High Rate Of Lactate Production Research Articles

Understanding the impact of bioactive coating materials for human mesenchymal stromal cells and implications for manufacturing

Bioactive materials interact with cells and modulate their characteristics which enable the generation of cell-based products with desired specifications. However, their evaluation and impact are often overlooked when establishing a cell therapy manufacturing process. In this study, we investigated the role of different surfaces for tissue culture including, untreated polystyrene surface, uncoated Cyclic Olefin Polymer (COP) and COP coated with collagen and recombinant fibronectin. It was observed that human mesenchymal stromal cells (hMSCs) expanded on COP-coated plates with different bioactive materials resulted in improved cell growth kinetics compared to traditional polystyrene plates and non-coated COP plates. The doubling time obtained was 2.78 and 3.02 days for hMSC seeded in COP plates coated with collagen type I and recombinant fibronectin respectively, and 4.64 days for cells plated in standard polystyrene treated plates. Metabolite analysis reinforced the findings of the growth kinetic studies, specifically that cells cultured on COP plates coated with collagen I and fibronectin exhibited improved growth as evidenced by a higher lactate production rate (9.38 × 105 and 9.67 × 105 pmol/cell/day, respectively) compared to cells from the polystyrene group (5.86 × 105 pmol/cell/day). This study demonstrated that COP is an effective alternative to polystyrene-treated plates when coated with bioactive materials such as collagen and fibronectin, however COP-treated plates without additional coatings were found not to be sufficient to support cell growth. These findings demonstrate the key role biomaterials play in the cell manufacturing process and the importance of optimising this selection.

Assessment of Mitochondrial Cell Metabolism by Respiratory Chain Electron Flow Assays.

Cellular energy metabolism is regulated by complex metabolic pathways. Although anaerobic glycolysis was reported as a primary source of energy in cancer leading to a high rate of lactate production, current evidence shows that the main energy source supporting cancer cell metabolism relies on mitochondrial metabolism. Mitochondria are the key organelle maintaining optimal cellular energy levels. MitoPlate™ S-1 provides a highly reproducible bioenergetics tool to analyze the electron flow rate in live cells. Measuring the rates of electron flow into and through the electron transport chain using different NADH and FADH2-producing metabolic substrates enables the assessment of mitochondrial functionality. MitoPlate™ S-1 are 96-well microplates pre-coated with different substrates used as probes to examine the activity of mitochondrial metabolic pathways based on a colorimetric assay. A comparative metabolic analysis between cell lines or primary cells allows to establish a specific metabolic profile and to detect possible alterations of the mitochondrial function of a tumor cell. Moreover, the direct measurements of electron flux triggered by metabolic pathway activation could highlight targets for potential drug candidates.

Lactate Production Precedes Inflammatory Cell Recruitment in Arthritic Ankles: an Imaging Study

PurposeInflammation is involved in many disease processes. However, accurate imaging tools permitting diagnosis and characterization of inflammation are still missing. As inflamed tissues exhibit a high rate of glycolysis, pyruvate metabolism may offer a unique approach to follow the inflammatory response and disease progression. Therefore, the aim of the study was to follow metabolic changes and recruitment of inflammatory cells after onset of inflammation in arthritic ankles using hyperpolarized 1-13C-pyruvate magnetic resonance spectroscopy (MRS) and 19F magnetic resonance imaging (MRI), respectively.ProcedureExperimental rheumatoid arthritis (RA) was induced by intraperitoneal injection of glucose-6-phosphate-isomerase-specific antibodies (GPI) containing serum. To monitor pyruvate metabolism, the transformation of hyperpolarized 1-13C-pyruvate into hyperpolarized 1-13C-lactate was followed using MRS. To track phagocytic immune cell homing, we intravenously injected a perfluorocarbon emulsion 48 h before imaging. The animals were scanned at days 1, 3, or 6 after GPI-serum injection to examine the different stages of arthritic inflammation. Finally, to confirm the pyruvate metabolic activity and the link to inflammatory cell recruitment, we conducted hematoxylin-eosin histopathology and monocarboxylase transporter (MCT-1) immune histochemistry (IHC) of inflamed ankles.ResultsHyperpolarized 1-13C-pyruvate MRS revealed a high rate of lactate production immediately at day 1 after GPI-serum transfer, which remained elevated during the progression of the disease, while 19F-MRI exhibited a gradual recruitment of phagocytic immune cells in arthritic ankles, which correlated well with the course of ankle swelling. Histopathology and IHC revealed that MCT-1 was expressed in regions with inflammatory cell recruitment, confirming the metabolic shift identified in arthritic ankles.ConclusionsOur study demonstrated the presence of a very early metabolic shift in arthritic joints independent of phagocytic immune cell recruitment. Thus, hyperpolarized 1-13C-pyruvate represents a promising tracer to monitor acute arthritic joint inflammation, even with minor ankle swelling. Furthermore, translated to the clinics, these methods add a detailed characterization of disease status and could substantially support patient stratification and therapy monitoring.

Abstract 713: Targeting glycolytic pathway with 2-Deoxy-glucose enhances lapatinib sensitivity in HER2-amplified breast cancer

Abstract Background: Aerobic glycolysis, commonly used by cancer cells, converts glucose to lactate regardless of available oxygen. 2-Deoxyglucose (2DG), a glucose metabolism inhibitor, inhibits hexokinase enzyme activity, and can be used in functional tumor imaging, which can enhance efficacy of chemotherapy and radiotherapy in solid tumors. Lapatinib, a HER kinase inhibitor, is used for HER2+ breast cancer treatment, but is limited in efficacy due to emergence of therapeutic resistance. However, data investigating combination of 2DG + lapatinib in HER2+ BC are limited. In this study, we evaluated whether 2DG enhances lapatinib sensitivity in lapatinib-sensitive and resistant HER2+ cells. Materials and methods: SKBR3 and BT474 cells were treated with increasing dose of lapatinib to develop lapatinib-resistant cells: SKBR3-LapR and BT474-LapR. Potential synergism between 2DG and lapatinib was evaluated by viability assays, and calculated via CalcuSyn software or relative risk ratios (RRRs). Glycolysis was monitored in response to drug treatments via western blots and assays for lactate production, glucose uptake, and hexokinase activity. Results: Lapatinib treatment significantly inhibited glycolysis in SKBR3 and BT474 cell lines (P < 0.01). Combination lapatinib + 2DG was synergistic in inhibiting SKBR3 and BT474 proliferation, with average IC50 values of 0.53±0.16 and 0.55±0.01, respectively. SKBR3-LapR cells exhibited higher rates of lactate production, glucose uptake and hexokinase activity compared to lapatinib-sensitive cells (P < 0.05), and BT474-LapR cells exhibited higher hexokinase activity (P < 0.001) and glucose uptake (P < 0.05). RRRs were less than 1 with 2DG + lapatinib combination in SKBR3-LapR (0.88-0.98) and BT474-LapR (0.76-0.96) cells. Moreover, 2DG + lapatinib induced greater apoptosis, especially in lapatinib-resistant cells (P = 0.004 and 0.003 for SKBR3-LapR and BT474-LapR cells, respectively). Finally, 2DG + lapatinib inhibited several steps of the glycolytic pathway, namely: reduction of hexokinase II expression levels, inhibition of lactate production, decreased glucose uptake and impaired hexokinase activity, in both lapatinib-sensitive and resistant cells. Conclusion: 2DG can enhance lapatinib sensitivity by inhibiting aerobic glycolysis in both naïve and lapatinib-resistant HER2+ breast cancer cells. 2DG impinges on multiple steps of glycolysis, and combination with lapatinib may serve to prevent and/ or ameliorate therapeutic resistance in patients treated with lapatinib. Citation Format: Xiaosong Chen, Junjun Liu, Toby Ward, Xiaofei Liu, Yan Mao, Jessica Bockhorn, Kunwei Shen, Mark Pegram. Targeting glycolytic pathway with 2-Deoxy-glucose enhances lapatinib sensitivity in HER2-amplified breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 713. doi:10.1158/1538-7445.AM2015-713

Metabolic effects of hypoxia in colorectal cancer by 13C NMR isotopomer analysis.

13C NMR isotopomer analysis was used to characterize intermediary metabolism in three colorectal cancer cell lines (WiDr, LS1034, and C2BBe1) and determine the “metabolic remodeling” that occurs under hypoxia. Under normoxia, the three colorectal cancer cell lines present high rates of lactate production and can be seen as “Warburg” like cancer cells independently of substrate availability, since such profile was dominant at both high and low glucose media contents. The LS1034 was the less glycolytic of the three cell lines and was the most affected by the event of hypoxia, raising abruptly glucose consumption and lactate production. The other two colorectal cell lines, WiDr and C2BBe1, adapted better to hypoxia and were able to maintain their oxidative fluxes even at the very low levels of oxygen. These differential metabolic behaviors of the three colorectal cell lines show how important an adequate knowledge of the “metabolic remodeling” that follows a given cancer treatment is towards the correct (re)design of therapeutic strategies against cancer.

A Genetically Encoded FRET Lactate Sensor and Its Use To Detect the Warburg Effect in Single Cancer Cells

Lactate is shuttled between and inside cells, playing metabolic and signaling roles in healthy tissues. Lactate is also a harbinger of altered metabolism and participates in the pathogenesis of inflammation, hypoxia/ischemia, neurodegeneration and cancer. Many tumor cells show high rates of lactate production in the presence of oxygen, a phenomenon known as the Warburg effect, which has diagnostic and possibly therapeutic implications. In this article we introduce Laconic, a genetically-encoded Forster Resonance Energy Transfer (FRET)-based lactate sensor designed on the bacterial transcription factor LldR. Laconic quantified lactate from 1 µM to 10 mM and was not affected by glucose, pyruvate, acetate, betahydroxybutyrate, glutamate, citrate, α-ketoglutarate, succinate, malate or oxalacetate at concentrations found in mammalian cytosol. Expressed in astrocytes, HEK cells and T98G glioma cells, the sensor allowed dynamic estimation of lactate levels in single cells. Used in combination with a blocker of the monocarboxylate transporter MCT, the sensor was capable of discriminating whether a cell is a net lactate producer or a net lactate consumer. Application of the MCT-block protocol showed that the basal rate of lactate production is 3–5 fold higher in T98G glioma cells than in normal astrocytes. In contrast, the rate of lactate accumulation in response to mitochondrial inhibition with sodium azide was 10 times lower in glioma than in astrocytes, consistent with defective tumor metabolism. A ratio between the rate of lactate production and the rate of azide-induced lactate accumulation, which can be estimated reversibly and in single cells, was identified as a highly sensitive parameter of the Warburg effect, with values of 4.1 ± 0.5 for T98G glioma cells and 0.07 ± 0.007 for astrocytes. In summary, this article describes a genetically-encoded sensor for lactate and its use to measure lactate concentration, lactate flux, and the Warburg effect in single mammalian cells.

Engineering a Cyanobacterial Cell Factory for Production of Lactic Acid

Metabolic engineering of microorganisms has become a versatile tool to facilitate production of bulk chemicals, fuels, etc. Accordingly, CO(2) has been exploited via cyanobacterial metabolism as a sustainable carbon source of biofuel and bioplastic precursors. Here we extended these observations by showing that integration of an ldh gene from Bacillus subtilis (encoding an l-lactate dehydrogenase) into the genome of Synechocystis sp. strain PCC6803 leads to l-lactic acid production, a phenotype which is shown to be stable for prolonged batch culturing. Coexpression of a heterologous soluble transhydrogenase leads to an even higher lactate production rate and yield (lactic acid accumulating up to a several-millimolar concentration in the extracellular medium) than those for the single ldh mutant. The expression of a transhydrogenase alone, however, appears to be harmful to the cells, and a mutant carrying such a gene is rapidly outcompeted by a revertant(s) with a wild-type growth phenotype. Furthermore, our results indicate that the introduction of a lactate dehydrogenase rescues this phenotype by preventing the reversion.

Evaluation of the Impact of Diabetes on Retinal Metabolites by NMR Spectroscopy

Purpose/Aim of the study: Diabetic retinopathy (DR) is a leading cause of blindness in working age adults in developed countries. Changes in metabolites and in metabolic pathways of the retina caused by hyperglycemia may compromise the physiology of the retina. Using nuclear magnetic resonance (NMR) spectroscopy, we aimed to investigate the effect of diabetes on the levels of intermediate metabolites in rat retinas and the metabolic pathways that could be affected.Materials and Methods: Diabetes was induced in male Wistar rats with a single injection of streptozotocin (65 mg/Kg, i.p.). Metabolic alterations were analyzed in streptozotocin-induced diabetic rat retinas by 1H NMR spectroscopy. Glucose uptake was measured with 2-deoxy-d-[1-3H]glucose. Lactate production was evaluated by 1H NMR spectroscopy using [U-13C]glucose.Results: Tissue levels of several metabolic intermediates were quantified, but no significant changes in the levels of most metabolites were detected, with the exceptions of glucose, significantly increased, and lactate, significantly reduced in diabetic rat retinas, as compared to age-matched controls. The cytosolic redox ratio, indirectly evaluated by lactate-to-pyruvate ratio, was significantly reduced in diabetic rat retinas, as well as glucose uptake. Parallel studies demonstrated that lactate production rates were significantly diminished, suggesting a reduction in the glycolytic flux.Conclusions: These results suggest that diabetes may significantly decrease glycolysis in the retina since higher intracellular glucose levels do not translate into higher intracellular lactate levels or into higher rates of lactate production. These changes may alter the normal functioning of the retina during diabetes and may contribute for vision loss in DR.

VALIDATION OF THEIN VITROINCUBATION OF EXTENSOR DIGITORUM LONGUS MUSCLE FROM MICE WITH A MATHEMATICAL MODEL

In vitro incubation of tissues; in particular, skeletal muscles from rodents, is a widely-used experimental method in diabetes research. This experimental method has previously been validated, both experimentally and theoretically. However, much of the method's experimental data remains unclear, including the high-rate of lactate production and the lack of an observable increase in glycogen content, within a given time. The predominant hypothesis explaining the high-rate of lactate production is that this phenomenon is dependent on a mechanism in glycolysis that works as a safety valve, producing lactate when glucose uptake is super-physiological. Another hypothesis is that existing anoxia forces more ATP to be produced from glycolysis, leading to an increased lactate concentration. The lack of an observable increase in glycogen content is assumed to be dependent on limitations in sensitivity of the measuring method used. We derived a mathematical model to investigate which of these hypotheses is most likely to be correct. Using our model, data analysis indicates that the in vitro incubated muscle specimens, most likely are sensing the presence of existing anoxia, rather than an overflow in glycolysis. The anoxic milieu causes the high lactate production. The model also predicts an increased glycogenolysis. After mathematical analyses, an estimation of the glycogen concentration could be made with a reduced model. In conclusion, central anoxia is likely to cause spatial differences in glycogen concentrations throughout the entire muscle. Thus, data regarding total glycogen levels in the incubated muscle do not accurately represent the entire organ. The presented model allows for an estimation of total glycogen, despite spatial differences present in the muscle specimen.

CD147 silencing inhibits lactate transport and reduces malignant potential of pancreatic cancer cells in in-vivo and in-vitro models

Background: CD147 (basigin, EMMPRIN) is a multifunctional, highly conserved glycoprotein enriched in pancreatic ductal adenocarcinomas (PDACs) which is associated with poor prognosis in many malignancies. The role of CD147 in pancreatic cancer, however, remains elusive. Methods and Results: Silencing of CD147 by RNA interference (RNAi) reduced the proliferation rate of MiaPaCa2 and Panc1 cells. CD147 is required for the function and expression of the monocarboxylate transporters MCT1 and MCT4 that are expressed in human PDAC cells as demonstrated by real-time reverse transcription-PCR (RT-PCR) as well as immunohistology. MCT1 and MCT4 are the natural transporters of lactate, and MiaPaCa2 cells exhibited a high rate of lactate production, which is characteristic for the Warburg effect, an early hallmark of cancer that confers a significant growth advantage. Further induction of lactate production by sodium azide in MiaPaCa2 cells increased MCT1 as well as MCT4 expression. CD147 silencing inhibited the expression and function of MCT1 and MCT4 and resulted in an increased intracellular lactate concentration. Addition of exogenous lactate inhibited cancer cell growth in a dose-dependent fashion. In vivo, knock-down of CD147 in MiaPaCa2 cells by inducible short hairpin RNA (shRNA)-mediated CD147 silencing reduced invasiveness through the chorioallantoic membrane of chick embryos (CAM assay) and inhibited tumourigenicity in a xenograft model in nude mice. Conclusion : The function of CD147 as an ancillary protein that is required to sustain the expression and function of MCT1 and MCT4 is involved in the association of CD147 expression with the malignant potential of pancreatic cancer cells exhibiting the Warburg effect.

CD147 silencing inhibits lactate transport and reduces malignant potential of pancreatic cancer cells in in vivo and in vitro models

Background:CD147 (basigin, EMMPRIN) is a multifunctional, highly conserved glycoprotein enriched in pancreatic ductal adenocarcinomas (PDACs) which is associated with poor prognosis in many malignancies. The role of CD147 in pancreatic...

Improvement of the energy metabolism of recombinant CHO cells by cell sorting for reduced mitochondrial membrane potential

One of the major problems in process performance of mammalian cell cultures is the production of lactic acid. Cell specific glucose uptake rates usually correlate to glucose concentration and approximately 80% of the metabolised glucose is converted into lactic acid. As the mitochondrial membrane potential was shown to correlate to cell specific glucose uptake rates, we used Rhodamine 123, a lipophilic cationic dye for cell sorting to improve the energy metabolism of existing production cell lines. Two recombinant CHO cell lines with known differences in lactic acid production rate were used to evaluate Rhodamine 123 staining as a descriptor for glucose uptake rates and to determine whether it is possible to isolate subclones with altered metabolic properties. Such subclones would exhibit an improved process performance, and in addition could be used as models for genomic and metabolic studies. From the cell line with high lactate production, a subclone sorted for reduced mitochondrial membrane potential was found to have a lower specific lactate formation rate compared to the parental cell line in batch cultures. In addition, the glucose consumption rate was also reduced, while both the growth rate and the final cell concentration were increased. A subclone sorted for high mitochondrial membrane potential, on the other hand, had a higher glucose consumption rate, a higher lactate production rate and reduced growth. The potential of using flow cytometric cell sorting methods based on physiological activity for cell line optimisation is discussed.

Metabolic approaches to the treatment of ischemic heart disease: the clinicians' perspective.

This review article discusses pharmacological approaches to optimizing myocardial metabolism during ischemia. Fatty acids are the main fuel for the healthy heart, with a lesser contribution coming from the oxidation of glucose and lactate. Myocardial ischaemia dramatically alters fuel metabolism, causing an accelerated rate of glucose conversion to lactate and a switch from lactate uptake by the heart to lactate production. This causes a dramatic disruption in cell homeostasis (e.g. lactate accumulation and a decrease in pH and ATP). Paradoxically, moderately ischemic tissue (approximately 50% of normal flow) continues to derive most of its energy (50-70%) from the oxidation of fatty acids despite a high rate of lactate production. This ischaemia-induced disruption in cardiac metabolism can be minimized by metabolic agents that reduce fatty acid oxidation and increase the combustion of glucose and lactate, resulting in clinical benefit to the ischemic patient. Agents that inhibit fatty acid beta-oxidation, such as ranolazine and trimetazidine, have proven to be effective in the treatment of stable angina. Treatment of acute myocardial infarction patients with an infusion of the glucose-insulin-potassium, which results in suppression of myocardial fatty acid oxidation and greater glucose combustion, has proven effective in reducing mortality. These metabolic therapies are free of direct hemodynamic or chronotropic effects, and thus are well positioned for use alongside traditional agents such as beta-adrenergic receptor antagonists or calcium channel antagonists.

Changes in cardiac metabolism: a critical step from stable angina to ischaemic cardiomyopathy

Cardiac work requires a high rate of adenosine triphosphate (ATP) breakdown. ATP is resynthesized in the mitochondria with energy from the combustion of fatty acids, glucose and lactate. Fatty acids are the main fuel for the heart, supplying 60–90% of the energy, with the balance (10–40%) from pyruvate oxidation (formed from glycolysis and lactate). Fatty acid oxidation inhibits pyruvate oxidation in the mitochondria. During myocardial ischaemia, oxygen consumption and ATP production is reduced, causing accelerated glycolysis and lactate production; the pH falls and cell function is impaired. Paradoxically, with a partial reduction in coronary flow, the myocardium continues to derive most of its energy from the oxidation of fatty acids despite a high rate of lactate production; this fatty acid oxidation during ischaemia inhibits pyruvate oxidation, and drives pyruvate conversion to lactate. Partial inhibition of fatty acid oxidation in ischaemic myocardium, such as with the long-chain 3-ketoacyl thiolase inhibitor trimetazidine, reduces lactate production and H+ accumulation during ischaemia, and results in clinical benefit in patients with angina pectoris.

Estimation of the intracellular fluxes for a hybridoma cell line by material balances

In general, quantitative analysis of intracellular metabolic fluxes can be a useful tool for the investigation and understanding of mammalian cell physiology and its changes in response to environmental variations. To estimate these fluxes, a stoichiometric matrix taking into account the most relevant pathways involved in energy and mass transformations in a hybridoma cell line was constructed. This matrix, combined with the specific rates of consumption or production for the extracellular metabolites, was used to estimate the intracellular fluxes by means of a least squares procedure. To corroborate the estimated fluxes, experiments with 13C-labelled glucose were performed, and the 12C/ 13C ratio in the excreted lactate was determined experimentally by nuclear magnetic resonance (NMR). This ratio was compared to the predicted value using the calculated fluxes and showed good agreement. The interpretation of the results obtained allows two trends to be established in the metabolism of the cell line investigated. Firstly, the high rate of lactate production in the hybridoma cell line studied may be due to the lack of activity of the malate-aspartate shuttle, meaning that the only way to oxidize quickly the NADH produced in the glycolysis was the synthesis of lactate. Secondly, the high consumption of glutamine unbalances the tricarboxylic acid cycle and the cell has to export some of the carbon excess in the TCA using the malate shunt.

Relationship between intracellular phosphate, proton motive force, and rate of nongrowth energy dissipation (energy spilling) in Streptococcus bovis JB1.

When the rate of glucose addition to nongrowing Streptococcus bovis cell suspensions was increased, the fermentation was homolactic, fructose-1,6-diphosphate (FDP) increased, intracellular inorganic phosphate (P(i)) declined, and the energy-spilling rate increased. ATP and ADP were not significantly affected by glucose consumption rate, but the decrease in P(i) was sufficient to cause an increase in the free energy of ATP hydrolysis (delta G'p). The increase in delta G'p was correlated with an increase in proton motive force (delta p). S. bovis continuous cultures (dilution rate of 0.65 h-1) that were provided with ammonia as the sole nitrogen source also had high rates of lactate production and energy spilling. When Trypticase was added as a source of amino acids, lactate production decreased; a greater fraction of the glucose was converted to acetate, formate, and ethanol; and the energy-spilling rate decreased. Trypticase also caused a decrease in FDP, an increase in P(i), and a decrease in delta p. The change in delta p could be explained by P(i)-dependent changes in the delta G'p. When P(i) declined, delta G'p and delta p increased. The ratio of delta G'p to delta p (millivolt per millivolt) was always high (> 4) at low rates of energy spilling but declined when the energy-spilling rate increased. Based on these results, it appears that delta p and the energy-spilling rate are responsive to fluctuations in the intracellular P(i) concentration.

Cultivation of the hybridoma cell line MN12 in a homogeneous continuous culture system: effect of culture age.

The stability of the hybridoma cell line MN12 in a long-term homogeneous continuous culture was studied using a panel of analytical methods. These include two flow cytometry methods, for the determination of relative cytoplasmic and membrane IgG content. In addition, the antibody production was determined by an ELISA, and the metabolic state of the cells was determined by means of glucose consumption and lactate production. These results indicate a possible selection of variants of MN12 hybridoma cells with an overall aerobic metabolism, but with a higher glucose consumption rate and a higher lactate production rate. These variants are mainly characterized by a different membrane IgG content and cytoplasmic antibody content. These changes may possibly be affected by the culture age.

The impairment of respiration by glycolysis in the Lewis lung carcinoma

Isolated vegetative tumour cells from mice bearing the Lewis lung carcinoma showed low rates of basal respiration with both low oxygen uptake rates and cytochrome-c oxidase activity. The cells were affected by a marked Crabtree effect and a high rate of lactate production in the presence of 10 mM glucose. The glycolytic capacity of the tumour was also assessed through the measurement of the maximum activities for hexokinase, phosphofructokinase, pyruvate kinase and lactate dehydrogenase. These activities were similar to the ones found in other fast-growing, undifferentiated tumours. The concentration of fructose-2,6-bisphosphate in the tumour was 2,3 nmoles/g fresh tissue wt., a value which is of the same order of magnitude as that found in other types of highly glycolytic cells. It is concluded that the Lewis lung carcinoma follows the same pattern as other undifferentiated tumours with a high capacity for both glucose and amino acid utilization.

A comparison of oxygen debt in small and large rainbow trout, Salmo gairdneri Richardson

The potential for anaerobic metabolism in fish white muscle appears to increase with body size. Larger fish have higher white muscle glycolytic potential (Somero & Childress, 1980) and display both higher rates of lactate production and higher maximum concentrations of lactate following exhaustive exercise (Goolish, in press). The post-exercise elevation in oxygen consumption by fish (' oxygen debt ') is considered to be the result of processes involved in the recovery from anaerobic metabolism (Gaesser & Brooks, 1984; Milligan & McDonald, 1988) and therefore may also be influenced by body size. The present study compares the oxygen debt of small and large rainbow trout following exhaustive exercise. The respirometer consisted of flow-through tubular chambers (75 and 4200 ml) immersed in a constant water bath (15 C). Oxygen consumption rates were calculated from the flow rate and the change in dissolved oxygen as measured with a Camlab $8543 oxygen meter (Cambridge, U.K.). Mean sizes (*s.E.) of the large and small fish were 32.7cmf3.1 (n = 4) and 7.8 cm 0.6 (n = 3), respectively. Individual post-absorptive fish were placed in the respirometer and monitored until stable control levels of oxygen consumption were observed. The fish were then removed, exercised for 6 min, and returned to the respirometer. Maximal burst activity was induced using a net in tanks of approximately the same relative size (3 by 4 body lengths). Rates of oxygen consumption were determined after 1.2,3,4,5,6 and 8 h, and expressed as a percentage of the mean pre-exercise level. The relative increase in oxygen consumption following exhaustive swimming was higher for the larger fish at each sampling time (Fig. I). The duration of the response was also longer for the large fish (8 h) than for the small fish (5 h). The relative increase in oxygen consumption was significantly higher for the larger fish when the data were combined for the duration of the response (P < 0.02), but individual sampling times were not significantly different. The higher relative increase for large fish was partly due to their lower standard metabolic rate; 58.6 and 96.1 mg 0, kg-' h-' for large and small fish, respectively. The area under each curve was used to calculate the total amount of oxygen consumed above pre-exercise levels. Averaged over the duration of the response, the absolute increases in respiration rates following exercise were similar for the large and small fish; 40.4 and 49.5 mg O2 kg- h-', respectively. Post-exercise oxygen consumption expressed per gramme of white muscle was also similar for large (0.69 mg 0, g-') and small fish (0.65 mg Having white muscle lactate production (Goolish, in press) and post-exercise oxygen consumption data from the same group of fish also provides an opportunity to examine the relationship between these variables and the cost of glycogen restoration. The oxygen debt data from the group of large fish (33 cm) are used here because of their lower variability. Lactate production for fish of the length, subtracting the resting level, is 34.7 pmol g-l (Goolish, in press). This is equivalent to energy production of 1.94 J g-l of white muscle tissue, assuming a 2 : 1 ratio between lactate and each glucosyl unit (Stevens & Black, 1966; Wardle, 1978) and that 558 J are obtained per gramme of glycogen (Wardle, 1975). The

Glycolytic and oxidative metabolism in relation to retinal function.

Measurements of lactate production and ATP concentration in superfused rat retinas were compared with extracellular photoreceptor potentials (Fast PIII). The effect of glucose concentration, oxygen tension, metabolic inhibition, and light were studied. Optimal conditions were achieved with 5-20 mM glucose and oxygen. The isolated retina had a high rate of lactate production and maintained the ATP content of a freshly excised retina, and Fast PIII potentials were similar to in vivo recordings. Small (less than 10%) decreases in aerobic and anaerobic lactate production were observed after illumination of dark-adapted retinas. There were no significant differences in ATP content in dark- and light-adapted retinas. In glucose-free medium, lactate production ceased, and the amplitude of Fast PIII and the level of ATP declined, but the rates of decline were slower in oxygen than in nitrogen. ATP levels were reduced and the amplitude of Fast PIII decreased when respiration was inhibited, and these changes were dependent on glucose concentration. Neither glycolysis alone nor Krebs cycle activity alone maintained the superfused rat retina at an optimal level. Retinal lactate production and utilization of ATP were inhibited by ouabain. Mannose but not galactose or fructose produced lactate and maintained ATP content and Fast PIII. Iodoacetate blocked lactate production and Fast PIII and depleted the retina of ATP. Pyruvate, lactate, and glutamine maintained ATP content and Fast PIII reasonably well (greater than 50%) in the absence of glucose, even in the presence of iodoacetate. addition of glucose, mannose, or 2-deoxyglucose to medium containing pyruvate and iodoacetate abolished Fast PIII and depleted the retina of its ATP. It is suggested that the deleterious effects of these three sugars depend upon their cellular uptake and phosphorylation during the blockade of glycolysis by iodoacetate.