31P-saturation-transfer nuclear-magnetic-resonance measurements of phosphocreatine turnover in guinea-pig brain slices.

The technique of 31P saturation-transfer n.m.r. was used to determine the forward and the reverse rate constants of creatine phosphotransferase in superfused guinea-pig cerebral tissues in vitro. The calculated forward rate constant of 0.22 +/- 0.03s-1 compared well with a previously reported value for rat brain in vivo [Shoubridge, Briggs & Radda (1982) FEBS Lett. 140, 288-292]. The reverse rate constant was found to be 0.55 +/- 0.10s-1. 3. By using concentrations of ATP and phosphocreatine estimated previously for this superfused preparation [Cox, Morris, Feeney & Bachelard (1983) Biochem. J. 212, 365-370], forward and reverse flux rates were calculated to be 0.68 and 0.72 mumol X s-1 X g-1 respectively. The concordance of forward and reverse fluxes contrasts with the situation observed in vitro in other tissues, and suggests that the creatine phosphotransferase reaction is at equilibrium under the conditions used here. 4. Lowering the concentration of glucose in the superfusing medium from 10mM to 0.5mM had no significant effect on phosphocreatine concentration or on the forward (ATP-generating) flux through creatine phosphotransferase. The results indicate that a normal phosphocreatine content in the presence of lowered glucose availability is reflected by an unchanged turnover rate.     

Kinetic analysis of the cerebral creatine kinase reaction under hypoxic and hypoglycaemic conditions in vitro. A 31P-n.m.r. study.

1. The tissue concentration of phosphocreatine (PCr) and the pseudo-first-order rate constant of creatine kinase (kf) were monitored in superfused guinea-pig brain slices in vitro by using 31P-n.m.r. techniques. 2. Superfusion of slices in low oxygen partial pressure (pO2 approx. 16 kPa) decreased tissue PCr concentrations by 48% but ATP concentrations were unchanged. Regression analysis revealed a significant negative correlation between the PCr concentration in hypoxic tissue and the increase in the rate constant, kf. Nevertheless the forward flux through the enzyme (Jf = kf.[PCr]) declined under these conditions. 3. Lowering the glucose concentration to 0.2 or 0.1 mM decreased PCr concentrations by 29% and 48% respectively; here ATP concentrations as well as PCr concentrations also decreased. Only in the presence of the lower glucose concentration (0.1 mM) was kf increased. However, unlike the situation in hypoxic tissue, Jf was maintained at control rates. 4. In spectra obtained in the presence of low oxygen or low glucose concentrations, a resonance attributable to tissue inorganic phosphate became dectectable. This observation is discussed in terms of known changes in tissue phosphate concentrations and possible alterations in cytoplasmic pH.     

Effects of Ammonia and β-Methylene-dl-Aspartate on the Oxidation of Glucose and Pyruvate by Neurons and Astrocytes in Primary Culture

Both ammonia and beta-methylene-DL-aspartate (beta-MA), an irreversible inhibitor of aspartate aminotransferase activity and thus of the malate-aspartate shuttle, were found previously to decrease oxidative metabolism in cerebral cortex slices. In the present work, the possibility that ammonia and beta-MA affect energy metabolism by a common mechanism (i.e., via inhibition of the malate-aspartate shuttle) was investigated using primary cultures of neurons and astrocytes. Incubation of astrocytes for 30 min with 5 mM beta-MA resulted in a decreased production of 14CO2 from [U-14C]glucose, but did not affect 14CO2 production from [2-14C]pyruvate. Conversely, incubation of astrocytes with 3 mM ammonium chloride resulted in decreased 14CO2 production from [2-14C]pyruvate, but 14CO2 production from [U-14C]glucose was not significantly affected. Ammonium chloride had no significant effect on 14CO2 production from either [U-14C]glucose or [2-14]pyruvate by neurons. However, incubation of neurons with beta-MA or beta-MA plus ammonium chloride resulted in a approximately 45% decrease of 14CO2 production from both [U-14C]glucose and [2-14C]pyruvate. A 2-h incubation of astrocytes with beta-MA resulted in no change in ATP levels, but a 35% decrease in phosphocreatine. Similar treatment of neurons resulted in greater than 50% decrease in ATP, but had little effect on phosphocreatine. beta-MA also caused a decrease in glutamate and aspartate content of neurons, but not of astrocytes. The different metabolic responses of neurons and astrocytes towards beta-MA were probably not due to a differential inhibition of aspartate aminotransferase which was inhibited by approximately 45% in astrocytes and by approximately 55% in neurons.     

A 31P-n.m.r. study of the acute effects of beta-blockade on the bioenergetics of skeletal muscle during contraction.

1. The effects of beta-adrenoceptor antagonist administration on skeletal muscle contractile performance and bioenergetics in vivo have been investigated during unilateral sciatic nerve stimulation in the rat. 2. Two muscle stimulation protocols have been used: supramaximal stimulation at 4 Hz, or incremental supramaximal stimulation at 1, 2 and 4 Hz. Changes in high-energy phosphate concentrations were followed using 31P-n.m.r., and gastrocnemius muscle twitch characteristics were monitored continuously. 3. Under all conditions investigated, DL-propranolol administration (2.5 mg/kg body wt.) caused a significant decrease in cyclic AMP concentrations in resting and stimulated gastrocnemius muscle, prevented an increase in heart rate upon muscle stimulation, but did not affect plasma glucose, fatty acid or lactate concentrations in comparison with values obtained in control experiments. 4. Administration of DL-propranolol 5 min or 35 min before unilateral stimulation of 4 Hz had no effect on changes in muscle phosphocreatine, ATP or Pi concentrations, intracellular pH or contractile performance. 5. In contrast, animals receiving DL-propranolol 5 min before unilateral stimulation of 1, 2 and 4 Hz showed a significant deterioration in gastrocnemius muscle tension development during 2 and 4 Hz stimulation compared with control animals. Concurrent with this change in contractile performance was a higher muscle concentration of phosphocreatine, a lower concentration of Pi and no significant change in intramuscular pH compared with control experiments. 6. The changes in muscle performance and bioenergetics observed during the incremental stimulation protocol were not observed when D-propranolol was administered and could be completely circumvented by a short period of muscle stimulation of 4 Hz prior to initiation of the incremental stimulation protocol. 7. Mechanisms are discussed which may account for the failure of gastrocnemius muscle to generate the expected force during the incremental stimulation protocol in the presence of beta-blockade.     

Experimentally induced defects of mitochondrial metabolism in rat skeletal muscle. Biological effects of the NADH: coenzyme Q reductase inhibitor diphenyleneiodonium.

An animal model for the human condition of mitochondrial myopathy has been established and characterized physiologically and biochemically. The NADH: coenzyme Q reductase inhibitor diphenyleneiodonium [Bloxham (1979) Biochem. Soc. Trans. 7, 103-106] was either infused acutely in vivo into rat hind limb or injected chronically into rats. Both modes of delivery resulted in a reduced muscle oxidative capacity and increased fatigue. Analysis of muscle metabolites by h.p.l.c. and 31P-n.m.r. indicated that ATP concentrations were similar to control values during periods of stimulation and these were maintained by the phosphocreatine pool. During the recovery period after muscle stimulation in the experimental animals the muscle pH remained depressed and the rate of phosphocreatine synthesis was markedly delayed as compared with controls. Factors thought to be involved in the fatigue response are discussed in relation to this model.     

The metabolic state of muscle in the isolated perfused rat hemicorpus in relation to rates of protein synthesis.

Measures of perfusion adequacy in perfused rat hemicorpus preparations were investigated as potential indices of tissue function during studies of muscle protein metabolism. Perfusion under normal conditions for up to 80 min resulted in rates of protein synthesis and concentrations of ATP in muscle that were similar to those in vivo, but phosphocreatine in muscle gradually decreased and muscle lactate increased. Hypoxic conditions led to lower rates of protein synthesis, lower phospho-creatine and raised lactate contents in muscle compared with normal perfusions, and ATP was slightly decreased. Hypoxic preparations also released more lactate and K+ into the medium and had higher perfusion pressures, but glucose uptake and muscle water content were not altered. In totally ischaemic muscle, concentrations of ATP and phosphocreatine were even lower than in hypoxic muscle, and that of lactate was higher. From 11 preparations perfused for 60 min under normal conditions, three were selected on the basis of lower muscle ATP content than the others. Preparations with low ATP also showed lower muscle phosphocreatine concentrations, O2 uptake and CO2 output, as well as higher perfusion pressure and muscle lactate concentrations than in the remaining preparations, but muscle water, ADP and AMP concentrations and lactate and K+ flux were no different. In perfusions extended to 3 h, deterioration of function was more apparent. There were significant correlations between rates of protein synthesis and the concentrations of ATP, phosphocreatine and lactate in two different muscles (r = 0.756-0.929), but not with any of the other indices investigated. Taken overall, these experiments showed that concentrations of ADP, AMP and water in muscle, rates of lactate and glucose metabolism, K+ output, perfusion pressure and blood gas parameters were unsuitable for distinguishing unsound from sound preparations, because they did not consistently demonstrate differences, or could not be ascribed to only muscle metabolism. It was found that ATP, phosphocreatine and lactate concentrations in muscle were the best indicators of impaired metabolic state in studies of protein synthesis. Measurements of these could be used on a routine basis for rejecting unsatisfactory preparations.     

Platelet-derived-growth-factor-stimulated heterogeneous polyphosphoinositide metabolism and phosphate uptake in C3H fibroblasts.

1. Gated 31P-n.m.r. spectra were obtained from the ankle flexor muscles of the rat at various times after 3 s isometric tetanic contraction. This allowed the time course of changes in phosphocreatine (PCr), Pi and free ADP concentrations and intracellular pH to be monitored in skeletal muscle in vivo with 1 s time resolution. 2. ATP concentration did not change significantly, either during the recovery from a 3 s tetanus or during the overall protocol. 3. The calculated rate of recovery of ADP towards pre-stimulation levels was very rapid (t1/2 less than 5 s). The rate of Pi disappearance (t1/2 = 14 s) was more rapid than the rate of PCr synthesis (t1/2 = 24 s), resulting in a significant transient decrease in n.m.r.-visible PCr + Pi between 25 and 45 s after tetanic contraction. 4. The rates of PCr, Pi and ADP recovery are higher than those previously reported for recovery from steady-state exercise in humans or twitch isometric contraction in animals.     

Effects of N-Methyl-d-Aspartate on [Ca2+]i and the Energy State in the Brain by 19F- and 31P-Nuclear Magnetic Resonance Spectroscopy

The effects of N-methyl-D-aspartate (NMDA) on the free intracellular Ca2+ concentration [( Ca2+]i) and the energy state in superfused cerebral cortical slices have been studied using 19F- and 31P-nuclear magnetic resonance spectroscopy. [Ca2+]i was measured using the calcium indicator 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA). NMDA (10 microM) in the absence of extracellular Mg2+ caused the expected rise in [Ca2+]i but produced an impairment of the energy state: the phosphocreatine (PCr) content was decreased by 42%, and the Pi/PCr ratio was increased by 55%. There was no detectable change in ATP or free intracellular Mg2+ concentration. Increasing the NMDA concentration in the superfusing medium to 100 or 400 microM caused no further increase in [Ca2+]i or further decrease in PCr content, but the Pi/PCr ratio continued to rise. The impairment of the energy state preceded the effect on [Ca2+]i, and these changes were irreversible on return to control conditions. Repeating the experiments in the presence of 1.2 mM extracellular Mg2+ resulted in similar changes in the energy state, with no change in [Ca2+]i. The possibilities that the effects were due to membrane depolarisation or to the presence of 5FBAPTA within the tissues were eliminated. The results suggest that low concentrations (10 microM) of NMDA produce an impaired energy state independent of the presence of extracellular Mg2+ and that the decreased energy state is not due to the changes in [Ca2+]i, which are seen only in the absence of extracellular Mg2+.     

Counter-transport in chick embryo fibroblasts. A significant factor in measurement of glucose entry.

Enhanced rates of carrier-mediated 3-O-methyl-D-glucose (0.1 mM) transport were observed in primary cell cultures of chicken embryo fibroblasts deprived of glucose for 1 day. The addition of 5.5 mM-glucose, glucosamine or 2-deoxy-D-glucose for 15 min (37 degrees C) to glucose-starved cultures followed by washing and immediate measurement of 3-O-methyl-D-glucose transport resulted in an apparent further stimulation of transport. Transport stimulation increased with increasing concentrations of the added preincubation sugar and was observed at test concentrations ranging from 0.1 mM- to 10 mM-3-O-methyl-D-glucose. This enhancement occurred when the preloaded sugar was rapidly effluxing from cells and was eliminated by allowing cultures to incubate in buffer without sugar for 30 min (37 degrees C) after the removal of hexose and before measuring transport. A transient overshoot in the cumulative uptake of 3-O-methyl-D-glucose was observed in glucose-starved cultures that were pre-incubated in the presence of 55 mM-glucose or -glucosamine for 15 min (37 degrees C). These data suggest that counter-transport accounts for the apparent enhancement of glucose-transport capability observed in glucose-starved cells when they are briefly re-exposed to hexose.     

Futile substrate cycles in the glycolytic pathway of boar and rat spermatozoa and the effect of alpha-chlorohydrin

In boar spermatozoa incubated with 0.1 mM-glucose about 20 nmol glucose were converted to lactate and CO2 and the rate of futile substrate cycling between glucose and glucose 6-phosphate was about 6 nmol/10(8) spermatozoa/30 min. Futile cycling was increased in the presence of 0.05 or 1 mM-alpha-chlorohydrin but not to an extent sufficient to account for the rapid decline in ATP concentration observed under these conditions. These estimates include a substantial rate of fructose formation from fructose phosphates. The addition of 10 mM-L-lactate plus 1 mM-pyruvate protected the spermatozoa against the effect of alpha-chlorohydrin and glucose on the ATP concentration but increased futile substrate cycling. Substrate cycling between fructose 6-phosphate and fructose 1,6-bisphosphate could not be measured in boar spermatozoa but in rat spermatozoa its rate (nmol/10(8) spermatozoa/30 min) was about 10 under control condition and about 25 in the presence of 1 mM-alpha-chlorohydrin. This increase was insufficient to account for the decline in ATP concentration. In both species futile substrate cycling consumed a significant proportion of the ATP synthesis during lactate production but only about 5% of that produced in the oxidation of glucose to acetyl carnitine and CO2.     

Interactions of glucose, acetoacetate and insulin in mammary-gland slices of lactating rats.

1. Utilization of 5mM-glucose by slices of lactating mammary gland was decreased 33% on addition of acetoacetate (2mM) to the incubation medium. This inhibition was accompanied by increases in the intracellular concentrations of citrate and glucose 6-phosphate. 2. In the presence of acetoacetates the accumulation of pyruvate in the medium approximately doubled. 3. Insulin completely reversed the inhibitory effect of acetoacetates on glucose utilization, without altering the amount of acetoacetate removed or pyruvate formed. 4. Similar results were obtained with mammary-gland slices from diabetic rats, except that insulin did not completely reverse the effects of acetoacetates. 5. Acetoacetate inhibited the formation of 14CO2 from [1-14C]pyruvate; this effect was not overcome by insulin. 6. Insulin increased the proportion of [3-14C]acetoacetate that was converted into lipid and decreased that oxidized to CO2.7. The physiological significance of these findings is discussed.     

31P NMR Relaxation Does Not Affect the Quantitation of Changes in Phosphocreatine, Inorganic Phosphate, and ATP Measured In Vivo During Complete Ischemia in Swine Brain

Abstract: Ischemia-induced changes in ³¹P NMR relaxation were examined in 16 piglets. NMR spectra were acquired under control conditions and during complete cerebral ischemia induced via cardiac arrest. Changes in T ₁ were assessed directly in six animals during control conditions and after 30–45 min of complete ischemia when changes in brain P₁ levels had reached a plateau. The T ₁ for P₁ did not change, i.e., 2.3 ± 0.5 s during control conditions versus 2.4 ± 1.0 s during ischemia. To evaluate phosphocreatine and ATP, two types of spectra, with a long (25-s) or short (1-s) interpulse delay time, were collected during the first 10 min of ischemia (n = 10). Both types of spectra showed the same time course of changes in phosphocreatine and ATP levels, implying that the T ₁ relaxation times do not change during ischemia. There were no changes in the linewidths of phosphocreatine, ATP, or P₁ during ischemia, implying that the T *₂ values remain constant. Our results suggest that the ³¹P T ₁ and T *₂ for phosphocreatine, P_i, and ATP do not change during ischemia, and therefore changes in ³¹P NMR peak intensity accurately reflect changes in metabolite concentrations.     

The pentose cycle and insulin release in isolated mouse pancreatic islets during starvation.

When islets from mice were incubated with 16.7 mM-glucose, previous starvation for 48 h decreased the rate of insulin release by approx. 50% and glucose utilization was decreased by approx. 35%. The maximally extractable activity of glucose 6-phosphate dehydrogenase was diminished by 28% after starvation. The formation of 14CO2 from both [1-14C]glucose was, however, higher than the rate of oxidation of [6-14C]-glucose in islets from both fed and starved mice. The fraction of glucose utilized that was oxidized (specific 14CO2 yield) ranged from one-fifth to one-third and was higher in islets from starved mice with both [1-14C]glucose and [6-14C]glucose as substrate. The contribution of pentose-cycle oxidation to total glucose metabolism was small (3% in the fed state and 4% in the starved state). The absolute rates of glucose carbon metabolism via the pentose-cycle oxidation to total glucose metabolism was small (3% in the fed state and 4% in the starved state). The absolute rates of glucose carbon metabolism via the pentose cycle and the turnover of NADPH in this pathway were identical in islets from fed and starved animals. After incubation at 16.7 mM-glucose for 30 min the contents of glucose (6-phosphate and 6-phosphogluconate were both unchanged by starvation. It is concluded that there is no correlation between the decreased sensitivity of the insulin secretory mechanism during starvation and the metabolism of glucose via the pentose cycle, the islet content of glucose 6-phosphate or 6-phosphogluconate.     

Direct effects of CO on cerebral energy metabolism in bloodless rats

Cerebrocortical b-cytochromes have been found to be sensitive to reduction in the presence of CO and O2 in vivo. CO-mediated cytochrome b reduction responses in "bloodless" rats were correlated in this study with changes in concentrations of high energy and glycolytic intermediates measured in cortex after rapid brain freezing. Cytochrome redox state and metabolite concentrations also were compared with cerebral blood flow (CBF) and cerebral metabolic rate for O2 (CMRo2) measured before and after CO administration. No definite biochemical evidence of energy limitation was found in parietal cortex after the fluorocarbon-for-blood exchange; however, CO had direct effects on brain metabolite concentrations. Fifteen-minute CO exposures at inspired CO/O2 of 0.003-0.06 increased cerebrocortical phosphocreatine and ADP and decreased creatine concentration. CO exposure produced no significant changes in either ATP concentration or CMRo2, although CBF increased slightly. These findings may be interpreted to indicate that CO binding to cytochrome aa3 at low CO/O2 in vivo increases extramitochondrial pH relative to that within the mitochondrial matrix. In the process, cytochrome b reduction levels increase, possibly signaling an increased efficiency of oxidative phosphorylation relative to O2 uptake by unblocked respiratory chains.     

The role of insulin in the intestinal absorption of glucose in the rat.

1. Acute pre-treatment with either mannoheptulose or streptozotocin--both compounds acting as powerful suppressors of insulin secretion--caused a significant decrease on the in vivo rate of intestinal glucose absorption following an intragastric [U-14C]glucose administration. 2. Mannoheptulose treatment also lowered the rate of whole-body oxidation of the administered tracer. 3. Insulin had no effect on the metabolic fate of [U-14C]glucose by isolated enterocytes. 4. However, the rate of glucose uptake, measured by the oxidation of [1-14C]glucose to 14CO2 in the presence of phenazine methosulphate, was decreased by insulin at concentrations of 50-200 munits/ml. 5. In addition, the rate of transport of [U-14C]glucose by brush-border membrane vesicles was also inhibited by insulin at high concentrations (100-1000 munits/ml). 6. This indicated that insulin acts by inhibiting glucose transport in isolated in vitro preparations. 7. Acute pre-treatment with either mannoheptulose or streptozotocin caused a significant decrease in the rate of gastric emptying, measured as the distribution of [3H]insulin along the gastrointestinal tract, following an intragastric glucose load. 8. It is concluded that insulin secretion modulates intestinal glucose absorption in vivo by enhancing gastric emptying in spite of the inhibitory effects of glucose transport observed with in vitro preparations.     

Abnormal phosphocreatine metabolism in perfused diabetic hearts. A 31P nuclear-magnetic-resonance study.

31P n.m.r. analysis of control and diabetic hearts perfused for 1 h with a glucose buffer showed constant and normal levels of phosphocreatine and ATP. Supplementing the buffer with 0.5, 1.2 or 2.0 mM-palmitic acid had little or no effect on high-energy-phosphate levels in control hearts. In contrast, increases in palmitate concentration produced significant decreases in ATP in diabetic hearts, despite normal and constant levels of phosphocreatine. This 31P n.m.r. study suggests a defect in phosphocreatine metabolism in the perfused diabetic heart that might be related to creatine kinase kinetics.     

Effect of glucose on the intracellular pH of pancreatic islet cells.

To characterize the effect of glucose on the intracellular pH (pHi) of pancreatic islet cells, we measured the accumulation of 14C-labelled 5,5-dimethyloxazolidine-2,4-dione ( [14C]DMO) in beta-cell-rich islets from ob/ob mice. D-Glucose (20 mM) stimulated insulin release and enhanced the [14C]DMO equilibrium uptake corresponding to an increase of pHi by about 0.15 unit. The glucose effect on DMO uptake was concentration-dependent, with half-maximal effect at about 4 mM-glucose and maximum effect at about 10 mM-glucose. It was inhibited by 20 mM-mannoheptulose and potentiated by 4 mM-L-5-hydroxytryptophan, but not affected by 2 mM-theophylline. Mannoheptulose is an inhibitor and L-5-hydroxytryptophan and theophylline are potentiators of glucose-stimulated insulin release. The glucose-induced increase in pHi appeared rapidly (7 min) and persisted for at least 30 min and it was observed both in bicarbonate/CO2-buffered and in Hepes [4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid]-buffered media. Addition of extracellular bicarbonate buffer lowered the pHi, but did not affect basal insulin release, whereas 5 mM-NH4+ increased pHi and induced a 4-fold increase of basal insulin release. We conclude that, in contrast with previous assumptions, glucose increases intracellular pH in the islet cells. This effect may be coupled to the glucose metabolism and associated with triggering of insulin release.     

Role of fructose 2,6-bisphosphate in the stimulation of glycolysis by anoxia in isolated hepatocytes.

1. Incubation of hepatocytes from fed or starved rats with increasing glucose concentrations caused a stimulation of lactate production, which was further increased under anaerobic conditions. 2. When glycolysis was stimulated by anoxia, [fructose 2,6-bis-phosphate] was decreased, indicating that this ester could not be responsible for the onset of anaerobic glycolysis. In addition, the effect of glucose in increasing [fructose 2,6-bisphosphate] under aerobic conditions was greatly impaired in anoxic hepatocytes. [Fructose 2,6-bisphosphate] was also diminished in ischaemic liver, skeletal muscle and heart. 3. The following changes in metabolite concentration were observed in anaerobic hepatocytes: AMP, ADP, lactate and L-glycerol 3-phosphate were increased; ATP, citrate and pyruvate were decreased: phosphoenolpyruvate and hexose 6-phosphates were little affected. Concentrations of adenine nucleotides were, however, little changed by anoxia when hepatocytes from fed rats were incubated with 50 mM-glucose. 4. The activity of ATP:fructose 6-phosphate 2-phosphotransferase was not affected by anoxia but decreased by cyclic AMP. 5. The role of fructose 2,6-bisphosphate in the regulation of glycolysis is discussed.     

Glutamine metabolism in isolated incubated adipocytes of the rat.

1. Phosphate-dependent glutaminase activity in the epididymal fat-pad was 15.1 nmol/min per mg of protein. Glutaminase activity demonstrated differences with respect to adipose-tissue sites. Considerable variation was found in different sites of adipose tissue from lean control and Zucker obese animals. 2. Adipocytes incubated in the presence of 2 mM-glutamine utilized glutamine at a rate of 1.8 mumol/h per g dry wt., and glutamate, ammonia, lactate and alanine were produced. Addition of glucose plus insulin increased the rates of glutamine utilization and glutamate, ammonia, lactate and alanine production. Isoprenaline alone or plus glucose further stimulated the rate of glutamine utilization and formation of end products. 3. The rate of incorporation of 14C from glutamine into CO2 was similar to that of glucose, but the rate of incorporation into triacylglycerol was much less. Addition of unlabelled glucose or glucose plus insulin stimulated the rate of incorporation of [14C]glutamine into triacylglycerol, but had no effect on that of 14CO2 formation. Isoprenaline plus glucose increased the rate of incorporation of [14C]glutamine into CO2, but decreased the rate of incorporation into triacylglycerol. 4. In the absence of insulin, the rate of [14C]glutamine incorporation into triacylglycerol was related to the glucose concentration (0-10 mM). However, in the presence of insulin, the rate of incorporation of [14C]glutamine was maximal at 1 mM-glucose.     

Estimation of intracellular sugar phosphate concentrations in Saccharomyces cerevisiae using 31P nuclear magnetic resonance spectroscopy

A systematic procedure has been formulated for estimating the relative intracellular concentrations of sugar phosphates in Saccharomyces cerevisiae based upon (31)P nuclear magnetic resonance (NMR) measurements. The sugar phosphate region of the (31)P NMR spectrum is first decomposed by computer analysis, and the decomposition consistency and identification of individual sugar phosphate resonances are established based on in vitro chemical shift calibrations determined in separate experiments. Numerous evaluations of intracellular S. cerevisiae compositions for different strains and different cell environments provide the basis for in vivocorrelations of inorganic phosphate chemical shift with the chemical shifts of 3-phosphoglycerate, beta;-fructose 1,6-diphosphate, fructose 6-phosphate, and glucose 6 phosphate. Relative intracellular sugar phosphate concentrations are obtained by correcting peak areas for partial saturation during transient in vivo experiments. In vivo concentrations estimated by this method agree well with estimates for similar systems based on other techniques. This approach does not require costly la belled compounds, and has the advantage that other important metabolic state variables such-as internal and external pH and intracellular levels of phosphate, ATP, ADP, NAD(H), and polyphosphate may be determined from the same (31)P spectrum. Extension of this strategy to other cellular systems should be straightforward.