METABOLISM OF HEXOSES IN RAT CEREBRAL CORTEX SLICES

Abstract—

• 1 The metabolism of two ¹⁴C-labelled hexoses and one hexose analogue, viz. mannose, fructose and glucosamine, has been compared with that of glucose for slices of rat cerebral cortex incubated in vitro.
• 2 The metabolism of [U-¹⁴C]mannose was essentially identical to that of glucose; oxygen consumption and CO₃ production were similar and maximal at a substrate concentration of 2·75 mM. Incorporation of label into lactate, aspartate, glutamate and GABA was similar for the two substrates at 5·5 mM substrate concentration.
• 3 With [U-¹⁴C]fructose, maximal oxygen consumption and CO₃ production were obtained at a substrate concentration of 11 mM. At 5·5 mM, incorporation into lactate was 5 per cent, into glutamate and GABA 30 per cent, into alanine 63 per cent and into aspartate 152 per cent of that from glucose. Increasing substrate concentration to 27·5 mm was without effect on incorporation into amino acids from glucose and raised incorporation from fructose into glutamate, GABA and alanine to a level similar to that found with glucose; at the higher substrate concentration aspartate incorporation from fructose was 200 per cent and lactate 42 per cent of that with glucose. Unlabelled fructose was without effect on incorporation of radioactivity from [3-¹⁴C]pyruvate into CO₂ or amino acids; it increased incorporation into lactate by 36 per cent. Unlabelled glucose diminished incorporation into CO₂ from [U-¹⁴C]fructose to 35 per cent; incorporation into lactate was stimulated 178 per cent at 5·5 mM fructose; at 27·5 mM it was diminished to 75 per cent.
• 4 By comparison with [1-¹⁴C]glucose, incorporation of radioactivity from [1-¹⁴C]-glucosamine into lactate, CO₂, alanine, GABA and glutamine was very low; incorporation into aspartate was similar to glucose. Thus the metabolism of glucosamine resembled that of fructose. Glucosamine-1-phosphate, glucosamine-6-phosphate, and an unidentified metabolite, all accumulated.

     

GLUCOSE AND AMINO ACID METABOLISM IN ISOLATED NEURONAL AND GLIAL CELL FRACTIONS IN VITRO

Abstract—

• 1 The metabolism of three substrates, [U-¹⁴C]glucose, [U-¹⁴C]pyruvate and [U-¹⁴C]glutamate has been studied in vitro in neuronal and glial cell fractions obtained from rat cerebral cortex by a density gradient technique.
• 2 The mixed cell suspension, after washing, metabolized glucose and glutamate in a manner essentially similar to the tissue slice. Exceptions were a reduced ability to generate lactate from glucose and alanine from glutamate, and a lowered effect of added glucose in suppressing the production of aspartate from glutamate.
• 3 After 2 hr incubation with [U-¹⁴C]glucose, the concentration of the amino acids glutamate, glutamine, GABA, aspartate and alanine were raised in the neuronal, compared to the glial fraction to 234 per cent, 176 per cent, 202 per cent, 167 per cent and 230 per cent respectively although both were lower than in the tissue slice. Incorporation of radio-activity was absolutely lower in the neuronal fraction, however, and the specific activities of the amino acids were: glutamate 12 per cent, GABA 18 per cent, aspartate 34 per cent, and alanine 33 per cent of those in the glial fraction.
• 4 After the incubation with [U-¹⁴C]pyruvate, the pool size of the amino acids were higher than after incubation with glucose, except for GABA, which was reduced to one-third. The concentrations of the amino acids glutamate, glutamine, GABA, aspartate, and alanine in the neuronal fraction were respectively 46 per cent, 143 per cent, 105 per cent, 97 per cent, and 57 per cent of those in the glial. Thus, with the exception of alanine, the specific activity of the neuronal amino acids compared to the glial was little increased when pyruvate replaced glucose as substrate.
• 5 After 2 hr incubation with [U-¹⁴C]glutamate in the presence of non-radioactive glucose, the pool sizes of all the amino acids were increased in both neuronal and glial fractions, with the exception of neuronal alanine and glial glutamine. The concentrations of the amino acids glutamine, GABA, aspartate and alanine were raised in the neuronal fraction, compared to the glial, to 425 per cent, 187 per cent, 222 per cent, and 133 per cent respectively. The specific activities of all the amino acids were higher than with glucose alone with the exception of alanine, and neuronal GABA. Neuronal glutamine and aspartate had specific activities respectively 102 per cent and 84 per cent of glial.
• 6 An unidentified amino acid, with R_F comparable to that of alanine and specific activity close to that of glutamate, was also present after incubation. It was relatively concentrated in the neuronal fraction.
• 7 The distribution of the enzymes glutamate dehydrogenase, aspartate aminotransferase, glutamate decarboxylase and glutamine synthetase between the cell fractions was studied. With the exception of glutamine synthetase, none of the enzymes was lost from the cell fractions during their preparation. Only 14 per cent of the glutamine synthetase, compared with 75 per cent of total protein, was recovered in the fractions. Of the enzymes, glutamate dehydrogenase activity was 406 per cent, and glutamate synthetase activity 177 per cent in the neuronal fraction compared to the glial in the absence of detergent. In the presence of detergent, glutamate dehydrogenase control was 261 per cent, aspartate aminotransferase activity 237 per cent is the neuronal as compared to the glial fraction.
• 8 Incorporation of radioactivity into acid-insoluble material from either glutamate or pyruvate was twice as high into the neuronal as the glial fraction.
• 9 The extent to which these differences may be extrapolated back to the intact tissue is considered, and certain correction factors calculated. The significance of the observations for an understanding of the compartmentation of amino acid pools and metabolism in the brain, and the possible identification of such compartments, is discussed.

     

EFFECT OF INSULIN ON LEVELS AND TURNOVER OF INTERMEDIATES OF BRAIN CARBOHYDRATE METABOLISM IN VIVO

Abstract— –The rates of incorporation of ¹⁴C from [U-^l4C]glucose into intermediary metabolites have been measured in rat brain in vivo. The time course of labelling of glycogen was similar to that of glutamate and of glucose, which were all maximally labelled between 20 and 40min, but different from lactate, which lost radioactivity rapidly after 20min. The extent of labelling of glycogen (d.p.m./ μ mol of glucose) was of the same order as that of glutamate at 20 and 40 min after injection of [¹⁴C]glucose. However, calculations of turnover rates showed that glutamate turns over some 8-10 times faster than glycogen. Insulin, intracisternally applied, produced after 4-5 h a 60 per cent increase in glucose-6-P and a 50 per cent increase in glycogen. There was no change in the levels of glucose, glutamate or lactate, nor in the activity or properties of the particulate and soluble hexokinase of the brain. The injection of insulin affected neither the glycogen nor glucose contents of skeletal muscle from the same animals. The effects of insulin on the incorporation of ^l4C into the metabolites contrasted with its effects on their levels. The specific activities of glycogen and glucose were unchanged and there was a slight but non-significant increase in the specific activity of glutamate. The time course of incorporation into lactate was unaffected up to 20 min, but a significant delay in the loss of ¹⁴C after 20 min occurred as a result of the insulin injection. At 40 min, the specific activity of cerebral lactate was 60 per cent higher in insulin-treated animals than in control animals. The results are interpreted in terms of an effect of insulin on glucose uptake to the brain, with possibly an additional effect on a subsequent stage in metabolism, which involves lactate.     

THE COMPARTMENTATION OF GLUTAMATE AND ITS METABOLITES IN FRACTIONS OF NEURON CELL BODIES AND NEUROPIL; STUDIED BY INTRAVENTRICULAR INJECTION OF [U-14C]GLUTAMATE

Abstract—

• 1 The in vivo metabolism of glutamate in rat neuron cell bodies and neuropil was studied after intraventricular injection of (U-¹⁴C)glutamic acid followed by separation of the tissue into neuronal and neuropil fractions.

• 2 The losses of amino acid and of radioactivity during the fractionation were equivalent. Recoveries were: glutamate, 32; glutamine, 15; aspartate, 25; GABA, 41; alanine, 30 per cent. In the washed cell fractions glutamine was 45 per cent and alanine 132 per cent higher in the neuronal fraction, glutamate was 62, GABA 77 and aspartate 95 per cent of neuropil levels. This contrasted with results obtained previously for in vitro incorporation. Calculation from these results indicated that 28 per cent of the original cell suspension was neuronal, 72 per cent neuropil. In the final cell preparations, 29 per cent of the neuron cell bodies and 26 per cent of the neuropil were recovered.

• 3 Specific activity of glutamate in the neuronal fraction 15 min after injection was higher than in the original suspension, but had declined to 30 per cent of its initial value by 2 h. In the neuropil, specific activity of glutamate was below that of the cell suspension at 15 min, but at later times rose above it by up to 40 per cent.

• 4 Radioactivity was detected in aspartate and glutamine 15 min after injection and GABA by 60 min after injection. In the original cell suspension the specific activity of glutamine was higher than that of glutamate at all times (the Waelsch effect) but aspartate and GABA were lower than glutamate.

• 5 In the neuronal fraction the specific activity of glutamine was below that of glutamate at all times, indicating a precursor-product relationship. In the neuropil fraction, glutamine specific activity remained above glutamate for the first hour.

• 6 These results are discussed in relation to the interpretation of the Waelsch effect in terms of metabolic compartmentation.

     

THE EFFECT OF BREATHING OXYGEN ON THE METABOLISM OF THE RAT BRAIN UNDER NORMAL AND ISCHAEMIC CONDITIONS

Abstract—

• 1 Breathing oxygen (1 atm.) for 2 hr increased the glycogen content of the rat brain from 3·38 to 4·35 μmoles glucosyl residues/g wet wt. At the same time the glucose and lactate concentrations were significantly decreased.
• 2 Both under normal conditions and when breathing oxygen, the sum (glycogen + glucose) × 2 + lactate, with which the balance of carbohydrate breakdown and lactate formation was assessed, was 13·5 μmoles/g wet wt.
• 2 Oxygen breathing effected a significant decrease in this sum after an ischaemic period of 1–15 min. In the control group breathing normal air, the sum, after all periods of ischaemia, ranged from 98 to 106 per cent of the starting value.
• 3 An increased partial pressure of oxygen did not change the breakdown rate of the high-energy phosphate compounds. This result was not consistent with an oxidation of the carbohydrates which were missing in the balance. It is probable that other metabolites, which were not tested for, accumulated.
• 5 0 We failed to find any indication of storage of oxygen which the ischaemic brain could use for oxidative energy production.

     

The effects of undernutrition upon the energy reserve of the brain and upon other selected metabolic intermediates in brains and livers of infant rats

—Rats undernourished from the first to the ninth day of life exhibited no decrease in the energy reserve (P-creatine, ATP, glucose and glycogen) of the brain, although they underwent a 41 per cent decrease in body weight. The apparent increase in the cerebral levels of glucose-6-phosphate and the decreases in hepatic glucose and lactate in the starved animals were probably a consequence of the fact that they froze faster than the control animals rather than of any essential differences in vivo. However, decreases in cerebral glutamate (11 per cent) and hepatic glutamate (33 per cent) in the undernourished animals cannot be explained on this basis. Possible explanations for this decrease in cerebral glutamate content are: a decreased supply of glutamate from the liver, a decreased synthesis of glutamate by the brain, or an increased use of glutamate as an energy source. Since levels of glutamate in the brain increase progressively during the first weeks of life, another interesting possibility is that the lower level of cerebral glutamate in undernourished rats represents a biochemical indicator of a delay in the maturation of specific morphological components which are rich in glutamate and are characteristic of the brain.     

Glycogen, ammonia and related metabolities in the brain during seizures evoked by methionine sulphoximine

Abstract— The levels of ATP, P-creatine, glucose, glycogen, lactate, glutamate and ammonia were measured in mouse brain after administration of the convulsive agent methionine sulphoximine (MSO). No changes were observed in ATP and P-creatine levels either before or during the seizures. Lactate levels were unchanged until the onset of seizures (4–5 hr) at which time the levels increased an average of 65 per cent. Glucose and glycogen levels increased progressively. Just before the onset of seizures the levels had increased 95 and 62 per cent, respectively. During the seizures both substances had increased a total of 130 per cent. Comparable changes were found in cerebral cortex, cerebellum and subcortical forebrain. Through the use of quantitative histochemical methods it was found that the greatest increases in glycogen occurred in layers I and III (layers II and IV were not analysed). Progressively smaller changes were found in layers V and VI and no increase at all was found in the subjacent white matter. Glucose, in contrast to glycogen, increased to about the same degree in all cerebral layers and in subjacent white matter. The increase in glycogen after MSO administration may be related to the fact that MSO also causes an increase in the ratio of brain to serum glucose levels. This would indicate that an increase in intracellular glucose had occurred. Ammonia levels were increased 300–400 per cent in both cerebrum and cerebellum. A time study in cerebellum showed that the increase begins early and reaches maximal levels long before the onset of seizures. Glutamate levels were reduced by small but statistically significant amounts in both cerebrum and cerebellum. Administration of methionine sulphoximine completely prevented seizures and the increase in lactate, but did not prevent the increases in glycogen and glucose. The rise in ammonia was reduced but not prevented. During 20 sec of complete ischaemia (decapitation) ATP, P-creatine and glucose fell somewhat more rapidly than normal in brain of animals undergoing MSO seizures. From the changes it was calculated that the metabolic rate had been increased about 20 per cent by the seizure. A new sensitive and specific enzymic method for determination of tissue ammonia is presented together with evised enzymic procedures for lactate and glutamate.     

Extraction, purification and turnover of rat brain glycogen

Abstract— Glycogen was prepared from rapidly frozen rat brain by the usual techniques and found to contain considerable amounts of non-glycogen carbohydrate. The crude glycogen was partially purified by extraction with hot or cold water and reprecipitation. Enzymic estimation showed that the carbohydrate extracted into hot water contained only 50 per cent of glucose after hydrolysis; of the hot water insoluble material, namely some 30 per cent of the total carbohydrate present in the crude glycogen, less than half of the carbohydrate was released by hydrolysis in 1 M-HC1. The glycogen soluble in hot water incorporated ¹⁴C from [¹⁴C]glucose at considerably higher rates than the residual material and also decreased more rapidly during post-mortem autolysis. Glycogen extracted into cold water was of higher purity than that extracted by hot water; although the material behaved as glycogen during precipitation and re-extraction it contained only 75 per cent of its carbohydrate as glucose. Contaminants included fucose, galactose and hexuronic acid. The rates of metabolism of the partially purified glycogen are compared with published rates; it is suggested that the observed rates are inaccurate due to the impurities present in brain glycogen prepared by classical techniques.     

METABOLISM OF GLUCOSE, AMINO ACIDS, AND SOME RELATED METABOLITES IN THE BRAIN OF TOADS (BUFO BOREAS) ADAPTED TO FRESH WATER OR HYPEROSMOTIC ENVIRONMENTS

The levels and specific radioactivities (SA) of glucose, lactate, pyruvate, α-oxoglutarate and seven amino acids in the brain of toads adapted to fresh water or to an hyperosmotic environment were analysed at various times (5 min–4 h) after an injection of [U-¹⁴C]glucose into the bloodstream. The concentrations and SA of glucose, lactate and five amino acids in blood plasma also were measured. In addition, the SA of glutamine, glutamate, aspartate and GABA in brain were determined 30 min after an injection of [1,5-¹⁴C]citrate into the cisterna magna. The flow of labelled carbon atoms from glucose to amino acids and related metabolites in the toad brain was qualitatively similar to that in the mammalian brain, but quantitatively less than one-tenth of the rate in the brain of rats. Hyperosmotic adaptation induced a large increase in the levels of glucose and amino acids in the brain without affecting the rate of glucose utilization. The SA of several amino acids relative to the SA of glucose were initially lower in hyperosmotically-adapted toads than in toads adapted to fresh water, presumably because of a greater dilution of isotope by the larger amino acid pools in the hyperosmotically-adapted toads. The rates of synthesis of alanine and glutamine from pyruvate and glutamate, respectively, appeared to increase with hyperosmotic adaptation, but the rate of GABA synthesis from glutamate was unaltered. The SA of α-oxoglutarate and glutamate were similar at all time periods in both groups of toads, an indication that these compounds were interconverted much more rapidly than the rate at which α-oxoglutarate was formed from isocitrate. The SA of lactate in comparison to that of glucose varied but was always considerably lower, even at 4 h after the [¹⁴C]glucose injection. After[U-¹⁴C]glucose, glutamine had a SA lower than that of glutamate, whereas after the injection of [1⁴C]citrate, glutamine was formed with a SA much higher than that of glutamate. Hence, glutamate in the toad brain exhibited metabolic compartmentation similar to that in rat brain.     

GLYCOGEN AND GLYCOGEN PHOSPHORYLASE IN THE CEREBRAL CORTEX OF MICE UNDER THE INFLUENCE OF METHIONINE SULPHOXIMINE

Abstract— Glucose and glycogen levels in the mouse cerebral cortex in vivo were studied after recovery from methionine sulphoximine seizures. The animals appeared normal 24 h after methionine sulphoximine administration but both glucose and glycogen still persisted at higher levels 72 h after injection (by 64 and 275 per cent, respectively). When seizures were prevented by methionine, the increase in glucose and glycogen at the longer time intervals was significantly smaller than in animals treated with methionine sulphoximine only; glucose reached normal values at 48 or 72 h; the accumulation of glycogen was reduced by about three to five times, but after 72 h the levels were still significantly higher than in control animals (67 or 32 per cent increase, depending on the administered dose of methionine). In contrast to the considerable accumulation of glycogen after administration of methionine sulphoximine in vivo, it had no effect on the level of glycogen in brain cortex slices in vitro. After 3 h incubation in the absence of methionine sulphoximine, glycogen was resynthesized to a level of about 4 μmol/g wet tissue and this value was not significantly affected by the presence of various concentrations of methionine sulphoximine in the incubation medium (10^-5 to 10^-2 M). The total (a+b forms) phosphorylase activity of mouse cerebral cortex in vivo after methionine sulphoximine administration was not affected. The fraction of active phosphorylase was reduced by about 50 per cent at the time of seizures. When seizures were prevented by methionine, the decrease in active phosphorylase was also completely prevented. In the preconvulsive period (1-2 h) and after recovery from the seizures (48 h after methionine sulphoximine administration) active phosphorylase was normal. The possible mechanisms involved in the increased accumulation of glycogen after methionine sulphoximine administration are discussed.     

Influence of handling and/or anaesthesia on stress response in rainbow trout. Effects on liver primary metabolism

• 1.1. The overall effect of handling, anaesthesia and sham injection on some blood metabolites, liver glycogen and several key enzymes involved in liver carbohydrates and nitrogen metabolism was studied in rainbow trout. In addition, the possible role of anaesthesia (MS222) itself as a stress-inductor or suppressor was also studied.
• 2.2. Stress resulted in hyperglycaemia and initially in liver glycogen depletion, as well as increasing plasma amino acid levels.
• 3.3. Glycogen stores subsequently recovered while amino acid concentration fell.
• 4.4. These changes seemed to correlate with the increased activity of liver fructose 1,6-bisphosphatase, glucose 6-phosphate dehydrogenase, alanine aminotransferase and glutamate dehydrogenase, thus supporting the hypothesis that gluconeogenic flux from amino acids increases in stressed trouts.
• 5.5. Anaesthesia, under the same experimental conditions, did not seem to mediate in stress production, but rather resulted in stress suppression.

     

The effect of time on physiological changes in eel Anguilla anguilla,induced by lindane

• 1.1. Eel were exposed to a sublethal concentration of lindane (0.335 ppm) for 6, 12, 24, 48, 72 and 96 hr.
• 2.2. Concentrations of glycogen, glucose, lactate, pyruvate and lipids were determined in gill tissue after lindane exposure.
• 3.3. Gill glycogen descreased and glucose levels increased at 6 hr of treatment, lactate and pyruvate concentration increased between 6 and 48 hr. Total lipid values decreased between 6 and 24 hr; thereafter, the levels increased up to 72 hr of exposure.
• 4.4. Clear changes were found in all parameters tested in gill tissues. The observed effects of lindane on metabolism in fish are discussed in relation to acute stress syndrome.

     

Poster Sessions BP01: Energy Metabolism. NMR investigation of metabolic alterations in the brain of thiamine‐deficient rats

Thiamine deficiency (TD) results in region‐selective impairment of brain metabolism. Since thiamine is a cofactor for enzymes involved in glucose metabolism, ¹H and ¹³C‐NMR was used to investigate metabolic fluxes through the major pathways of glucose metabolism in vulnerable (medial thalamus, MT; inferior colliculus, IC) and nonvulnerable brain structures of rats made thiamine deficient following treatment with the central thiamine antagonist pyrithiamine vs. pair‐fed controls. Symptomatic stages of TD resulted in decreased glutamate and GABA in MT an IC confirming previous biochemical studies. ¹³C‐isotopomer analysis revealed decreased de novo synthesis of [4–¹³C]glutamate (30%p < 0.02) and [2–¹³C]GABA (60%p < 0.01) in MT and IC consistent with decreased activities of pyruvate‐ and α‐ketoglutarate dehydrogenases. These changes were accompanied by decreased consumption of glucose and increased synthesis of lactate from [1–¹³C]glucose confirming decreased mitochondrial metabolism. Accumulation of glyceraldehyde‐3‐phosphate suggested inhibition of glucose flux through the thiamine‐deficient enzyme transketolase. Onset of symptoms of TD and significant cell death was accompanied by decreased neuronal marker molecules NAA and NAAG in MT. Focal lactate accumulation resulting from decreased activities of mitochondrial thiamine‐dependent enzymes appears to play a key role in the pathogenesis of selective neuronal cell death in TD. [funded by CIHR Canada].     

Antagonistic regulation of the glucose/glucose 6-phosphate cycle by insulin and glucagon in cultured hepatocytes.

Flux through the glucose/glucose 6-phosphate cycle in cultured hepatocytes was measured with radiochemical techniques. Utilization of [2-3H]glucose was taken as a measure of glucokinase flux. Liberation of [14C]glucose from [U-14C]glycogen and from [U-14C]lactate, as well as the difference between the utilization of [2-3H]glucose and of [U-14C]glucose, were taken as measures of glucose-6-phosphatase flux. At constant 5 mM-glucose and 2 mM-lactate concentrations insulin increased glucokinase flux by 35%; it decreased glucose-6-phosphatase flux from glycogen by 50%, from lactate by 15% and reverse flux from external glucose by 65%, i.e. overall by 40%. Glucagon had essentially no effect on glucokinase flux; it enhanced glucose-6-phosphatase flux from glycogen by 700%, from lactate by 45% and reverse flux from external glucose by 20%, i.e. overall by 110%. At constant glucose concentrations cellular glucose 6-phosphate concentrations were essentially not altered by insulin, but were increased by glucagon by 230%. In conclusion, under basic conditions without added hormones the glucose/glucose 6-phosphate cycle showed only a minor net glucose uptake, of 0.03 mumol/min per g of hepatocytes; this flux was increased by insulin to a net glucose uptake of 0.21 mumol/min per g and reversed by glucagon to a net glucose release of 0.22 mumol/min per g. Since the glucose 6-phosphate concentrations after hormone treatment did not correlate with the glucose-6-phosphatase flux, it is suggested that the hormones influenced the enzyme activity directly.     

THE ENZYMIC HYDROLYSIS OF PHOSPHATIDYL INOSITOL BY GUINEA PIG BRAIN:

Abstract—

• 1 Phosphatidylinositol hydrolase activity of homogenates of guinea pig brain was studied by using [2-³H]inositol labelled substrate and measuring the release of radioactivity into the acid soluble fraction.
• 2 Inositol phosphate and diglyceride were found to be the main hydrolysis products. The principal enzyme involved, therefore, is a phosphatidylinositol inositolphosphohydrolase.
• 3 Most of the enzymic activity (61 per cent) was found in the soluble fraction. Osmotic shock of the high speed particulate fraction resulted in release of an additional 23 1 per cent into the soluble fraction. However, as contrasted to lactate dehydrogenase, significant activity remained particulate bound.

     

The metabolism of glucose in diaphragm muscle from normal rats, from streptozotocin-treated diabetic rats and from rats treated with anti-insulin serum

1. The metabolism of [U-¹⁴C]glucose by the isolated diaphragm muscle of normal rats, rats rendered diabetic with streptozotocin and rats with transitory insulin deficiency after an injection of anti-insulin serum was studied. 2. The incorporation of [¹⁴C]glucose into glycogen and oligosaccharides was significantly decreased in the diabetic diaphragm muscle and in the muscle from rats treated with anti-insulin serum. 3. Neither diabetes nor transitory insulin deficiency influenced the oxidation of glucose, or the formation of lactate and hexose phosphate esters from glucose. 4. Insulin fully restored the incorporation of glucose into glycogen and maltotetraose in the diabetic muscle, but the incorporation into oligosaccharides, although increased in the presence of insulin, was significantly lower than the values obtained with normal diaphragm in the presence of insulin.     

On-line optimization of glutamate production based on balanced metabolic control by RQ

In glutamate fermentations by Corynebacterium glutamicum, higher glutamate concentration could be achieved by constantly controlling dissolved oxygen concentration (DO) at a lower level; however, by-product lactate also severely accumulated. The results of analyzing activities changes of the two key enzymes, glutamate and lactate dehydrogenases involved with the fermentation, and the entire metabolic network flux analysis showed that the lactate overproduction was because the metabolic flux in TCA cycle was too low to balance the glucose glycolysis rate. As a result, the respiratory quotient (RQ) adaptive control based “balanced metabolic control” (BMC) strategy was proposed and used to regulate the TCA metabolic flux rate at an appropriate level to achieve the metabolic balance among glycolysis, glutamate synthesis, and TCA metabolic flux. Compared with the best results of various DO constant controls, the BMC strategy increased the maximal glutamate concentration by about 15% and almost completely repressed the lactate accumulation with competitively high glutamate productivity.     

Glutamine metabolism in AS-30D hepatoma cells. Evidence for its conversion into lipids via reductive carboxylation

A study was undertaken to assess the role of a physiological concentration of glutamine in AS-30D cell metabolism. Flux of¹⁴C-glutamine to¹⁴CO₂ and of¹⁴C-acetate to glutamate was detected indicating reversible flux between glutamate and TCA cycle -ketoglutarate. These fluxes were transaminase dependent. A flux analysis was compared using data from three tracers that label -ketoglutarate carbon 5, [2-¹⁴C]glucose, [1-¹⁴C]acetate and [5-¹⁴C]glutamine. The analysis indicated that the probability of flux of TCA cycle -ketoglutarate to glutamate was, at minimum, only slightly less than the probability of flux of -ketoglutarate through -ketoglutarate dehydrogenase. The apparent Km for oxidative flux of [¹⁴C]glutamine to¹⁴CO₂, 0.07 mM, indicated that this flux was at a maximal rate at physiological, 0.75 mM, glutamine. Although oxidative flux through -ketoglutarate dehydrogenase was the major fate of glutamine, flux of glutamine to lipid via reductive carboxylation of -ketoglutarate was demonstrated by measuring incorporation of [5-¹⁴C]glutamine into¹⁴C-lipid. In media containing glucose (6 mM), and glutamine (0.75 mM) 47 per cent of the lipid synthesized from substrates in the media was derived from glutamine via reductive carboxylation and 49 per cent from glucose. These findings of nearly equal fluxes suggest that lipogenesis via reductive carboxylation may be an important role of glutamine in hepatoma cells.     

Relation between energy production and adenine nucleotide metabolism in human blood platelets

The relation between ATP production and adenine nucleotide metabolism was investigated in human platelets which were starved by incubation in glucose-free, CN^?-containing medium and subsequently incubated with different amounts of glucose. In the absence of mitochondrial energy production (blocked by CN^?) and glycogen catabolism (glycogen almost completely consumed during starvation), lactate production increased proportionally with increasing amounts of glucose. The generated ATP was almost completely consumed in the various ATP-consuming processes in the cell except for a fixed portion (about 7%) that was reserved for restoration of the adenylate energy charge. During the first 10 min after glucose addition, the adenine nucleotide pool remained constant. Thereafter, when the glycolytic flux, measured as lactate formation, was more than 3.5 μmol · min^?1 · 10^?11 cells, the pool increased slightly by resynthesis from hypoxanthine-inosine and then stabilized; at a lower flux the pool decreased and metabolic ATP and energy charge declined to values found during starvation. Between moments of rising and falling adenylate energy charges, periods of about 10 min remained in which the charge was constant and ATP supply and demand had reached equilibrium. This enabled comparison between the adenylate energy charge and ATP regeneration velocity. A linear relation was obtained for charge values between 0.4 and 0.85 and ATP regeneration rates between 0.6 and 3.5 ATP equiv. · min^?1 · 10^?11 cells. These data indicate that in starved platelets ATP regeneration velocity and energy charge are independent and that each appears to be subject to the availability of extracellular substrate.