Effect of cholera toxin on glutamine metabolism and transport in rabbit ileum

The aim of the present study was to evaluate the effect of cholera toxin on energy balance from intestinal glutamine metabolism and oxidation, glutamine-dependent sodium absorption, and cholera toxin-dependent ion flux. Cholera toxin-stimulated sodium and L-glutamine ileal transport and metabolism were studied in Ussing chambers. Glutamine (10 mM) transport and metabolism were simultaneously studied using (14)C flux and HPLC. In the same tissues, the flux of each amino acid was studied by HPLC, and glutamine metabolism and oxidation were studied by the determination of amino acid specific activity and (14)CO(2) production. In control tissues, glutamine stimulated sodium absorption and was mainly oxidized. The transepithelial flux of intact glutamine represented 45% of glutamine flux across the luminal membrane. The other metabolites were glutamate and, to a lesser degree, citrulline, ornithine, and proline. Cholera toxin did not alter glutamine-stimulated sodium absorption, glutamine oxidation, transport, and metabolism. In conclusion, the present results indicate that cholera toxin does not alter glutamine intestinal function and metabolism. In addition, approximately 95% of the energy provided by glutamine oxidation remains available to the enterocyte.     

Low oxygen reduces the modulation to an oxidative phenotype in monolayer‐expanded chondrocytes

Autologous chondrocyte implantation requires a phase of in vitro cell expansion, achieved by monolayer culture under atmospheric oxygen levels. Chondrocytes reside under low oxygen conditions in situ and exhibit a glycolytic metabolism. However, oxidative phosphorylation rises progressively during culture, with concomitant reactive oxygen species production. We determine if the high oxygen environment in vitro provides the transformation stimulus. Articular chondrocytes were cultured in monolayer for up to 14 days under 2%, 5%, or 20% oxygen. Expansion under 2% and 5% oxygen reduced the rate at which the cells developed an oxidative phenotype compared to 20% oxygen. However, at 40 ± 4 fmol cell⁻¹ h⁻¹ the oxygen consumption by chondrocytes expanded under 2% oxygen for 14 days was still 14 times the value observed for freshly isolated cells. Seventy‐five to 78% of the increased oxygen consumption was accounted for by oxidative phosphorylation (oligomycin sensitive). Expansion under low oxygen also reduced cellular proliferation and 8‐hydroxyguanosine release, a marker of oxidative DNA damage. However, these parameters remained elevated compared to freshly isolated cells. Thus, expansion under physiological oxygen levels reduces, but does not abolish, the induction of an oxidative energy metabolism. We conclude that simply transferring chondrocytes to low oxygen is not sufficient to either maintain or re‐establish a normal energy metabolism. Furthermore, a hydrophobic polystyrene culture surface which promotes rounded cell morphology had no effect on the development of an oxidative metabolism. Although the shift towards an oxidative energy metabolism is often accompanied by morphological changes, this study does not support the hypothesis that it is driven by them. J. Cell. Physiol. 222:248–253, 2010. © 2009 Wiley‐Liss, Inc.     

Respiration and carbohydrate energy metabolism of the lung-dwelling parasite Rhabdias bufonis (Nematoda: Rhabdiasoidea)

An investigation of the carbohydrate energy metabolism of Rhabdias bufonis, the lung-dwelling nematode parasite of the African toad, Bufo regularis, indicates that the nematode stores very little glycogen (0.137 +/- 0.003% on a fresh weight basis) but does utilize oxygen in vitro. The intracellular distribution and high levels of activity observed for the enzymes phosphoenolpyruvate carboxykinase, pyruvate kinase, lactate dehydrogenase, malate dehydrogenase, malic enzyme and fumarate reductase suggest two alternative pathways of carbohydrate energy metabolism.     

The obligate intracellular bacterium Chlamydia trachomatis is auxotrophic for three of the four ribonucleoside triphosphates

Using we11-characterized mutant host cell lines, deficient in specific enzymes of energy and nucleotide metabolism, we addressed numerous questions regarding nucleotide metabolism in the obligate intracellular bacterium Chlamydia trachomatis. The results presented indicate that C. trachomatis: (i) does not absolutely depend on mitochondrial generated ATP for survival; (ii) does have a significant draw on host-cell NTP pools but does not have a detrimental effect on the ability of the host cell to maintain its energy charge; (iii) lacks the ability to synthesize purine and pyrimidine nucleotides de novo; (iv) is not capable of interconverting purine nucleotides; and (v) possesses the pyrimidine metabolic-pathway enzymes CTP synthetase and deoxycytidine nucleotide deaminase. In total our results indicate that C. trachomatis is auxotrophic for host-cell ATP, GTP and UTP. In contrast, CTP can be obtained from the host cell or it can be synthesized from UTP by the parasite.     

Pyruvate and lactate metabolism by Shewanella oneidensis MR-1 under fermentation, oxygen limitation, and fumarate respiration conditions

Shewanella oneidensis MR-1 is a facultative anaerobe that derives energy by coupling organic matter oxidation to the reduction of a wide range of electron acceptors. Here, we quantitatively assessed the lactate and pyruvate metabolism of MR-1 under three distinct conditions: electron acceptor-limited growth on lactate with O(2), lactate with fumarate, and pyruvate fermentation. The latter does not support growth but provides energy for cell survival. Using physiological and genetic approaches combined with flux balance analysis, we showed that the proportion of ATP produced by substrate-level phosphorylation varied from 33% to 72.5% of that needed for growth depending on the electron acceptor nature and availability. While being indispensable for growth, the respiration of fumarate does not contribute significantly to ATP generation and likely serves to remove formate, a product of pyruvate formate-lyase-catalyzed pyruvate disproportionation. Under both tested respiratory conditions, S. oneidensis MR-1 carried out incomplete substrate oxidation, whereby the tricarboxylic acid (TCA) cycle did not contribute significantly. Pyruvate dehydrogenase was not involved in lactate metabolism under conditions of O(2) limitation but was required for anaerobic growth, likely by supplying reducing equivalents for biosynthesis. The results suggest that pyruvate fermentation by S. oneidensis MR-1 cells represents a combination of substrate-level phosphorylation and respiration, where pyruvate serves as an electron donor and an electron acceptor. Pyruvate reduction to lactate at the expense of formate oxidation is catalyzed by a recently described new type of oxidative NAD(P)H-independent d-lactate dehydrogenase (Dld-II). The results further indicate that pyruvate reduction coupled to formate oxidation may be accompanied by the generation of proton motive force.     

Energetic cost of a meal in a frequent feeding lizard

Specific dynamic action (SDA) describes the rise in metabolism following feeding in animals and represents the energetic cost of digesting and assimilating a meal. The overall energetic cost of feeding may depend on whether or not an animal is post-absorptive at the time of feeding. The aim of this study was to compare the energetic cost of SDA due to feeding frequently compared with infrequently in the eastern water skink, Eulamprus quoyii. For similar quantities of food, repeated feeding incurred an energetic cost equal to 8.8% of the metabolizable energy of the meal (25,220 J), while single feeding incurred an energetic cost of 9.4% of the metabolizable energy of the meal (26,072 J). Experimental lizards maintained a rise in (VO2) that was on average 1.8 times greater than the (VO2) of the unfed controls over a 50-h interval as a result of feeding frequently. This prolonged rise in metabolism resulting from frequent feeding does not result in a higher energetic cost of SDA compared with that resulting from infrequent single feeding.     

Integrated Assessment of Large-Scale Biofuel Production

This paper proposes a different framework for discussing the possibility of replacing a significant fraction of fossil energy consumption of modern economies with biofuels. The proposed analysis is not based on the two classic feasibility checks—land availability and output/input energy ratio—debated in the majority of the literature in this field. Rather, the focus is on the desirability of an energy sector powered by biomass energy. Discussing of desirability requires introducing a multicriteria approach, that in turn requires a definition of a set of criteria of performance for such an energy sector. The concepts of societal metabolism and ecosystem metabolism are introduced to define five criteria of performance for an energy sector powered by alternative sources.

This paper does not tell the society whether or not biofuels should be used to replace fossil energy. Rather, it proposes a method of characterization of pros and cons for the option biofuel which can be used to make more informed choices. An analysis of three systems of production—corn-ethanol, sunflower-biodiesel, and SRWC-methanol—is provided to indicate the existence of systemic characteristics associated with an energy sector powered by biofuels. These characteristics are likely to persist even when different technical coefficients will be achieved. The conclusion is that, at the moment, it is not possible to replace the actual performance of an energy sector based on fossil energy with an energy sector running on biofuel. Biomass energy can and will have to play an important role in the sustainability of humankind, but this will require a better understanding of (i) the role that an energy sector plays within a given structure of societal metabolism; and (ii) the impact generated on ecosystem metabolism by societal metabolism, plus a lot of wisdom.     

Freezing and cellular metabolism in the gall fly larva,Eurosta solidaginis

Summary The effects of extracellular freezing on intracellular metabolism were monitored over both a short (9 h) and long (12 weeks) time course using the freeze tolerant larvae of the gall fly,Eurosta solidaginis.The process of freezing, monitored over the short time course, had no effect upon cellular energy levels (adenylates, arginine phosphate) but initiated a rise in glucose-6-P and lactate levels. This suggests that freezing initiates a shift towards glycolysis as the predominant mode of energy production. The process of thawing at 3°C (after 24 h at –16°C) also had no effect, even transient, on cellular energy levels demonstrating that thawing and the rapid redistribution of water and solutes which must accompany it does not disrupt cellular metabolism. During thawing accumulated lactate was quickly cleared with a t 1/2 of 20–30 min.Long term freezing at –16°C had dramatic effects on energy metabolism. Freezing for up to 1 week had minimal effects with only a small drop in arginine phosphate reserves and an increase in lactate content noted. Between 1 and 2 weeks of freezing, however, larvae showed strong signs of energy stress. The arginine phosphate pool fell from 75% to 30% of control levels, ATP content dropped by 50% and energy charge dropped to 0.75. This state, with continued lactate accumulation, was maintained through 4 weeks of freezing. Between 6 and 12 weeks of freezing energy stress became even greater. Phosphagen and ATP contents dropped to 5 and 25% of control values and energy charge decreased to about 0.50. Despite this stress, however, 94% of larvae survived 12 weeks of freezing with an 86% hatch rate of adults. The data demonstrate that the larvae can survive prolonged periods of winter freezing drawing upon glycolysis and phosphagen reserves to supply the continued basal energy demands of the cell.     

Pyruvate dehydrogenase activity and energy metabolite levels following bilateral common carotid artery occlusion in rat brain

The influence of chronic cerebral hypoperfusion on cerebral energy metabolism was studied. The bilateral common carotid arteries of Wistar rats were occluded for 0, 2, 7, and 28 days. Cerebral energy metabolism was evaluated by assaying adenosine triphosphate (ATP), phosphocreatine (PCr), and lactate levels and measuring pyruvate dehydrogenase (PDH) activity (each time point, n = 6). Pathological changes were assessed light-microscopically by Klüver-Barrera staining and immunohistochemical labeling for astroglia (each time point, n = 3). There were no changes in ATP and PCr levels or PDH activity; there was slight but significant transient lactate accumulation at 2 days. Myelin pallor and increase in immuno-reactive astroglia were only observed at 28 days. These results indicate that chronic cerebral hypoperfusion induces delayed white matter changes in the corpus callosum of rat brain, but does not affect energy production.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin increases reliance on fats as a fuel source independently of diet: evidence that diminished carbohydrate supply contributes to dioxin lethality

The environmental toxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes a wasting syndrome marked by hypophagia, loss of body fat, changes in intermediary metabolism and death. Use of conventional laboratory animals has not resolved whether or not TCDD affects intermediary metabolism independently of hypophagia. We used the chick embryo, which does not require an exogenous food supply for energy, to answer this question. Our results show that TCDD treatment increases dependence on fats as a fuel source independently of changes in food intake and therefore can affect intermediary metabolism independently of hypophagia. Results of experiments using aminocarnitine to inhibit fatty acid oxidation suggest that TCDD treatment impairs carbohydrate production rather than its utilization and that the former effect contributes to TCDD lethality.     

Direct and indirect calorimetry measurements during the annual growth cycle of juvenile frogs Rana ridibunda P

A thermodynamic study of the energetic metabolism in juvenile amphibians was conducted over an annual cycle of growth. The total energy flux (delta H = enthalpy variation) was measured with direct microcalorimetry and accounted for the sum of all metabolic pathways. Indirect calorimetry, oxygen consumption measurements (VO2), provides the values for the oxidative metabolism-dependent energy. A comparison between the results of both techniques simultaneously performed, provides for the calculation of the energetic efficiency, which indicates the respective importance of aerobic and anaerobic metabolisms during the annual biological cycle. It is clear that oxidative metabolism does not represent all, nor even a constant percentage of the total energy production of the frog, Rana ridibunda. The ration (Formula: see text) varied between 0.72 and 1.00, according to the season and the feeding or fasting periods. These results are discussed with special reference to the ecophysiology and behaviour of this species.     

Oocyte metabolism predicts the development of cat embryos to blastocyst in vitro

Current methods for detecting complete oocyte maturation and developmental competence are inadequate. The objectives of this study were to (1) examine the relationship between cat oocyte energy metabolism and development in vitro after fertilization and (2) determine if cumulus cell metabolism could be used to predict development of individual oocytes after fertilization in vitro. The hanging drop method was used to assess metabolism of three different types of cat oocytes: immature (IMO), in vitro matured (IVM), and in vivo matured (IVOM). Stage of oocyte nuclear maturation or developmental competence was assessed after metabolic analysis. Glycolysis and oxidation of glucose, glutamine, palmitate, and lactate increased with the resumption of oocyte meiotic maturation (P<0.05). Pyruvate was the preferred substrate, but uptake was not linked to maturation. IVM oocytes had impaired glucose and palmitate metabolism compared to IVOM oocytes (P<0.05). Oocyte glycolytic activity and oocyte glucose oxidation correlated well with embryo development after insemination in vitro (P<0.05). Furthermore, oocytes that had similar glucose metabolism and that were grouped together for culture on this basis had higher (P<0.05) overall rates of development than oocytes grouped randomly. There was no correlation (P>0.05) between cumulus cell metabolism and individual oocyte development after in vitro fertilization. The data reveal that energy metabolism is linked to oocyte maturation in the cat and that glucose metabolic activity can indicate those oocytes most likely to fertilize and develop in vitro. Measuring cumulus cell metabolism does not accurately predict individual oocyte development after insemination in vitro.     

Genetic demonstration that the mutationally expressed nucleobase transporter of mouse S49 cells is nonconcentrative

Somatic cell genetic analysis of purine base transporters in mouse S49 cells has demonstrated the existence of a unique high-affinity purine base transporter, which is mutationally expressed and is not found in wild-type S49 cells or any other cells of the animal kingdom (B. Aronow, et al. (1986) Mol. Cell. Biol. 6, 2957). In order to determine whether this nucleobase transport system is active and concentrative, a secondary mutation in hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) was inserted into the cell line expressing this novel base transporter. The HGPRTase-deficient cells were capable of transporting hypoxanthine at increased rates but did not accumulate the base to concentrations in excess of that in the culture medium. Moreover, neither sodium azide nor ouabain had significant effects on hypoxanthine transport rates, indicating that energy metabolism and the maintenance of a sodium gradient were not required for transport function. These studies suggest that the novel mutationally expressed base transporter is independent of subsequent metabolism and does not require energy or a functioning Na+-K+-dependent ATPase activity.     

Effect of aeration intensity on the biochemical composition of baker's yeast. I. Factors affecting the type of metabolism

Efforts were made to eliminate the influence of other factors as far as possible in order to obtain reliable results on the effects of oxygen on the growth of baker's yeast. A cultivation method is presented which permits the study of the effects of aeration intensity under conditions where the influence of catabolite repression is eliminated. A completely synthetic medium with glucose as the only carbon and energy source is also described. The capacity of yeast to perform aerobic metabolism varies when cultivated under different intensities of aeration. A clear maximum is observed for growth with 10% oxygen in the aerating gas mixture. Under conditions where catabolite repression does not function yeast has the potential for oxidative metabolism even under oxygen-limited growth. The main agent controlling the ability of yeast to support growth using only the oxidative metabolism is the available oxygen. At high oxygen tensions the metabolism is disturbed.     

Stress‐induced peak (but not resting) metabolism correlates with mating display intensity in male guppies

Recent empirical and conceptual papers have highlighted the potential for metabolism to act as a proximate mechanism for behavior that could explain animal personality (consistency over time). Under this hypothesis, individuals with consistently high levels of behavioral activity should also have high resting metabolic rate (RMR) as it can reflect capacity to process food and generate energy. We tested for the predicted positive covariance between RMR and three behaviors that differ in energy demands in 30 male guppies, using multivariate mixed models; we repeatedly measured their activity (10 times each), courtship displays (nine times), voracity (10 times), and metabolism (four‐times). Resting metabolic rate (measured overnight in respirometry trials) did not consistently differ among males, whereas initial peak metabolism measured during those same trials (R = 0.42), and all behaviors were repeatable (R = 0.33–0.51). RMR declined over time suggesting habituation to the protocol, whereas peak metabolism did not. Initial peak metabolism was negatively correlated with courtship display intensity, and voracity was positively correlated with activity, but all other among‐individual correlations were not significant. We conclude that RMR does not provide a proximate explanation for consistent individual differences in behavior in male guppies, and therefore the potential for independent evolution of these physiological and behavioral traits seems possible. Finally, we identify peak metabolism as a potential measure of the stress response to confinement, which highlights the value of considering various aspects of metabolic rates recording during respirometry trials.     

Central energy metabolism remains robust in acute steatotic hepatocytes challenged by a high free fatty acid load

Overnutrition is one of the major causes of non-alcoholic fatty liver disease (NAFLD). NAFLD is characterized by an accumulation of lipids (triglycerides) in hepatocytes and is often accompanied by high plasma levels of free fatty acids (FFA). In this study, we compared the energy metabolism in acute steatotic and non-steatotic primary mouse hepatocytes. Acute steatosis was induced by pre-incubation with high concentrations of oleate and palmitate. Labeling experiments were conducted using [U-(13)C(5),U-(15)N(2)] glutamine. Metabolite concentrations and mass isotopomer distributions of intracellular metabolites were measured and applied for metabolic flux estimation using transient 13C metabolic flux analysis. FFAs were efficiently taken up and almost completely incorporated into triglycerides (TAGs). In spite of high FFA uptake rates and the high synthesis rate of TAGs, central energy metabolism was not significantly changed in acute steatotic cells. Fatty acid β-oxidation does not significantly contribute to the detoxification of FFAs under the applied conditions.     

Energy metabolism of the rat renal cortex, as measured by incubation in various substrates

Different substrate mixtures were investigated for their effect on energy metabolism using sections of the rat renal cortex. Simultaneous determination of adenine nucleotide concentrations and determination of the damage quotient of oxidative phosphorylation proved to be appropriate parameters for selecting substrate mixtures that have a favorable effect on energy metabolism. A mixture of albumin (1 mM) and octanoate (5.6 mM) with electrolytes proved to be adequate. The extent of oxygen consumption (60%) and 14CO2 formation (75%) argues in favor of the metabolization of this mixture; a damage quotient of 23% and general constancy of the concentration of high-energy compounds render prospective their testing in animal experiments. Addition of dicarbosylic acids increase the antimycin A resistant oxygen consumption without any energy conservation being demonstrable. Therefore, these substrates should not be used for conservation.     

Effects of NH4+ and K+ on the energy metabolism in Sp2/0-Ag14 myeloma cells

Potassium ions decrease the transport rate of ammonium ions into myeloma and hybridoma cells, one effect of the involved transport processes being an increased energy demand (Martinelle and Häggström, 1993; Martinelle et al., 1998b). Therefore, the effects of K+ and NH4+ on the energy metabolism of the murine myeloma cell line, Sp2/0-Ag14, were investigated. Addition of NH4Cl (10 mM) increased the metabolism via the alanine transaminase (alaTA) pathway, without increasing the consumption of glutamine. As judged by the alanine production, the energy formation from glutamine increased by 155%. The presence of elevated concentrations of KCl (10 mM) was positive, resulting in a decreased uptake of glutamine (45%), and an even larger suppression of ammonium ion formation (70%), while the same throughput via the alaTA pathway (and energy production from glutamine) was retained as in the control culture. However, the simultaneous presence of 10 mM K+ and 10 mM NH4+ was more inhibitory than NH4Cl alone; an effect that could not be ascribed to increased osmolarity. Although the culture with both K+ and NH4+ consumed 60% more glutamine than the culture with NH4+ alone, the energy generation from glutamine could not be increased further, due to the suppression of the glutamate dehydrogenase pathway. Furthermore, the data highlighted the importance of evaluating the metabolism via different energy yielding pathways, rather than solely considering the glutamine consumption for estimating energy formation from glutamine.     

Respiratory Studies on the Giant Amoeba Pelomyxa palustris1

ABSTRACT. The large fresh-water microaerobic amoeba Pelomyxa palustris does not contain mitochondria, but three types of bacterial endosymbionts are always present. Thus, it is of interest in the discussion of the possible origin of mitochondria from primitive prokaryotes. Gas exchanges (O2 , CO2 ) and concentration of endosymbionts were determined in individual amoebae, in which the life cycle stage was noted. Grey type (stationary phase) amoebae had a lower O2 uptake and lower endosymbiont concentration than light type (growth phase) amoebae, and highest O2 uptake was found in centrifugal pieces of light type Pelomyxa, centrifuged in vivo, which contained nearly all of the endosymbionts. In light type amoebae, the respiratory activity was independent of O2 concentration between 1 and 21%, and, when compared on the basis of dry weight and protein, of the same order as that of other free-living protozoa. The R.Q. was slightly higher than 1, indicating that glycolysis does not play a significant role in energy metabolism. It is concluded that P. palustris is fully aerobic, and suggestions are presented as to the role of the endosymbionts in its respiratory metabolism.