Substrate utilization by Campylobacter jejuni and Campylobacter coli

An attempt was made to elucidate in Campylobacter spp. some of the physiologic characteristics that are reflected in the kinetics of CO2 formation from four 14C-labeled substrates. Campylobacter jejuni and C. coli were grown in a biphasic medium, and highly motile spiral cells were harvested at 12 h. Of the media evaluated for use in the metabolic tests, minimal essential medium without glutamine, diluted with an equal volume of potassium sodium phosphate buffer (pH 7.2), provided the greatest stability and least competition with the substrates to be tested. The cells were incubated with 0.02 M glutamate, glutamine, alpha-ketoglutarate, or formate, or with concentrations of these substrates ranging from 0.0032 to 0.125 M. All four substrates were metabolized very rapidly by both species. A feature of many of these reactions, particularly obvious with alpha-ketoglutarate, was an immediate burst of CO2 production followed by CO2 evolution at a more moderate rate. These diphasic kinetics of substrate utilization were not seen in comparable experiments with Escherichia coli grown and tested under identical conditions. With C. jejuni, CO2 production from formate proceeded rapidly for the entire period of incubation. The rate of metabolism of glutamate, glutamine, and alpha-ketoglutarate by both species was greatly enhanced by increased substrate concentration. The approach to the study of the metabolism of campylobacters here described may be useful in detecting subtle changes in the physiology of cells as they are maintained past their logarithmic growth phase.     

Substrate utilization by Campylobacter jejuni and Campylobacter coli.

An attempt was made to elucidate in Campylobacter spp. some of the physiologic characteristics that are reflected in the kinetics of CO2 formation from four 14C-labeled substrates. Campylobacter jejuni and C. coli were grown in a biphasic medium, and highly motile spiral cells were harvested at 12 h. Of the media evaluated for use in the metabolic tests, minimal essential medium without glutamine, diluted with an equal volume of potassium sodium phosphate buffer (pH 7.2), provided the greatest stability and least competition with the substrates to be tested. The cells were incubated with 0.02 M glutamate, glutamine, alpha-ketoglutarate, or formate, or with concentrations of these substrates ranging from 0.0032 to 0.125 M. All four substrates were metabolized very rapidly by both species. A feature of many of these reactions, particularly obvious with alpha-ketoglutarate, was an immediate burst of CO2 production followed by CO2 evolution at a more moderate rate. These diphasic kinetics of substrate utilization were not seen in comparable experiments with Escherichia coli grown and tested under identical conditions. With C. jejuni, CO2 production from formate proceeded rapidly for the entire period of incubation. The rate of metabolism of glutamate, glutamine, and alpha-ketoglutarate by both species was greatly enhanced by increased substrate concentration. The approach to the study of the metabolism of campylobacters here described may be useful in detecting subtle changes in the physiology of cells as they are maintained past their logarithmic growth phase.     

The influence of diabetes on glutamate metabolism in retinas

Excised retinas from euglycemic and diabetic Sprague-Dawley rats were studied to evaluate differences in glutamate metabolism related to diabetes. Reports suggest, neuronal cell death possibly caused by glutamate excitotoxicity, is an early consequence of diabetes. To monitor the influence of diabetes on glutamate metabolism, we measured glutamatergic neurotransmission, anaplerotic glutamate synthesis from (14) CO(2) and pyruvate as well as rates of glutamate cataplerosis ([U-(14) C]glutamate to (14) CO(2) and (14) C-pyruvate). The data suggest the presence of a glutamate buffering anaplerotic/cataplerotic metabolic cycle in controls which is uncoupled by diabetes. For cycle operation, anaplerosis is initiated by a small pyruvate pool which is also the product of cataplerosis. In the cataplerotic pathway, glutamate conversion to α-ketoglutarate and then to CO(2) and pyruvate is reduced by 90% in diabetic retinal Müller cells because glutamate transamination by branched chain aminotransferase is competitively inhibited by branched chain amino acids (BCAAs). BCAAs, but not the ketoacids, were almost twice as high in diabetic compared to euglycemic rat retinas. The data suggest the hypothesis that glutamate levels in retinal Müller cells from diabetic rats are elevated because of the presence of excess BCAAs, and that elevated glutamate in Müller cells causes glutamate excitotoxicity.     

Hepatocyte heterogeneity in glutamate metabolism and bidirectional transport in perfused rat liver

1. The metabolic fate of infused [1-14C]glutamate was studied in perfused rat liver. The 14C label taken up by the liver was recovered to 85 +/- 2% as 14CO2 and [14C]glutamine. Whereas 14CO2 production accounted for about 70% of the [1-14C]glutamate taken up under conditions of low endogenous rates of glutamine synthesis, stepwise stimulation of glutamine synthesis by NH4Cl increased 14C incorporation into glutamine at the expense of 14CO2 production. Extrapolation to maximal rates of hepatic glutamine synthesis yielded an about 100% utilization of vascular glutamate taken up by the liver for glutamine synthesis. This was observed in both, antegrade and retrograde perfusions and suggests an almost exclusive uptake of glutamate into perivenous glutamine-synthetase-containing hepatocytes. 2. Glutamate was simultaneously taken up and released from perfused rat liver. At a near-physiological influent glutamate concentration (0.1 mM), the rates of unidirectional glutamate influx and efflux were similar (about 100 and 120 nmol g-1 min-1, respectively). 3. During infusion of [1-14C]oxoglutarate (50 microM), addition of glutamate (2 mM) did not affect hepatic uptake of [1-14C]oxoglutarate. However, it increased labeled glutamate release from the liver about 10-fold (from 9 +/- 2 to 86 +/- 20 nmol g-1 min-1; n = 4), whereas 14CO2 production from labeled oxoglutarate decreased by about 40%. This suggests not only different mechanisms of oxoglutarate and glutamate transport across the plasma membrane, but also points to a glutamate/glutamate exchange. 4. Oxoglutarate was recently shown to be taken up almost exclusively by perivenous glutamine-synthetase-containing hepatocytes [Stoll, B & H?ussinger, D. (1989) Eur. J. Biochem. 181, 709-716] and [1-14C]oxoglutarate (9 microM) was used to label selectively the intracellular glutamate pool in this perivenous cell population. The specific radioactivity of this intracellular (perivenous) glutamate pool was assessed by measuring the specific radioactivity of newly synthesized glutamine which is continuously released from these cells into the perfusate. Comparison of the specific radioactivities of glutamine and glutamate released from perivenous cells indicates that about 60% of total glutamate release from the liver is derived from the perivenous glutamine-synthetase-containing cell population. Following addition of unlabeled glutamate (0.1 mM), unidirectional glutamate efflux from perivenous cells increased from about 30 to 80 nmol g-1 min-1, whereas glutamate efflux from non-perivenous (presumably periportal) hepatocytes remained largely unaltered (i.e. 20-30 nmol g-1 min-1). 5. It is concluded that, in the intact liver, vascular glutamate is almost exclusively taken up by the small perivenous hepatocyte population containing glutamine synthetase.     

Hepatocyte heterogeneity in glutamate uptake by isolated perfused rat liver

Glutamate is simultaneously taken up and released by perfused rat liver, as shown by 14CO2 production from [1-14C]glutamate in the presence of a net glutamate release by the liver, turning to a net glutamate uptake at portal glutamate concentrations above 0.3 mM. 14CO2 production from portal [1-14C]glutamate is decreased by about 60% in the presence of ammonium ions. This effect is not observed during inhibition of glutamine synthetase by methionine sulfoximine. 14CO2 production from [1-14C]glutamate is not influenced by glutamine. Also, when glutamate accumulates intracellularly during the metabolism of glutamine (added at high concentrations, 5 mM), 14CO2 production from [1-14C]glutamate is not affected. If labeled glutamate is generated intracellularly from added [U-14C]proline, stimulation of glutamine synthesis by ammonium ions did not affect 14CO2 production from [U-14C]proline. After induction of a perivenous liver cell necrosis by CCL4, i.e. conditions associated with an almost complete loss of perivenous glutamine synthesis but no effect on periportal urea synthesis, 14CO2 production from [1-14C]glutamate is decreased by about 70%. The results are explained by hepatocyte heterogeneity in glutamate metabolism and indicate a predominant uptake of glutamate (that reaches the liver by the vena portae) by the small perivenous population of glutamine-synthesizing hepatocytes, whereas glutamate production from glutamine or proline is predominantly periportal. In view of the size of the glutamine synthetase-containing hepatocyte pool [Gebhardt, R. and Mecke, D. (1983) EMBO J. 2, 567-570], glutamate transport capacity of these hepatocytes would be about 20-fold higher as compared to other hepatocytes.     

The metabolism of amino acids in the bovine lens. Their oxidation as a source of energy

1. The metabolism by the bovine lens of nine (14)C-labelled l-amino acids was studied. These were: alanine, aspartate, glutamate, leucine, lysine, proline, serine, tyrosine and tryptophan. 2. All were taken up by the tissue and incorporated into protein. 3. Aspartate and glutamate, although poorly taken up, were readily metabolized to CO(2). Radioactivity from glutamate was also found in glutathione, glutamine, proline and ophthalmic acid. Aspartate was converted into glutamate, glutathione, proline, alanine and lactate. 4. Alanine was largely converted into lactate, which was released into the medium, but incorporation of radioactivity into CO(2), glutamate, glutathione, aspartate and lipids also occurred. 5. Radioactivity from leucine was detected in CO(2), lipids, glutamate, glutathione, proline and glutamine. 6. Lysine was only slightly broken down by the bovine lens; radioactivity was observed in CO(2), glutamate, glutathione, proline and two unidentified compounds. 7. Proline was metabolized to glutamate from which CO(2), glutathione and glutamine were formed. Hydroxyproline in the capsule collagen was labelled. 8. Radioactivity from serine was found in CO(2), lipids, glutathione, glycine, cystine, ATP, lactate and three unidentified compounds, one of which was probably taurine. 9. Neither tyrosine nor tryptophan were metabolized by the bovine lens. 10. The ability of the lens to metabolize amino acids was also shown by measurement of NH(3) production: more NH(3) was formed when glucose was absent from the incubation medium. 11. These experiments suggest that oxidation of amino acids is a source of energy for the lens.     

Antioxidative effects of lovastatin in cultured human endothelial cells

The effects of lovastatin on glutathione peroxidase activity, hydrogen peroxide consumption, [3H]cholesterol uptake and [14C]acetate incorporation were investigated in cultured human endothelial cells. Treatment of endothelial cells with lovastatin in a medium without serum for 4 hr significantly increased both glutathione peroxidase activity and hydrogen peroxide consumption. This treatment also significantly inhibited cholesterol synthesis and cholesterol esterification. However, lovastatin stimulated cholesterol uptake by the cells. These alterations produced by lovastatin continued up to 24 hr. When serum was present in the culture medium, only decreased cholesterol synthesis and esterification were detected. We suggest that the in vitro antioxidative ability of lovastatin resulted, in part at least, from its activating effect on glutathione peroxidase, its stimulative effect on the ability of endothelial cell to scavenge H(2)O(2), and its hypolipidemic effect.     

The effects in vitro of hypoglycaemia and recovery from anoxia on synaptosomal metabolism.

Synaptosomes from several regions of the rat brain were found to exhibit half-maximal rates of 14CO2 output and [14C]acetylcholine synthesis from D-[U-14C]glucose at glucose concentrations approx. 50-fold lower than those required by the brain in situ. However, synaptosomal acetylcholine synthesis was found not to be directly proportional to substrate oxidation as measured by 14CO2 output. When synaptosomes had been exposed to anoxia in vitro, their metabolic indices (14CO2 and [14C]acetylcholine synthesis, and adenine nucleotide levels) were found not to be significantly different from control aerobic values, unless they had been subjected to veratridine depolarization. This is in accord with previous findings that neither the absolute metabolic rates nor the vulnerability to hypoxic damage exhibited by brain in situ is reflected by brain slices in vitro, unless these are stimulated by depolarization. The use of synaptosomes as a model for synaptic damage in vivo is discussed.     

Transamination of glutamate to tricarboxylic acid-cycle intermediates in cultured neurons correlates with the ability of oxo acids to support neuronal survival in vitro.

Cultures of central-nervous-system neurons at low densities require for their survival exogenous pyruvate, alpha-oxoglutarate or oxaloacetate, even in the presence of high glucose concentrations. Most other alpha-oxo acids support cell survival only in the presence of alpha-amino acids which transaminate to alpha-oxoglutarate, oxaloacetate or pyruvate. The alpha-oxo acids therefore operate as acceptors of amino groups from appropriate donors to generate tricarboxylic acid-cycle-relevant substrates, and these alpha-oxo acids provide for neuronal support only insofar as they make it possible for exogenously supplied alpha-amino acid precursors to generate intracellularly one of the three critical metabolites. To examine more closely the relationship between transamination activity and neuronal survival, we measured 14CO2 production from [14C]glutamate in the presence of appropriate alpha-oxo acid partners by using 8-day-embryonic chick forebrain, dorsal-root-ganglion and ciliary-ganglion neurons. Neuronal survival was measured concurrently in monolayer neuronal cultures maintained with the corresponding amino acid/oxo acid pairs. Forebrain and ganglionic cell suspensions both produced 14CO2 from [14C]glutamate, which accurately correlated with 24 h neuronal survival. Concentrations of glutamate or alpha-oxo acid which provide for maximal neuronal survival also produced maximal amounts of 14CO2. The same ability to generate CO2 from glutamate (in the presence of the appropriate alpha-oxo acids) can ensure neuronal survival in 24 h cultures and therefore must meet energy or other metabolic needs of those neurons which glucose itself is unable to satisfy.     

Substrate utilization by Legionella cells after cryopreservation in phosphate buffer

The objective of this study was to evaluate by relatively simple metabolic tests the usefulness of buffers and energy sources commonly used in Legionella growth media. Legionella pneumophila serogroups 1 to 6, Legionella micdadei, and Legionella bozemanii were grown in an enriched charcoal-yeast extract diphasic medium. The cells were washed thrice, suspended in various buffers (pH 6.9) with 1 or 5 mM MgSO4, and used immediately or after controlled-rate cryopreservation. CO2 produced and C incorporated into the cold trichloracetic acid-insoluble fractions from 14C-labeled substrates were determine. Potassium phosphate buffer (0.02 M) was as satisfactory as organic buffers for glutamate metabolism, but the addition of KCl or NaCl reduced activity. Metabolic activity for glutamate was not lost upon cryopreservation, and cryopreserved cells were used to test the utilization of other single or paired substrates. Rates of activity for serine, glutamate, threonine, and pyruvate, in this descending order, were high, and those for alpha-ketoglutarate, succinate, and gamma-aminobutyrate were low. Although glutamine was not used as rapidly as glutamate, when added to glutamate it was preferentially metabolized, possibly because of more rapid transport. When glutamate and serine were combined, glutamate furnished more C for CO2 and less for incorporation, whereas the reverse was true of serine. In conclusion, glutamate as an energy source may in some cases spare other amino acids for synthesis. alpha-Ketoglutarate, a common constituent of Legionella media, may reduce oxygen toxicity but is probably not a chief energy source.     

Substrate utilization by Legionella cells after cryopreservation in phosphate buffer.

The objective of this study was to evaluate by relatively simple metabolic tests the usefulness of buffers and energy sources commonly used in Legionella growth media. Legionella pneumophila serogroups 1 to 6, Legionella micdadei, and Legionella bozemanii were grown in an enriched charcoal-yeast extract diphasic medium. The cells were washed thrice, suspended in various buffers (pH 6.9) with 1 or 5 mM MgSO4, and used immediately or after controlled-rate cryopreservation. CO2 produced and C incorporated into the cold trichloracetic acid-insoluble fractions from 14C-labeled substrates were determine. Potassium phosphate buffer (0.02 M) was as satisfactory as organic buffers for glutamate metabolism, but the addition of KCl or NaCl reduced activity. Metabolic activity for glutamate was not lost upon cryopreservation, and cryopreserved cells were used to test the utilization of other single or paired substrates. Rates of activity for serine, glutamate, threonine, and pyruvate, in this descending order, were high, and those for alpha-ketoglutarate, succinate, and gamma-aminobutyrate were low. Although glutamine was not used as rapidly as glutamate, when added to glutamate it was preferentially metabolized, possibly because of more rapid transport. When glutamate and serine were combined, glutamate furnished more C for CO2 and less for incorporation, whereas the reverse was true of serine. In conclusion, glutamate as an energy source may in some cases spare other amino acids for synthesis. alpha-Ketoglutarate, a common constituent of Legionella media, may reduce oxygen toxicity but is probably not a chief energy source.     

Processing of bisphenol A by plant tissues: glucosylation by cultured BY-2 cells and glucosylation/translocation by plants of Nicotiana tabacum

Bisphenol A (BPA, 4,4'-isopropylidenediphenol), an endocrine disrupter with estrogenic properties, was supplied to tobacco BY-2 cells in suspension culture and the chemical nature of its metabolites was investigated. The concentration of BPA in the culture medium decreased rapidly and became undetectable at 2.5 h after the application. Four metabolites of BPA were observed in a methanol extract of the cells when the culture was supplemented with [(14)C]BPA. The most abundant metabolite was determined to be 4,4'-isopropylidenediphenol-O-beta-D-glucopyranoside (BPAG) by mass spectrometry, nuclear magnetic resonance spectroscopy and by hydrolysis with beta-glucosidase. This identification was confirmed by synthesis. When [(14)C]BPA was administrated to tobacco seedlings from their roots, radioactivity was incorporated in BPAG and three unidentified metabolites. These metabolites were accumulated in the leaves after 4 h exposure, indicating that tobacco seedlings absorbed BPA through their root systems, metabolized to its beta-glucoside and translocated the metabolites to their leaves.     

Hormonal regulation of the alpha-ketoglutarate dehydrogenase complex in the isolated perfused rat liver

The metabolic flux through the alpha-ketoglutarate dehydrogenase reaction in perfused livers was monitored by measuring the rate of 14CO2 production from [1-14C]alpha-ketoglutarate. The rates of 14CO2 production and glucose production from [1-14C]alpha-ketoglutarate were increased with increasing perfusate alpha-ketoglutarate concentrations. Vasopressin, angiotensin II, and the alpha 1-adrenergic agonist phenylephrine stimulated transiently by 2.5-fold the metabolic flux through the alpha-ketoglutarate dehydrogenase reaction in the presence and absence of Ca2+ in the perfusion medium. High concentrations of glucagon (1 x 10(-8) M) and 8-p-chlorophenylthio-cAMP (100 microM) (data not shown) also stimulated transiently the metabolic flux through the alpha-ketoglutarate dehydrogenase reaction. However, lower glucagon concentrations (1 x 10(-9) M) stimulated the rate of 14CO2 production from [1-14C]alpha-ketoglutarate only under conditions optimized to fix the cellular oxidation-reduction state at an intermediate level, when glucagon (1 x 10(-9) M)-mediated elevation of cAMP content was greater than that observed under highly oxidizing and reducing conditions. These data indicate that agonists which increase cytosolic free Ca2+ levels stimulate the metabolic flux through the alpha-ketoglutarate dehydrogenase complex. Furthermore, the data presented here demonstrate for the first time that physiological glucagon concentrations stimulate the metabolic flux through the alpha-ketoglutarate dehydrogenase reaction only under conditions known to be optimal for glucagon-mediated Ca2+ mobilization in the isolated perfused rat liver.     

Metabolic Fate of 14C-Labeled Glutamate in Astrocytes in Primary Cultures

The metabolic fate of L-[U-14C]- and L-[1-14C]glutamate was studied in primary cultures of mouse astrocytes. Conversion of the uniformly labeled compound to glutamine and aspartate was followed by determination of specific activities after dansylation with [3H]dansyl chloride and subsequent thin layer chromatography of the dansylated amino acids. Metabolic fluxes were calculated from the alterations of specific activities and the pool sizes, which were likewise measured by a dansylation method. Formation of 14CO2 from [1-14C]glutamate was determined by the trapping of CO2 in hyamine hydroxide in a gas-tight chamber, which is, in the known absence of glutamate decarboxylase activity in the cultured astrocytes, an unequivocal expression of the metabolic flux via alpha-ketoglutarate to CO2 and succinyl-CoA. The metabolic fluxes determined by these procedures amounted to 2.4 nmol/min/mg protein for glutamine synthesis, 1.1 nmol/min/mg protein for aspartate production, and 4.1 nmol/min/mg protein for formation and subsequent decarboxylation of alpha-ketoglutarate. The latter process was unaffected by virtually complete inhibition of glutamate-oxaloacetic transaminase with aminooxyacetic acid, indicating that the formation of alpha-ketoglutarate occurs as an oxidative deamination rather than as a transamination. This suggests that the formation of alpha-ketoglutarate from glutamate represents a net degradation, not an isotopic exchange.     

Carbon dioxide production by red skeletal muscle of goldfish (Carassius auratus L.) aerobic and anaerobic metabolism of glucose and glutamate

Oxygen uptake and metabolic CO2 production by lateral red muscle of goldfish have been measured in vitro. Added glucose 6-phosphate depresses the rate of oxygen uptake by minced red muscle (Crabtree effect). Total CO2 production is stimulated resulting in a respiratory quotient which is considerably greater than one. 14CO2 release from [U-14C] glucose 6-phosphate and [U-14C] glutamate continues during anoxia. No activity of the hexose monophosphate shunt was observed. The results suggest that both aerobic and anaerobic CO2 production is of mitochondrial origin and, at least partially, derived from TCA cycle reactions.     

Metabolism, Macromolecular Synthesis, and Nuclear Behavior of Cryptococcus albidus at 37 C

The temperature-sensitive events which prevent Cryptococcus albidus from growing at 37 C were investigated. Cultures incubated at 37 C immediately after inoculation did not increase in optical density nor in cell numbers, and by 24 h 90% of cells in such cultures were deformed and dead. When cultures in log phase were shifted from 23 to 37 C the optical density increased but the cell numbers did not. Morphological observations revealed that the increase in turbidity at 37 C represented enlargement and distortion of cells without appreciable replication. Uptake and incorporation of (14)C-leucine were similar at 23 and 37 C. There was no difference in (14)CO(2) evolution from cells at either temperature. Uptake and incorporation of adenine-8-(14)C into RNA was slightly lower in cells incubated at 37 C. There was, however, a 60% reduction in incorporation of adenine-8-(14)C into DNA after 3 hr at 37 C. Nuclear staining revealed that nuclear migration did not occur in cells incubated at 37 C. Thus the data indicate that both adenine incorporation into DNA and nuclear migration prior to nuclear division by C. albidus are temperature sensitive.     

Influence of Gas Environment on Catabolic Activities and on Reoxidation of Reduced Nicotinamide Adenine Dinucleotide Phosphate in Chlamydia

We investigated the effect of the gas environment on the enzymatic reactions of intact isolated cells of the agents of trachoma and of meningopneumonitis of the host-dependent genus Chlamydia. In comparison with the reactions taking place in a gas phase of air, O(2) depressed CO(2) production from pyruvate and glutamate by trachoma and from glutamate by meningopneumonitis. O(2) enhanced the degradation of pyruvate by meningopneumonitis, but this effect was due to increased H(2)O(2), and was reversed by added catalase. Both dehydrogenation of alpha-ketoglutarate and was reversed by added catalase. Dehydrogenation of alpha-ketoglutarate by both agents and production of CO(2) from C(1) of glucose-6-phosphate were stimulated by O(2) and depressed in N(2). The latter activity was stimulated in air, O(2), and N(2) by nicotinamide adenine dinucleotide phosphate (NADP) in relation to the amount added, and also in air or O(2), but not in N(2), by moderate amounts of NADP and an excess of oxidized glutathione with concomitant formation of H(2)O(2). A small but significant amount of O(2) was consumed during the course of these reactions. It is suggested that glutathione reductase activity can occur only when accompanied by an oxidative reaction, and that this close link between the two reactions represents a mechanism of electron transport which transfers hydrogen to molecular O(2).     

Metabolism of phytol-U-14C and phytanic acid-U-14C in the rat

The metabolism of uniformly-labeled (14)C-phytol, (14)C-phytenic acid, and (14)C-phytanic acid was studied in the rat. Conversion of both phytol and phytenic acid to phytanic acid was demonstrated. Tracer doses of phytol-U-(14)C given orally were well absorbed (30-66%), and approximately 30% of the absorbed dose was converted to (14)CO(2) in 18 hr. After intravenous injection, 20% appeared in (14)CO(2) in 4 hr. Phytanic acid-U-(14)C given intravenously was oxidized at a comparable rate (22-37% in 4 hr) and was as rapidly oxidized as palmitic acid-1-(14)C (21% in 4 hr). Metabolism of these substrates was also studied in rats previously maintained on a diet containing 5% phytol by weight, which causes accumulation of phytanic acid, phytenic acid, and, to a lesser extent, phytol in blood and tissues. Despite the large body pools of preformed, unlabeled substrate in these animals, the fraction of an administered dose of phytol-U-(14)C or phytanic acid-U-(14)C converted to (14)CO(2) was not significantly diminished. These studies indicate that the rat has an appreciable capacity to degrade the highly branched carbon skeleton of phytol and its derivatives. Twenty-four hours after administration of phytol-U-(14)C, the lipid radioactivity remaining in the body was widely distributed among the tissues, highest concentrations being found in liver and adipose tissue. Four hours after intravenous administration of phytanic acid-U-(14)C, all of the major lipid classes in the liver contained radioactivity, most in triglycerides and phospholipids and least in cholesterol esters and lower glycerides. There was no demonstrable incorporation of mevalonate-2-(14)C or acetate-1-(14)C into liver phytanic acid when they were given intravenously to a rat previously fed phytol. Endogenous biosynthesis, if it occurs at all, must be extremely limited.     

Effects of insulin on the pattern of glucose metabolism in the perfused working and Lagendorff heart of normal and insulin-deficient rats

1. The metabolic pattern of [U-(14)C]glucose in the isolated rat heart has been studied, with both retrograde aortic (Langendorff) and atrially (working) perfused preparations in the presence and absence of insulin, in normal animals, animals rendered insulin-deficient (by injection of anti-insulin serum 1hr. before excision of the heart) and animals rendered diabetic by streptozotocin injection 7 days before use. 2. Radioautochromatograms of heart extracts show that the pattern of glucose metabolism in heart muscle is more complex than in diaphragm muscle. In addition to (14)CO(2), glycogen, oligosaccharides, phosphorylated sugars and lactate (the main metabolites formed from [(14)C]glucose in diaphragm muscle), (14)C label from [(14)C]glucose appears in heart muscle in glutamate, glutamine, aspartate and alanine, and in tricarboxylic acid-cycle intermediates. 3. By a quantitative scanning technique of two-dimensional chromatograms it was found that a mechanical work load stimulates glucose metabolism, increasing by a factor of 2-3 incorporation of (14)C into all the metabolites mentioned above except lactate and phosphorylated sugars, into which (14)C incorporation is in fact diminished; (14)CO(2) production is equally stimulated. 4. Addition of insulin to the perfusion fluid of the working heart causes increases in (14)C incorporation, by a factor of about 1.5 into (14)CO(2), by a factor of about 3-5 into glycogen, lactate and phosphorylated sugars, by a factor of about 2-3 into glutamate and tricarboxylic acid-cycle intermediates and by a factor of about 0.5 into aspartate, whereas incorporation into alanine and glutamine is not affected. The effect of a work load on the pattern of glucose metabolism is thus different from that of insulin. 5. Increasing the concentration of glucose in the perfusion fluid from 1 to 20mm leads to changes of the pattern of glucose metabolism different from that brought about by insulin. (14)CO(2) production steadily increases whereas [(14)C]lactate and glycogen production levels off at 10mm-glucose, at values well below those reached in the presence of insulin. 6. In Langendorff hearts of animals rendered insulin-deficient by anti-insulin serum or streptozotocin, glucose uptake, formation of (14)CO(2) and [(14)C]lactate, and (14)C incorporation into glycogen and oligosaccharides are decreased. In insulin-deficient working hearts, however, glucose uptake and (14)CO(2) production are normal, whereas incorporation of (14)C into glycogen and [(14)C]lactate production are greatly decreased. 7. Insulin added to the perfusion fluid restores (14)C incorporation from glucose into (14)CO(2), glycogen and lactate in the Langendorff heart from animals rendered insulin-deficient by anti-insulin serum; in hearts from streptozotocin-diabetic animals addition of insulin restores (14)C incorporation into glycogen and lactate, but (14)CO(2) production remains about 50% below normal. 8. The bearing of these results on the problem of the mode of action of insulin is discussed.     

Regulation of hepatic glutamate metabolism. Role of 2-oxoacids in glutamate release from isolated perfused rat liver

In isolated perfused rat liver, addition of the oxoanalogues of leucine, isoleucine, methionine and phenylalanine is followed by a rapid and reversible stimulation of glutamate release. This is not observed with the corresponding amino acids or 2-oxoisovalerate, 2-oxoglutarate or oxaloacetate. The increased glutamate release by the liver is accompanied by a decrease in the tissue contents of 2-oxoglutarate and glutamate by about 25% and 50%, respectively. During the metabolism of glutamine, i.e. conditions with elevated tissue glutamate concentrations, 2-oxoacid-induced glutamate release is stimulated. In the presence of glutamine (5 mM), 2-oxoisocaproate, 2-oxo-4-methylvalerate and 2-oxo-4-methylthiobutyrate were found to be most effective and glutamate release by the liver increased linearly from about 80 nmol g-1 min-1 to 600 nmol g-1 min-1 at increasing 2-oxoacid concentrations up to 1 mM. When glutamate tissue levels were decreased by phenylephrine, stimulation of glutamate release by 2-oxoisocaproate was markedly diminished. 2-Oxoacid-stimulated glutamate release is independent of oxoacid metabolism, indicating that the effect is probably not explained by a 2-oxoacid/glutamate exchange across the liver plasma membrane. 2-Oxoacid-induced glutamate export predominantly occurs in a sodium-independent way. At low concentrations of 2-oxoisocaproate (below 0.2 mM), the increased glutamate release was accompanied by a slight inhibition of 14CO2 production from added [14C]glutamate, indicating a simultaneous glutamate uptake and release also under these conditions. Stimulation of glutamate release by 2-oxoisocaproate is followed by a decreased rate of urea and glutamine synthesis from portal ammonia, as a consequence of an increased glutamate release.