Effect of Si-status on diel variation of intracellular free amino acids in Thalassiosira weissflogii under low-light intensity

Twenty-one intracellular free amino acids were analysed during a 12-12 h light-dark cycle, on duplicate axenic cultures of Thalassiosira weissflogii (clone Actin, Provasoli-Guillard CCMP) under either Si-sufficient or Si-starved conditions. Total concentrations ranged between 40 and 165 fmol/cell. Total level as well as individual levels of amino acids decreased during the dark period, and GLN/GLU ratio was lower during the dark period. All these results were correlated with the light-dark carbon metabolism of the algae and related to the protein synthesis at night. The Si-starved cultures showed a lower total level of FAA compare to the Si-sufficient cultures, especially in the light period. Silica status of the cells affected more the metabolites of the dark respiration than the photorespiratory metabolites SER and GLY. Si deprivation induced higher range of ALA and VAL, and a decrease of the TCA metabolites GLU & ASP. Additionally, the relative percentage of ASP increased under Si starvation, at the expense of GLU, and this shift was emphasized in the dark period.     

Carbohydrate metabolism in brain during convulsions and its modification by phenobarbitone

Abstract— Assays of citric acid cycle substrates and metabolites of the second stage of the glycolytic pathway have completed a series of studies of glucose metabolism in brains of mice rapidly frozen at intervals during electrically-induced, tonic-clonic convulsions. Citric acid cycle metabolism reached a new equilibrium at a significantly higher rate. However, oxidative metabolism did not keep up with the demand for energy supplies, as indicated by an increasing lactate level and an increasing lactate: pyruvate ratio. Administration of a sub-anaesthetic but anticonvulsant dose of phenobarbitone prior to convulsive electrical stirnulation was associated with as great an increase in anaerobic glycolysis as in mice given no drug prior to stimulation; but oxidative metabolism was not enhanced, as reflected by even greater lactate: pyruvate ratios in mice given phenobarbitone than in mice given no drug prior to convulsive stimulation.     

Fermentation of Xylose Causes Inefficient Metabolic State Due to Carbon/Energy Starvation and Reduced Glycolytic Flux in Recombinant Industrial Saccharomyces cerevisiae

In the present study, comprehensive, quantitative metabolome analysis was carried out on the recombinant glucose/xylose-cofermenting S. cerevisiae strain MA-R4 during fermentation with different carbon sources, including glucose, xylose, or glucose/xylose mixtures. Capillary electrophoresis time-of-flight mass spectrometry was used to determine the intracellular pools of metabolites from the central carbon pathways, energy metabolism pathways, and the levels of twenty amino acids. When xylose instead of glucose was metabolized by MA-R4, glycolytic metabolites including 3- phosphoglycerate, 2- phosphoglycerate, phosphoenolpyruvate, and pyruvate were dramatically reduced, while conversely, most pentose phosphate pathway metabolites such as sedoheptulose 7- phosphate and ribulose 5-phosphate were greatly increased. These results suggest that the low metabolic activity of glycolysis and the pool of pentose phosphate pathway intermediates are potential limiting factors in xylose utilization. It was further demonstrated that during xylose fermentation, about half of the twenty amino acids declined, and the adenylate/guanylate energy charge was impacted due to markedly decreased adenosine triphosphate/adenosine monophosphate and guanosine triphosphate/guanosine monophosphate ratios, implying that the fermentation of xylose leads to an inefficient metabolic state where the biosynthetic capabilities and energy balance are severely impaired. In addition, fermentation with xylose alone drastically increased the level of citrate in the tricarboxylic acid cycle and increased the aromatic amino acids tryptophan and tyrosine, strongly supporting the view that carbon starvation was induced. Interestingly, fermentation with xylose alone also increased the synthesis of the polyamine spermidine and its precursor S-adenosylmethionine. Thus, differences in carbon substrates, including glucose and xylose in the fermentation medium, strongly influenced the dynamic metabolism of MA-R4. These results provide a metabolic explanation for the low ethanol productivity on xylose compared to glucose.     

紫杉二烯人工酵母的代谢组学分析

为了更深入地从代谢角度研究萜类合成人工酵母的内在差异,以紫杉二烯人工酵母为例,利用代谢组学的方法对其发酵指数中期胞内代谢物的变化进行了测定。结果表明,与对照菌W303-1A相比,紫杉二烯的生产会对胞内糖酵解、三羧酸循环中间物及一些氨基酸的含量产生不同程度的影响,进而对其生长产生一定抑制作用。其中柠檬酸因紫杉二烯功能模块的引入下降明显,降幅达90%以上,因此可以作为后续功能酵母研究的标志性代谢物。紫杉二烯人工酵母细胞代谢组的研究可以为萜类化合物异源合成的优化提供更多的信息和帮助。     

Effects of 4-Aminopyridine on Extracellular Concentrations of Glutamate in Striatum of the Freely Moving Rat

4-aminopyridine (4-AP) is a voltage-sensitive K⁺-channel blocker extensively used in in vitro experiments as a depolarizing agent for the release of glutamate (GLU). This research investigated whether 4-AP could be used in in vivo experiments using microdyalisis. For that, the effects of 4-AP on the extracellular concentrations of glutamate (GLU), glutamine (GLN), taurine (TAU) and citrulline (CIT) in striatum of the freely moving rat were investigated. The effects of 4-AP were compared with those produced by perfusion with a high K⁺ (100 mM) medium. Intrastriatal perfusion with 4-AP (1, 5 and 10 mM) produced no effects on extracellular [GLU], [TAU] and [CIT], but decreased extracellular [GLN]. Perfusion with a high K⁺ (100 mM) medium increased extracellular [GLU] and [TAU], decreased extracellular [GLN], and had no effects on [CIT]. To test whether the lack of effects of 4-AP on extracellular [GLU] was due to GLU uptake mechanisms, 4-AP was perfused after a previous inhibition of GLU uptake with L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC). Under the effects of PDC (1 mM), 4-AP (1 mM) had no effects on extracellular [GLU], [TAU] and [CIT], but decreased extracellular [GLN]. These results show that 4-AP decreased extracellular [GLN] but failed to produce a significant release of GLU in striatum of the freely moving rat. Thus, 4-AP can not be used as a depolarizing agent for stimulating the release of GLU in in vivo studies using microdialysis.     

Monitoring hybridoma metabolism in continuous suspension culture at the intracellular level

A model mouse hybridoma cell line was grown in continuous culture experiments in a serum-free low-protein lipid-free medium. The steady-state responses of cell numbers, extra- and intracellular metabolite concentrations, substrate and (by) product consumption/production rates, and yield coefficients were investigated as a function of step changes in the glutamine concentration of the feed medium. In addition to the commonly performed analysis of metabolites in culture supernatants, we prepared perchloric acid extracts of cells and determined the amount and the composition of intracellular amino acids and organic acids. Significant differences were found with respect to intracellular metabolite pools for cells growing at nearly identical specific growth rates. To our knowledge this is the first time that data on the intracellular concentrations (pools) of amino acids and Krebs cycle intermediates are reported in the literature that were obtained under carefully defined culture conditions such as those attained in continuous culture experiments.     

Glycolysis Is Governed by Growth Regime and Simple Enzyme Regulation in Adherent MDCK Cells

Due to its vital importance in the supply of cellular pathways with energy and precursors, glycolysis has been studied for several decades regarding its capacity and regulation. For a systems-level understanding of the Madin-Darby canine kidney (MDCK) cell metabolism, we couple a segregated cell growth model published earlier with a structured model of glycolysis, which is based on relatively simple kinetics for enzymatic reactions of glycolysis, to explain the pathway dynamics under various cultivation conditions. The structured model takes into account in vitro enzyme activities, and links glycolysis with pentose phosphate pathway and glycogenesis. Using a single parameterization, metabolite pool dynamics during cell cultivation, glucose limitation and glucose pulse experiments can be consistently reproduced by considering the cultivation history of the cells. Growth phase-dependent glucose uptake together with cell-specific volume changes generate high intracellular metabolite pools and flux rates to satisfy the cellular demand during growth. Under glucose limitation, the coordinated control of glycolytic enzymes re-adjusts the glycolytic flux to prevent the depletion of glycolytic intermediates. Finally, the model''s predictive power supports the design of more efficient bioprocesses.     

INTRACELLULAR AND EXTRACELLULAR AMINO ACID POOLS OF THE MARINE DIATOM PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE) GROWN ON UNENRICHED SEAWATER IN HIGH-CELL-DENSITY DIALYSIS CULTURE1

The consumption of inorganic macronutrients (NO₃^?+ NO₂^?, NH₄⁺, and PO₄^?3) and the composition of intra- and extracellular dissolved free amino acid pools (IDFAA and EDFAA, respectively) were determined in continuous-reservoir batch dialysis cultures of the marine diatom Phaeodactylum tricornutum Bohlin maintained on unenriched natural seawater as a growth medium. Nutrient diffusion (Nd), which equals the nutrient uptake of the culture, increased with the cell density and the age of the culture. A concentration of 6.77 × 10⁷ cells · mL^?1 was obtained in stationary phase, which coincided with the NO₃^?+ NO₂^? diffusion limit (Nd_max) of the dialysis apparatus. The Nd_max for NH₄⁺ occurred much earlier, at the end of exponential growth, whereas Nd_max for PO₄^?3 was not attained during the growth cycle of the culture, even in early stationary phase. A significant depletion (77%) of the IDFAA pool during exponential phase was followed by a reestablishment–to approximately 60% of the initial level–of internal pools during linear and stationary growth phases. This recovery occurred during the illuminated portion of the photoperiod (12:12 h LD) and involved principally the amino acids GLN, GLU, β-GLU, and ASN. The recovery of GLN and ASN levels was particularly significant, because the intracellular concentrations of these amino acids were higher at the end of the growth cycle than before. The EDFAA pool was generally dominated by the amino acids SER and GLY+THR; however, during active growth, ORN and LYS often constituted an important fraction. The EDFAA concentration increased until linear growth phase was reached, during which a higher concentration of total free amino acids was attained in darkness than under illumination. The EDFAA component diminished afterward, and in stationary phase this fraction returned to concentrations equivalent to those observed at the beginning of the growth cycle. The variations in EDFAA concentrations were expressed by a pronounced decrease in the cellular excretion of amino acids with increasing cell density. These cellular responses of Phaeodactylum tricornutum in dense culture, specifically the regulation of amino acid excretion and intracellular pool size, may affect the N-conversion coefficient (Y_N). Consequently, by prolonging the linear phase of growth and reducing the concentration of autoinhibitory metabolites by diffusion, a markedly enhanced final cell density can be achieved in cultures grown on natural unenriched seawater.     

Synthesis of oxaloacetate in Bacillus subtilis mutants lacking the 2-ketoglutarate dehydrogenase enzymatic complex.

Bacillus subtilis mutants deficient in the 2-ketoglutarate dehydrogenase enzymatic complex required aspartate for growth at wild-type rates on carbon sources for which synthesis of the degradative enzymes is sensitive to catabolite repression (e.g., poor carbon sources), but did not require aspartate for growth on carbon sources which exert catabolite repression (e.g., good carbon sources). Measurement of metabolite pools in a mutant lacking the 2-ketoglutarate dehydrogenase active complex showed that the aspartate requirement for growth on poor carbon sources resulted from a deficiency in intracellular oxaloacetate pools even through pyruvate carboxylase was present at levels corresponding to those in wild-type cells. The oxaloacetate deficiency most likely resulted from the inability of the mutant to regenerate oxaloacetate from citrate due to the enzymatic block in the tricarboxylic acid cycle. Mutants in the enzymes of the dicarboxylic acid half of the citric acid cycle similarly required aspartate for wild-type growth in minimal medium. These results suggested that the complete turning of the tricarboxylic acid cycle is involved in the maintainance of oxaloacetate levels in B. subtilis. The ability of the mutants lacking the 2-ketoglutarate dehydrogenase enzymatic complex to grow at wild-type rates on media containing good carbon sources in the absence of exogenous aspartate is not understood.     

Identification of metabolic fluxes in hepatic cells from transient 13C-labeling experiments: Part II. Flux estimation

This contribution addresses the identification of metabolic fluxes and metabolite concentrations in mammalian cells from transient (13)C-labeling experiments. Whilst part I describes experimental set-up and acquisition of required metabolite and (13)C-labeling data, part II focuses on setting up network models and the estimation of intracellular fluxes. Metabolic fluxes were determined in glycolysis, pentose-phosphate pathway (PPP), and citric acid cycle (TCA) in a hepatoma cell line grown in aerobic batch cultures. In glycolytic and PPP metabolite pools isotopic stationarity was observed within 30 min, whereas in the TCA cycle the labeling redistribution did not reach isotopic steady state even within 180 min. In silico labeling dynamics were in accordance with in vivo (13)C-labeling data. Split ratio between glycolysis and PPP was 57%:43%; intracellular glucose concentration was estimated at 101.6 nmol per 10(6) cells. In contrast to isotopic stationary (13)C-flux analysis, transient (13)C-flux analysis can also be applied to industrially relevant mammalian cell fed-batch and batch cultures.     

Glial uptake of neurotransmitter glutamate from the extracellular fluid studied in vivo by microdialysis and (13)C NMR

Glial uptake of neurotransmitter glutamate (GLU) from the extracellular fluid was studied in vivo in rat brain by (13)C NMR and microdialysis combined with gas-chromatography/mass-spectrometry. Brain GLU C5 was (13)C enriched by intravenous [2,5-(13)C]glucose infusion, followed by [(12)C]glucose infusion to chase (13)C from the small glial GLU pool. This leaves [5-(13)C]GLU mainly in the large neuronal metabolic pool and the vesicular neurotransmitter pool. During the chase, the (13)C enrichment of whole-brain GLU C5 was significantly lower than that of extracellular GLU (GLU(ECF)) derived from exocytosis of vesicular GLU. Glial uptake of neurotransmitter [5-(13)C]GLU(ECF) was monitored in vivo through the formation of [5-(13)C,(15)N]GLN during (15)NH(4)Ac infusion. From the rate of [5-(13)C,(15)N]GLN synthesis (1.7 +/- 0.03 micromol/g/h), the mean (13)C enrichment of extracellular GLU (0.304 +/- 0.011) and the (15)N enrichment of precursor NH(3) (0.87 +/- 0.014), the rate of synthesis of GLN (V'(GLN)), derived from neurotransmitter GLU(ECF), was determined to be 6.4 +/- 0.44 micromol/g/h. Comparison with V(GLN) measured previously by an independent method showed that the neurotransmitter provides 80-90% of the substrate GLU pool for GLN synthesis. Hence, under our experimental conditions, the rate of 6.4 +/- 0.44 micromol/g/h also represents a reasonable estimate for the rate of glial uptake of GLU(ECF), a process that is crucial for protecting the brain from GLU excitotoxicity.     

Intrafollicular amino acid concentration and the effect of amino acids in a defined maturation medium on porcine oocyte maturation, fertilization, and preimplantation development

The objective of this study was to determine the intrafollicular concentrations of free amino acids in pigs and to examine the effect of amino acids in a chemically defined maturation medium on oocyte maturation, in vitro fertilization (IVF), and embryo development in vitro. Pooled follicular fluid aspirated separately from small (<3mm in diameter), medium (3-8mm), and large follicles (>8mm) in three pairs of ovaries was analyzed for amino acid concentration. In addition, oocyte maturation, fertilization, and embryo development were examined after in vitro maturation (IVM) of oocytes in a defined maturation medium supplemented individually with glutamate (GLU), glutamine (GLN), glycine (GLY), aspartate (ASP), asparagine (ASN), arginine (ARG), alanine (ALA), leucine (LEU), lysine (LYS), proline (PRO), and valine (VAL). Irrespective of follicle size, GLY, GLU, ALA, GLN, and PRO were the most abundant amino acids in pig follicular fluid (pFF). Sperm penetration was not altered by amino acid treatment during IVM, but monospermic fertilization was increased (P<0.05) by GLN, ASP, and VAL. All amino acids except ASP and ASN stimulated (P<0.05) male pronuclear formation after IVF. ARG and ALA treatment during IVM improved (P<0.05) blastocyst formation. In conclusion, GLY, GLU, ALA, GLN, and PRO were the most abundant amino acids in pFF and amino acids in a defined medium improved porcine monospermic fertilization, male pronuclear formation, and preimplantation development.     

Pyrimidine Pools and Macromolecular Composition of Pyrimidine-Limited Escherichia coli

The growth rate of a pyrimidine-requiring strain was controlled by limiting the concentration of exogenous orotic acid. As the steady state, pyrimidine-limited growth rate was decreased, the intracellular pyrimidine pools and the total nucleic acid per unit mass of culture also decreased. The ratio of deoxyribonucleic acid to protein remained constant, whereas the ratio of ribonucleic acid to protein decreased 30% over a threefold variation in growth rate (50- to 150-min doubling times). The intracellular uridine triphosphate and cytosine triphosphate pools also decreased (although not coordinately), and the pyrimidine biosynthetic enzymes were derepressed. Cell size was unaffected by pyrimidine-mediated variation of the growth rate.     

The ratio of glutamine:glutamate in microalgae: a biomarker for N-status suitable for use at natural cell densities

A measure of the N-status of phytoplankton at natural cell densitiesmay be made by determining the ratio of intracellular glutamine:glutamate(GLN:GLU). The intracellular pool of free amino acids from 10⁴–10⁶cells is extracted in ultraclean conditions and analysed byreverse phase HPLC. A GLN:GLU ratio of >0.5 indicates N-repleteand <0.2 N-deplete cells.     

Inducible accumulation of alpha-ketoglutaric acid in cultures of Streptomyces hygroscopicus JA 6599 producing a macrolide antibiotic]

The excessive production of pyruvic and 2-oxoglutaric acid by S. hygroscopicus JA 6599 grown on a medium rich in complex carbon and nitrogen sources was studied. Towards the end of the first day of batch cultivation a maximum level of both keto acids in the medium was observed. By diluting the complete culture with water at 22nd hour, however, a further increase in 2-oxoglutarate concentration was induced and the antibiotic production was slightly stimulated. In diluted cultures the oxygen saturation was found to be distinctly higher than in non-diluted ones and, on the other hand, the mycelial activities of both pyruvate and 2-oxoglutarate decarboxylases were decreased. Since the 2-oxoglutarate level was strongly influenced by inhibitors of glycolysis and of citric acid cycle, it is suggested that the metabolite accumulation in diluted cultures is mainly caused by modifications of the metabolic control of carbohydrate catabolism due to an improved aeration. Furthermore, the macrolide antibiotic A 6599 produced by S. hygroscopicus JA 6599 itself was shown to interfere with the accumulation of 2-oxoglutaric acid.     

Effect of cadmium sulphate on the metabolism of carbohydrates in organism of rats of different ages

The influence of cadmium sulfate on concentration of glucose, lactate, piruvate, alpha-ketoglutarate, malate, oxaloacetate in blood of 3-, 6- and 18-month-old poisoned rats was established the results of our researches. It was found, that poisoning of rats by cadmium sulfate causes the rise of concentration of glucose, metabolites of citric acid cycle and glycolysis in blood of animals of all age groups explored. The research results prove that in blood of 3-month-old poisoned rats the level of glycolysis and citric acid cycle activation is considerably higher in comparison with that of 6- and 18-month-old animals. As a result, a comparison of age-specific dynamics of changes of carbohydrate metabolism indices in the blood of rats, poisoned by cadmium showed that the organism of 3-month-old rats is more sensitive to toxic influence of cadmium.     

A daily study of the diatom spring bloom at Roscoff (France) in 1985. III. Free amino acids composition studied by HPLC analysis

The growth of a natural phytoplankton population was studiedduring a monospecific spring bloom of the diatom Rhizosoleniadelicatula at Roscoff (western English Channel). Direct examinationof the intracellular pool of 19 free amino acids (FAA) was usedas an index of the physiological status of the cells. TotalFAA in the particulate matter shows a general decrease duringthe bloom, and FAA cell content varies from 200 mM 1 cell volume^–1to 10 mM at the time of maximum biomass. FAA-N/particulate Nis <3% during the study, slightly decreasing at the timeof maximum biomass. Individual free amino acids appear reliablefor the development of the diatom biomass and good indicatorsof the growing population. At maximum biomass, major compoundsare glutamic acid (30.8 mol%), glutamine (11.7%), alanine (9.9%),isoleucine (6.6%) and lysine (6.4%). Serine, glycine, arginineand aspartic acid appear also as major components during otherperiods of the study. We suggest that the observed changes arean indication of the physiological state of the cells duringthe bloom. In particular glutamine (GLN), glutamic acid (GLU)and the GLN/GLU ratio allow the metabolic evolution of the naturalpopulation to be characterized. GLN is strictly linked to theliving biomass (Chla) while GLU is present at high percentagethroughout the study. The ecological significance of ß-alanine(BALA) is also revealed during this study, being strongly correlatedto degraded matter represented by phaeopigments. These resultsdemonstrate the validity of using individual free amino acidsas an aid in understanding the physiological status of algaein natural bloom conditions.     

190 The peculiarities of regulation of Yarrowia lipolytica yeast and citric acid overproduction

The growth of Yarrowia lipolytica yeast as well the biosynthesis of citric acid on rapeseed oil were studied. It was indicated that the initial step of assimilation of rapeseed oil in the yeast Y. lipolytica is their hydrolysis by extracellular lipases with the formation of glycerol and fatty acids, which appear in the medium in the phase of active growth. The concentrations of these metabolites change insignificantly upon further cultivation. Lipase and the key enzymes of glycerol metabolism (glycerol kinase) and the glyoxylate cycle responsible for the metabolism of fatty acids (isocitrate lyase and malate synthase) are induced just at the beginning of the growth phase and remain active in the course of further cultivation. These results, taken together, suggest that glycerol and fatty acids according in the medium do not suppress the metabolism of each other. The fact that glycerol and fatty acids can be consumed simultaneously is of special importance for the development of the efficient regime of oil feeding, Y. lipolytica produced citric acid (175?g/L) with a yield of 150%. It should be noted that the simultaneous utilization of two different substrates is not typical of micro-organisms, which first assimilate one of the two available substrates (commonly, a carbohydrate), whereas the assimilation of the other substrate starts only after the first substrate is fully consumed from the medium. Indeed, upon the cultivation of Y. lipolytica on the mixture of glucose and oleic acid, the latter substrate began to be utilized only when the concentration of glucose decreased. The glycolytic enzyme pyruvate dehydrogenase was induced from the first hours of cultivation and remained at high levels until the exhaustion of glucose in the medium. At the same time, the activities of isocitrate lyase and malate synthase were very low during the metabolism of glucose, but were rapidly induced (approximately in 10 times) after the exhaustion of glucose in the medium. When Y. lipolytica was grown on the mixture of glucose and hexadecane, the dynamics of growth and substrate consumption was typical of the diauxie phenomenon: the utilization of hexadecane began only in several hours after the time when glucose was completely exhausted in the cultivation medium. In this case, the exhaustion of glucose arrested growth and the culture resumed growth only after a lag period. The assay of enzymes showed that the glycolytic enzyme pyruvate dehydrogenase was active during the phase of growth on glucose, whereas the enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase were active during the phase of growth on hexadecane. In recent years in the literature, there are data that the different sugars produce signals which modify the conformation of certain proteins that, in turn, directly or through a regulatory cascade affect the expression of the genes subject to catabolite repression. These genes are not all controlled by a single set of regulatory proteins (Cho et al. 2009), but there are different circuits of repression for different groups of genes (Gancedo 1990). We will discuss the possible metabolic regulation in the case of Y. lipolytica.     

Metabolism of dog gastric mucosa. Levels of glycolytic, citric acid cycle and other intermediates.

Several metabolites, including those of glycolysis, the citric acid cycle, the hexose monophosphate shunt, glutamate, aspartate, and Coenzyme A were measured in defined parietal cell-enriched freeze-dried sections of dog gastric biopsies derived from nonsecreting and secreting tissue. In addition, NH3, ribulose 5-phosphate, glycerol, and succinate were measured in perchloric acid extracts of biopsies. The onset of secretion increased the level of glycolytic intermediates including pyruvate and lactate with the most marked increase being in fructose 1,6-diphosphate levels. The level of 6-phosphogluconate and ribulose 5-phosphate also increased, in spite of a constant NADP+/NADPH ratio. The levels of all the citric acid cycle intermediates measured also rose, the most marked rise being in malate and fumarate. The levels of glycerol, acetyl-CoA, and CoA increased, but the ratio of the latter intermediates remained constant. Calculation of the ratio of the oxidized to reduced form of diphosphopyridine nucleotide indicated a fall of the ratio in the cytoplasm and a rise in the mitochondria. From these data, it is concluded that the major energy source for acid secretion is due to an increase in citric acid cycle activity and that glycolysis, and probably also fatty acid oxidation, is stimulated to provide mitochondrial substrate.