 +/- 8 vs. 263 +/- 47 microM, p < 0.001). Measurement of the tissue oxygenation in the ileum mucosal layer by the Erlangen micro-lightguide spectrophotometer (EMPHO) technique demonstrated marked heterogeneity of hemoglobin saturation, with appearance of low oxygenated areas. Norepinephrine, usually stabilizing blood pressure (99 +/- 7 vs. 67 +/- 4 mmHg 60 min after infusion, p < 0.01), increased lactate formation (7.9 +/- 0.2 vs. 3.7 +/- 0.5 mM, p < 0.001) and drastically increased low oxygenated regions in the ileum mucosal layer. L-NMMA similarly increased blood pressure (92 +/- 6 vs. 67 +/- 4 mmHg 60 min after infusion, p < 0.05), but did not enhance lactate acidosis. However, some further deterioration of mucosa oxygenation was again noted. 1400W forwarded stabilization of blood pressure (88 +/- 5 vs. 67 +/- 4 mmHg 60 min after injection, p < 0.05), reduced plasmatic nitrite and nitrate levels similar to L-NMMA, without an aggravation of lactate acidosis. In addition, mucosal oxygenation did not deteriorate in response to this agent. Thereby, we conclude that in a rat model of endotoxic shock selective iNOS inhibitors are superior to non-specific NOS inhibitors and in particular to norepinephrine for the treatment of macro- and microcirculatory abnormalities in experimental septic shock.

AMINO-ACID-DEPENDENT, DIFFERENTIAL EFFECTS OF ETHANOL ON GLUCOSE PRODUCTION IN RABBIT KIDNEY-CORTEX TUBULES

The effect of ethanol on glucose synthesis in kidney-cortex tubules of control and diabetic rabbits has been investigated. Both freshly isolated and grown in primary cultures, kidney-cortex tubules were incubated with alanine or aspartate plus lactate or glycerol plus octanoate in the absence and presence of 100 mmol/l ethanol. In freshly isolated renal tubules incubated in the presence of alanine plus lactate or glycerol plus octanoate, and in tubules grown in primary culture in the medium containing alanine plus lactate plus octanoate alcohol, resulted in about 30% decrease in glucose formation. A diminished glucose production in freshly isolated tubules was accompanied by: (i) a decrease in alanine utilization, (ii) an increase in lactate or glycerol consumptions and (iii) a decline in GSH:GSSG ratio. The ethanol action was not abolished by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase (ADH). In view of ethanol-induced changes in gluconeogenic intermediates it is likely that in the presence of alanine plus glycerol plus octanoate ethanol causes a decline in flux through phosphoenolpyruvate carboxykinase, probably due to either an increase in intracellular content of 2-oxoglutarate, inhibitor of this key gluconeogenic enzyme and/or an enhanced flux through pyruvate kinase, as concluded from an increased lactate formation in the presence of glycerol in the incubation medium. In renal tubules grown in primary cultures in the presence of alanine plus lactate plus octanoate a decrease in GSH:GSSG ratio was accompanied by elevated generation of reactive oxygen species (ROS). Upon replacement of alanine by aspartate ethanol affected neither glucose production, substrate uptake, ROS accumulation nor GSH:GSSG ratio. In the presence of alanine ethanol-induced decrease in glucose production and elevation of ROS might cause a limited NADPH generation resulting in a decrease in the intracellular GSH:GSSG ratio. On the contrary, aspartate might protect against ROS generation, so intensive gluconeogenesis supports NADPH generation and in consequence high values of the intracellular GSH:GSSG ratio are maintained.

Imbalance of plasma membrane ion leak and pump relationship as a new aetiological basis of certain disease states.

The basis for life is the ability of the cell to maintain ion gradients across biological membranes. Such gradients are created by specific membrane-bound ion pumps [adenosine triphosphatases (ATPases)]. According to physicochemical rules passive forces equilibrate (dissipate) ion gradients. The cholesterol/phospholipid ratio of the membrane and the degree of saturation of phospholipid fatty acids are important factors for membrane molecular order and herewith a determinant of the degree of non-specific membrane leakiness. Other operative principles, i.e. specific ion channels can be opened and closed according to mechanisms that are specific to the cell. Certain compounds called ionophores can be integrated in the plasma membrane and permit specific inorganic ions to pass. Irrespective of which mechanism ions leak across the plasma membrane the homeostasis may be kept by increasing ion pumping (ATPase activity) in an attempt to restore the physiological ion gradient. The energy source for this work seems to be glycolytically derived ATP formation. Thus an increase in ion pumping is reflected by increased ATP hydrolysis and rate of glycolysis. This can be measured as an accumulation of breakdown products of ATP and end-products of anaerobic glycolysis (lactate). In certain disease entities, the balance between ATP formation and ion pumping may be disordered resulting in a decrease in inter alia (i.a.) cellular energy charge, and an increase in lactate formation and catabolites of adenylates. Cardiac syndrome X is proposed to be due to an excessive leakage of potassium ions, leading to electrocardiographic (ECG) changes, abnormal Tl-scintigraphy of the heart and anginal pain (induced by adenosine). Cocksackie B3 infections, a common agent in myocarditis might also induce an ionophore-like effect. Moreover, Alzheimer's disease is characterized by the formation of extracellular amyloid deposits in the brain of patients. Perturbation of cellular membranes by the amyloid peptide during the development of Alzheimer's disease is one of several mechanisms proposed to account for the toxicity of this peptide on neuronal membranes. We have studied the effects of the peptide and fragments thereof on 45Ca2+-uptake in human erythrocytes and the energetic consequences. Treatment of erythrocytes with the beta 1-40 peptide, results in qualitatively similar nucleotide pattern and decrease of energy charge as the treatment with Ca2+-ionophore A23187. Finally, in recent studies we have revealed and published in this journal that a rare condition, Tarui's disease or glycogenosis type VII, primarily associated with a defect M-subunit of phosphofructokinase, demonstrates as a cophenomenon an increased leak of Ca2+ into erythrocytes.

COMPARING POST EXERCISE BLOOD LACTATE BETWEEN SEA LEVEL AND ALTITUDE NATIVES

Blood lactate appearance and clearance has been closely linked to fatigue (Grassi et al., 1996). At altitude and during intense levels of exercise, there is an increase in lactate formation, a drop in pH and an increased fatigue rate. PURPOSE: It is the purpose of this study to compare percent recovery, based on post-exercise (PE) blood lactate (La), with sea level and altitude natives. METHODS: Fourteen (N = 14) subjects living and training at sea level (n = 7) and altitude (n = 7) were matched by demographic measures. Each subject completed a cycle ergometer protocol with increasing workload every three (3) minutes until volitional fatigue. Resting La (mM), peak La and recovery La (20 min. PE) were taken via finger-stick to determine percent recovery. RESULTS: Percent recovery and maximal heart rate were significantly greater in the sea level subjects when compared to their altitude counterparts.Table 1: Mean (SD) Measures between Sea Level and Altitude NativesConclusion: The following conclusions were established: (1) lactate clearance is enhanced in sea level cyclists, and (2) sea level cyclists reach greater maximal heart rates when compared to altitude cyclists. Lactate removal is enhanced through a greater circulatory component and/or an enhanced oxidative metabolism of lactate, facilitated by an increased partial pressure of oxygen.

Metabolic aspects of continuous renal replacement therapies

Metabolic aspects of continuous renal replacement therapies

Glucose Catabolism ofEscherichia coliStrains with Increased Activity and Altered Regulation of Key Glycolytic Enzymes

This study investigates the effect of overexpression of key glycolytic enzymes exhibiting either native or alternative allosteric regulation on glucose bioconversion by restingEscherichia colicells previously engineered for ethanol production. Homologous and heterologous pyruvate kinases (Pyk) and phosphofructokinases (Pfk) were individually and simultaneously overexpressed. Overexpression of theE. coliPfk led to a shift from ethanol to lactate formation (threefold above the control level) while overexpression of Pyks accelerated lactate formation two-fold with less reduction in ethanol formation. Further increase in lactate formation (five-fold above the control level) resulted from overexpression of Pfk fromLactobacillus bulgaricuswhich, unlike theE. coliPfk, is not allosterically regulated by either phosphoenolpyruvate or ADP. These effects on the carbon flux distribution were accompanied by significant changes in the intracellular concentrations of several glycolytic intermediates. Increased Pfk levels led primarily to reduced levels of hexose phosphates. Increased Pyk activity resulted in more complex changes which were different for overexpressed native Pyk and for overexpressedBacillus stearothermophilusPyk, which differs fromE. coliPyk in lacking activation by fructose 1,6-diphosphate, but is allosterically activated by AMP and ribose 5-phosphate. Simultaneous overexpression of native Pfk and Pyk caused a Pfk-overexpression-like phenotype with lower levels of hexose phosphates and further increased lactate formation (nine-fold above the control level). The flux data demonstrate that overexpression of even single enzymes early in a central pathway can increase the fluxes to a particular metabolic product, although it may not affect the glucose uptake rate.

Amylin-mediated inhibition of insulin-stimulated glucose transport in skeletal muscle.

We examined the effects of amylin on 3-O-methyl-D-glucose (3-O-MG) transport in perfused rat hindlimb muscle under hyperinsulinemic (350 microU/ml, 2,100 pmol/l) conditions. Amylin at 100 nmol/l concentration inhibited 3-O-MG transport relative to control in all three basic muscle fiber types. Transport decreased in slow-twitch oxidative (from 5.65 +/- 1.13 to 3.46 +/- 0.71 micromol . g-1 . h-1), fast-twitch oxidative (from 6.84 +/- 0.90 to 4.84 +/- 0.76 micromol . g-1 . h-1), and fast-twitch glycolytic (from 1.27 +/- 0.20 to 0.60 +/- 0.05 micromol . g-1 . h-1) muscle. Amylin inhibition of insulin-stimulated glucose transport in skeletal muscle was accompanied by a 433 +/- 72% increase in intracellular glucose 6-phosphate (G-6-P) despite the absence of extracellular glucose. The source of hexose units for the formation and maintenance of G-6-P was likely glycogen. Amylin increased glycogenolysis, increased lactate formation, and decreased glycogen synthase activity. Furthermore, the kinetics of glycogen synthase suggest that this enzyme may control intracellular G-6-P concentration. Despite the large increase in G-6-P, no detectable increase in uridine diphosphate-N-acetylhexosamines occurred, suggesting that the proposed glucosamine pathway may not be involved in transport inhibition. However, decreases in uridine diphosphate hexoses were detected. Therefore, uridine or hexosamine-based metabolites may be involved in amylin action.