Functional hepatocyte heterogeneity. Vascular 2-oxoglutarate is almost exclusively taken up by perivenous, glutamine-synthetase-containing hepatocytes

1. In isolated perfused rat liver maximal rates of 2-[1-14C]oxoglutarate uptake were about 0.4 mumol.g-1 .min-1; half-maximal rates of 2-[14C]oxoglutarate uptake were observed with influent concentrations of about 100 microM. 2-[14C]Oxoglutarate uptake by the liver was not affected by the direction of perfusion, but was decreased by about 80-90% when Na+ in the perfusion fluid was substituted by choline+, suggesting a Na+-dependence of hepatic 2-oxoglutarate uptake. In the absence of added ammonia, [14C]oxoglutarate uptake by the liver was about twice the net oxoglutarate uptake, indicating a simultaneous release of unlabeled oxoglutarate from perfused rat liver. 2. 14C-Labeled metabolites derived from [1-14C]oxoglutarate and recovered in the effluent perfusate were 14CO2 and 14C-labeled glutamate and glutamine; they accounted for 85-100% of the radiolabel taken up by the liver. 14CO2 was the major product (more than 70%) from [1-14C]oxoglutarate taken up the liver, provided glutamine synthesis was either inhibited by methionine sulfoximine or the endogenous rate of glutamine production was below 40 nmol.g-1.min-1. 3. Stimulation of glutamine synthesis by ammonia did not affect [14C]oxoglutarate uptake by the liver, but considerably increased net hepatic oxoglutarate uptake, indicating a decreased release of unlabeled oxoglutarate from the liver. Stepwise stimulation of hepatic glutamine synthesis led to a gradual decrease of 14CO2 production and radiolabel was recovered increasingly as [14C]glutamine in the effluent. At high rates of glutamine formation (i.e. about 0.6 mumol.g-1.min-1), about 60% of the [1-14C]oxoglutarate taken up by the liver was recovered in the effluent as [14C]glutamine. 14CO2 and [14C]glutamine production from added [1-14C]oxoglutarate were dependent on the rate of hepatic glutamine synthesis but not on the direction of perfusion. Extrapolation of 14C incorporation into glutamine to maximal rates of hepatic glutamine synthesis yielded an about 100% utilization of the [14C]oxoglutarate taken up by the liver for glutamine synthesis. This was again true for both the antegrade and the retrograde perfusion directions. On the other hand, addition of ammonia did not affect 14CO2 production from labeled oxoglutarate, when glutamine synthetase was inhibited by methionine sulfoximine. 4. The data suggest that vascular oxoglutarate is almost exclusively taken up by the small perivenous hepatocyte population containing glutamine synthetase, i.e. a cell population comprising only 6-7% of all hepatocytes. Thus, the findings demonstrate the existence of a, to date, uniquely zonally distributed oxoglutarate transport system which is probably Na+-dependent in the plasma membrane.(ABSTRACT TRUNCATED AT 400 WORDS)     

Urea synthesis and CO2/HCO3- compartmentation in isolated perfused rat liver

With physiological portal HCO3- and CO2 concentrations of 25mM and 1.2mM in the perfusate, respectively, acetazolamide inhibited urea synthesis from NH4Cl in isolated perfused rat liver by 50-60%, whereas urea synthesis from glutamine was inhibited by only 10-15%. A decreased sensitivity of urea synthesis from glutamine to acetazolamide inhibition was also observed when the extracellular HCO3- and CO2 concentrations were varied from 0-50mM and 0-2.4mM, respectively. Stimulation of intramitochondrial CO2 formation at pyruvate dehydrogenase with high pyruvate concentrations (7mM) was without effect on the acetazolamide sensitivity of urea synthesis from NH4Cl. Urea synthesis was studied under conditions of a limiting HCO3- supply for carbamoyl-phosphate synthesis. In the absence of externally added HCO3- or CO2, when 14CO2 was provided intracellularly by [U-14C]glutamine or [1-14C]-glutamine oxidation, acetazolamide had almost no effect on label incorporation into urea, whereas label incorporation from an added tracer H14CO3- dose was inhibited by about 70%. 14CO2 production from [U-14C]glutamine was about twice as high as from [1-14C]glutamine, indicating that about 50% of the CO2 produced from glutamine is formed at 2-oxoglutarate dehydrogenase. The fractional incorporation of 14CO2 into urea was about 13% with [1-14C]-as well as with [U-14C]glutamine. Addition of small concentrations of HCO3- (1.2mM) to the perfusate increased urea synthesis from glutamine by about 70%. This stimulation of urea synthesis was fully abolished by acetazolamide. The carbonate-dehydratase inhibitor prevented the incorporation of added HCO3- into urea, whereas incorporation of CO2 derived from glutamine degradation was unaffected. Without HCO3- and CO2 in the perfusion medium, when 14CO2 was provided by [1-14C]-pyruvate oxidation, acetazolamide inhibited urea synthesis from NH4Cl as well as 14C incorporation into urea by about 50%. Therefore carbonate-dehydratase activity is required for the utilization of extracellular CO2 or pyruvate-dehydrogenase-derived CO2 for urea synthesis, but not for CO2 derived from glutamine oxidation. This is further evidence for a special role of glutamine as substrate for urea synthesis.     

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.     

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.     

Simultaneous synthesis and degradation of glutamine in isolated rat liver cells. Effect of vasopressin

When hepatocytes suspensions obtained from whole livers of 48-h-fasted rats were incubated in Krebs-Henseleit buffer with a near-physiological concentration (1 mM) of L-[1-14C]glutamine as substrate, the apparent removal of glutamine was low, but the release of 14CO2 was much larger than the enzymatically measured removal of glutamine. This indicates that glutamine was metabolized at rates much higher than those accounted for by the apparent removal of glutamine. This also suggests that glutamine utilization was, at least in part, masked by concomitant synthesis of glutamine from endogenous substrates via glutamine synthetase. Evidence that such synthesis occurred was obtained by: (i) addition of methionine sulfoximine, an inhibitor of glutamine synthetase, which caused a large increase in the apparent removal of glutamine; and (ii) measurement of the specific radioactivity of L-[1-14C]glutamine which was shown to decrease during incubation. Addition of vasopressin (10(-7) M) led to a marked increase in glutamine removal by a dual mechanism: it accelerated flux through glutaminase, the enzyme which initiates the hepatic degradation of glutamine, and inhibited flux through glutamine synthetase.     

Alteration in Neuronal-Glial Metabolism of Glutamate by the Neurotoxin Kainic Acid

The effect of the excitotoxin kainic acid on glutamate and glutamine metabolism was studied in cerebellar slices incubated with D-[2-14C]glucose, [U-14C]gamma-aminobutyric acid, [3H]acetate, [U-14C]glutamate, and [U-14C]glutamine as precursors. Kainic acid (1 mM) strongly inhibited the labeling of glutamine relative to that of glutamate from all precursors except [2-14C]glucose and [U-14C]glutamine. Kainic acid did not inhibit glutamine synthetase directly. The data indicate that in the cerebellum kainic acid inhibits the synthesis of glutamine from the small pool of glutamate that is thought to be associated with glial cells. Kainic acid also markedly stimulated the efflux of glutamate from cerebellar slices and this release was not sensitive to tetrodotoxin. Kainic acid stimulated efflux of both glucose- and acetate-labeled glutamate. In contrast, veratridine released glucose-labeled glutamate preferentially via a tetrodotoxin-sensitive mechanism. Kainic acid did not release [U-14C]glutamate from synaptosomal fractions. These results suggest that the bulk of the glutamate released from cerebellar slices by kainic acid comes from nonsynaptic pools.     

Enhanced intestinal synthesis of polyamines from proline in cortisol-treated piglets

This study was conducted to determine a role for cortisol in regulating intestinal ornithine decarboxylase (ODC) activity and to identify the metabolic sources of ornithine for intestinal polyamine synthesis in suckling pigs. Thirty-two 21-day-old suckling pigs were randomly assigned to one of four groups with eight animals each and received daily intramuscular injections of vehicle solution (sesame oil; control), hydrocortisone 21-acetate (HYD; 25 mg/kg body wt), RU-486 (10 mg/kg body wt, a potent blocker of glucocorticoid receptors), or HYD plus RU-486 for two consecutive days. At 29 days of age, pigs were killed for preparation of jejunal enterocytes. The cytosolic fraction was prepared for determining ODC activity. For metabolic studies, enterocytes were incubated for 45 min at 37 degrees C in 2 ml of Krebs-bicarbonate buffer (pH 7.4) containing 1 mM [U-(14)C]arginine, 1 mM [U-(14)C]ornithine, 1 mM [U-(14)C]glutamine, or 1 mM [U-(14)C]proline plus 1 mM glutamine. Cortisol administration increased intestinal ODC activity by 230%, polyamine (putrescine, spermidine, and spermine) synthesis from ornithine and proline by 75-180%, and intracellular polyamine concentrations by 45-83%. Polyamine synthesis from arginine was not detected in enterocytes of control pigs but was induced in cells of cortisol-treated pigs. There was no detectable synthesis of polyamines from glutamine in enterocytes of all groups of pigs. The stimulating effects of cortisol on intestinal ODC activity and polyamine synthesis were abolished by coadministration of RU-486. Our data indicate that an increase in plasma cortisol concentrations stimulates intestinal polyamine synthesis via a glucocorticoid receptor-mediated mechanism and that proline (an abundant amino acid in milk) is a major source of ornithine for intestinal polyamine synthesis in suckling neonates.     

Tricarboxylic acid-cycle metabolism in brain. Effect of fluoroacetate and fluorocitrate on the labelling of glutamate aspartate, glutamine and γ-amino butyrate

1. The effect of fluoroacetate and fluorocitrate on the compartmentation of the glutamate-glutamine system was studied in brain slices with l-[U-(14)C]glutamate, l-[U-(14)C]aspartate, [1-(14)C]acetate and gamma-amino[1-(14)C]butyrate as precursors and in homogenates of brain tissue with [1-(14)C]acetate. The effect of fluoroacetate was also studied in vivo in mouse brain with [1-(14)C]acetate as precursor. 2. Fluoroacetate and fluorocitrate inhibit the labelling of glutamine from all precursors but affect the labelling of glutamate to a much lesser extent. This effect is not due to inhibition of glutamine synthetase. It is interpreted as being due to selective inhibition of the metabolism of a small pool of glutamate that preferentially labels glutamine.     

Arginine synthesis from enteral glutamine in healthy adults in the fed state

Recent studies have documented transfer of labeled nitrogen from [2-(15)N]glutamine to citrulline and arginine in fasting human adults. Conversely, in neonates and piglets we have shown no synthesis of arginine from [2-(15)N]glutamate, and others have shown in mice that glutamine is a nitrogen, but not a carbon donor, for arginine synthesis. Therefore, we performed a multitracer study to determine whether glutamine is a nitrogen and/or carbon donor for arginine in healthy adult men. Two glutamine tracers, 2-(15)N and 1-(13)C, were given enterally to five healthy men fed a standardized milkshake diet. There was no difference in plasma enrichments between the two glutamine tracers. 1-(13)C isotopomers of citrulline and arginine were synthesized from [1-(13)C]glutamine. Three isotopomers each of citrulline and arginine were synthesized from the [2-(15)N]glutamine tracer: 2-(15)N, 5-(15)N, and 2,5-(15)N(2). Significantly greater enrichment was found of both [5-(15)N]arginine (0.75%) and citrulline (3.98%) compared with [2-(15)N]arginine (0.44%) and [2-(15)N]citrulline (2.62%), indicating the amino NH(2) from glutamine is mostly transferred to arginine and citrulline by transamination. Similarly, the enrichment of the 1-(13)C isotopomers was significantly less than the 2-(15)N isotopomers, suggesting rapid formation of α-ketoglutarate and recycling of the nitrogen label. Our results show that the carbon for 50% of newly synthesized arginine comes from dietary glutamine but that glutamine acts primarily as a nitrogen donor for arginine synthesis. Hence, studies using [2-(15)N]glutamine will overestimate arginine synthesis rates.     

Compartmentation of glutamate metabolism in brain. Evidence for the existence of two different tricarboxylic acid cycles in brain

1. (14)C from [1-(14)C]glucose injected intraperitoneally into mice is incorporated into glutamate, aspartate and glutamine in the brain to a much greater extent than (14)C from [2-(14)C]glucose. This difference for [1-(14)C]glucose and [2-(14)C]glucose increases with time. The amount of (14)C in C-1 of glutamate increases steadily with time with both precursors. It is suggested that a large part of the glutamate and aspartate pools in brain are in close contact with intermediates of a fast-turning tricarboxylic acid cycle. 2. (14)C from [1-(14)C]acetate and [2-(14)C]acetate is incorporated to a much larger extent into glutamine than into glutamate. An examination of the time-course of (14)C incorporated into glutamine and glutamate reveals that glutamine is not formed from the glutamate pool, labelled extensively by glucose, but from a small glutamate pool. This small glutamate pool is not derived from an intermediate of a fast-turning tricarboxylic acid cycle. 3. It is proposed that two different tricarboxylic acid cycles exist in brain.     

A cortisol surge mediates the enhanced polyamine synthesis in porcine enterocytes during weaning

This study was conducted to determine whether a cortisol surge mediates the enhanced expression of intestinal ornithine decarboxylase (ODC) in weanling pigs. Piglets were nursed by sows until 21 days of age, when 40 pigs were randomly assigned into one of four groups (10 animals/group). Group 1 continued to be fed by sows, whereas groups 2-4 were weaned to a corn and soybean meal-based diet. Weanling pigs received intramuscular injections of vehicle solvent (sesame oil), RU-486 (a potent blocker of glucocorticoid receptors; 10 mg/kg body wt), and metyrapone (an inhibitor of adrenal cortisol synthesis; 5 mg/kg body wt), respectively, 5 min before weaning and 24 and 72 h later. At 29 days of age, pigs were used to prepare jejunal enterocytes for ODC assay and metabolic studies. To determine polyamine (putrescine, spermidine, and spermine) synthesis, enterocytes were incubated for 45 min at 37 degrees C in 2 ml Krebs-bicarbonate buffer containing 1 mM [U-(14)C]arginine, 1 mM [U-(14)C]ornithine, 1 mM [U-(14)C]glutamine, or 1 mM [U-(14)C]proline plus 1 mM glutamine. Weaning increased intestinal ODC activity by 230% and polyamine synthesis from ornithine, arginine, and proline by 72-157%. Arginine was a quantitatively more important substrate than proline for intestinal polyamine synthesis in weaned pigs. Administration of RU-486 or metyrapone to weanling pigs prevented the increases in intestinal ODC activity and polyamine synthesis, reduced intracellular polyamine concentrations, and decreased villus heights and intestinal growth. Our results demonstrate an essential role for a cortisol surge in enhancing intestinal polyamine synthesis during weaning, which may be of physiological importance for intestinal adaptation and remodeling.     

Corticosteroids increase glutamine utilization in human splanchnic bed

Glutamine is the most abundant amino acid in the body and is extensively taken up in gut and liver in healthy humans. To determine whether glucocorticosteroids alter splanchnic glutamine metabolism, the effect of prednisone was assessed in healthy volunteers using isotope tracer methods. Two groups of healthy adults received 5-h intravenous infusions of l-[1-(14)C]leucine and l-[(2)H(5)]glutamine, along with q. 20 min oral sips of tracer doses of l-[1-(13)C]glutamine in the fasting state, either 1) at baseline (control group; n = 6) or 2) after a 6-day course of 0.8 mg.kg(-1).day(-1) prednisone (prednisone group; n = 8). Leucine and glutamine appearance rates (Ra) were determined from plasma [1-(14)C]ketoisocaproate and [(2)H(5)]glutamine, respectively, and leucine and glutamine oxidation from breath (14)CO(2) and (13)CO(2), respectively. Splanchnic glutamine extraction was estimated by the fraction of orally administered [(13)C]glutamine that failed to appear into systemic blood. Prednisone treatment 1) did not affect leucine Ra or leucine oxidation; 2) increased plasma glutamine Ra, mostly owing to enhanced glutamine de novo synthesis (medians +/- interquartiles, 412 +/- 61 vs. 280 +/- 190 mumol.kg(-1).h(-1), P = 0.003); and 3) increased the fraction of orally administered glutamine undergoing extraction in the splanchnic territory (means +/- SE 64 +/- 6 vs. 42 +/- 12%, P < 0.05), without any change in the fraction of glutamine oxidized (means +/- SE, 75 +/- 4 vs. 77 +/- 4%, not significant). We conclude that high-dose glucocorticosteroids increase in splanchnic bed the glutamine requirements. The role of such changes in patients receiving chronic corticoid treatment for inflammatory diseases or suffering from severe illness remains to be determined.     

Competition among oxidizable substrates in brains of young and adult rats. Dissociated cells.

The rates of conversion of D-(-)-3-hydroxy[3-14C]butyrate, [3-14C]acetoacetate, [6-14C]glucose and [U-14C]glutamine into 14CO2 were measured in the presence and absence of alternative oxidizable substrates in intact dissociated cells from the brains of young and adult rats. When unlabelled glutamine was added to [6-14C]glucose or unlabelled glucose was added to [U-14C]glutamine, the rate of 14CO2 production was decreased in both young and adult rats. The rate of oxidation of 3-hydroxy[3-14C]butyrate was also decreased by the addition of unlabelled glutamine in both age groups, but in the reverse situation, i.e. unlabelled 3-hydroxybutyrate added to [U-14C]glutamine, only the brain cells from young rats were affected. No significant effects were seen when glutamine and acetoacetate were combined. The addition of either of the two ketone bodies to [6-14C]glucose markedly lowered the rate of 14CO2 production in young rats, but in the adult only 3-hydroxybutyrate was effective and the magnitude of decrease in the rate of [6-14C]glucose oxidation was much lower than in young animals. Unlabelled glucose decreased the rate of [3-14C]acetoacetate oxidation to a minor extent in brain cells from both age groups; when added to 3-hydroxy[3-14C]butyrate, glucose had no effect in young rats and greatly enhanced 14CO2 production in adult brain cells. Many of these patterns of substrate interaction in dissociated brain cells differ from those in whole homogenates; they may be a function of the plasma membranes and the role of a carrier-mediated transport system or a reflection of a difference in the population of cell types or subcellular organelles in these two preparations.     

Carbon conversion efficiency and central metabolic fluxes in developing sunflower (Helianthus annuus L.) embryos

The efficiency with which developing sunflower embryos convert substrates into seed storage reserves was determined by labeling embryos with [U-(14)C6]glucose or [U-(14)C5]glutamine and measuring their conversion to CO2, oil, protein and other biomass compounds. The average carbon conversion efficiency was 50%, which contrasts with a value of over 80% previously observed in Brassica napus embryos (Goffman et al., 2005), in which light and the RuBisCO bypass pathway allow more efficient conversion of hexose to oil. Labeling levels after incubating sunflower embryos with [U-(14)C4]malate indicated that some carbon from malate enters the plastidic compartment and contributes to oil synthesis. To test this and to map the underlying pattern of metabolic fluxes, separate experiments were carried out in which embryos were labeled to isotopic steady state using [1-(13)C1]glucose, [2-(13)C1]glucose, or [U-(13)C5]glutamine. The resultant labeling in sugars, starch, fatty acids and amino acids was analyzed by NMR and GC-MS. The fluxes through intermediary metabolism were then quantified by computer-aided modeling. The resulting flux map accounted well for the labeling data, was in good agreement with the observed carbon efficiency, and was further validated by testing for agreement with gas exchange measurements. The map shows that the influx of malate into oil is low and that flux through futile cycles (wasting ATP) is low, which contrasts with the high rates previously determined for growing root tips and heterotrophic cell cultures.     

Metabolism of Proline, Glutamate, and Ornithine in Proline Mutant Root Tips of Zea mays (L.)

In excised pro_1-1 mutant and corresponding normal type roots of Zea mays L. the uptake and interconversion of [¹⁴C]proline, [¹⁴C]glutamic acid, [¹⁴C]glutamine, and [¹⁴C]ornithine and their utilization for protein synthesis was measured with the intention of finding an explanation for the proline requirement of the mutant. Uptake of these four amino acids, with the exception of proline, was the same in mutant and normal roots, but utilization differed. Higher than normal utilization rates for proline and glutamic acid were noted in mutant roots leading to increased CO₂ production, free amino acid interconversion, and protein synthesis. Proline was synthesized from either glutamic acid (or glutamine) or ornithine in both mutant and normal roots; it did not accumulate but rather was used for protein synthesis. Ornithine was not a good precursor for proline in either system, but was preferentially converted to arginine and glutamine, particularly in mutant roots. The pro_1-1 mutant was thus not deficient in its ability to make proline. Based on these findings, and on the fact that ornithine, arginine, glutamic acid and aspartic acid are elevated as free amino acids in mutant roots, it is suggested that in the pro_1-1 mutant proline catabolism prevails over proline synthesis.     

Evidence for cycloheximide acting as a glutamine analogue in plant tissue.

In growing maize root tissue [14C]asparagine formation in inhibited and [14C]glutamine accumulation stimulated by treatment with cycloheximide or glutamine analogs such as azaserine. In contrast, puromycin enhances the accumulation of [14C]asparagine but not [14C]glutamine. Cycloheximide and puromycin alone inhibit protein synthesis. This is interpreted to mean that the alteration in amide metabolism following cycloheximide treatment is a direct result of the antibiotic acting as a glutamine analog. While cycloheximide is often the cytoplasmic protein synthesis inhibitor of choice due to its potency and rapid action, its assumed specificity of action of eukaryotes is doubtful.     

Lipogenesis and cholesterogenesis in the liver of rats after cholesterol loading

Intensity of fatty acids and separate classes of lipids synthesis was studied in vitro in the liver of white rats at loading by cholesterol in the dose of 300 mg/kg once a day during 30 days by incubation of organ homogenate with [6-(14)C] glucose, [2-(14)C] lysine, [1-(14)C] palmitic acid with following determination of radioactivity of fatty acids, phospholipids, cholesterol, acylglycerols radioactivity was investigated. The inhibition of fatty acids and separate classes of lipids synthesis in vitro in the liver of white rats at loading by cholesterol at the use of [6-(14)C] of glucose and [2-(14)C] lysine, as predecessors of fatty acids and lipids and stimulation of lipids synthesis at the use of [1-(14)C] palmitic acid as the predecessor was established. The loading of white rats by cholesterol results in its synthesis inhibition in the liver during incubation of its homogenates with [6-(14)C] glucose and does not influence the cholesterol synthesis during incubation of homogenates with [2-(14)C] lysine and [1-(14)C] palmitic acid. Thus synthesis of fatty acids and their use in the phospholipids and acylglycerols synthesis in the liver of white rats with hypercholesterolemia sharply decreases during incubation of their homogenates with [6-(14)C] glucose and [2-(14)C] lysine, and the synthesis of cholesterol, phospholipids and acylglycerols - increases during incubation with [1-(14)C] palmitic acid.     

Incorporation of label from d-β-hydroxy-[14C]butyrate and [3-14C]acetoacetate into amino acids in rat brain in vivo

The metabolism of ketone bodies by rat brain was studied in vivo. Rats starved for 48h were given either d-beta-hydroxy[3-(14)C]butyrate or [3-(14)C]acetoacetate by intravenous injection and killed after 3 or 10min. Total radioactivity in the acid-soluble material of the brain and the specific radioactivities of the brain amino acids glutamate, glutamine, aspartate and gamma-aminobutyrate were determined. A group of fed animals were also given d-beta-hydroxy[3-(14)C]butyrate. In the brains of all animals (14)C was present in the acid-soluble material and the specific radioactivity of glutamate was greater than that of glutamine.     

Utilization of Citrate, Acetylcarnitine, Acetate, Pyruvate and Glucose for the Synthesis of Acetylcholine in Rat Brain Slices

Slices of rat caudate nuclei were incubated in saline media containing choline, paraoxon, unlabelled glucose, and [1,5-14C] citrate, [1-14C-acetyl]carnitine, [1-14C]acetate, [2-14C]pyruvate, or [U-14C]glucose. The synthesis of acetyl-labelled acetylcholine (ACh) was compared with the total synthesis of ACh. When related to the utilization of unlabelled glucose (responsible for the formation of unlabelled ACh), the utilization of labelled substrates for the synthesis of the acetyl moiety of ACh was found to decrease in the following order: [2-14C]pyruvate greater than [U-14C]glucose greater than [1-14C-acetyl]carnitine greater than [1,5-14C]citrate greater than [1-14C]acetate. The utilization of [1,5-14C]citrate and [1-14C]acetate for the synthesis of [14C]ACh was low, although it was apparent from the formation of 14CO2 and 14C-labelled lipid that the substrates entered the cells and were metabolized. The utilization of [1,5-14C]citrate for the synthesis of [14C]ACh was higher when the incubation was performed in a medium without calcium (with EGTA); that of glucose did not change, whereas the utilization of other substrates for the synthesis of ACh decreased. The results indicate that earlier (indirect) evidence led to an underestimation of acetylcarnitine as a potential source of acetyl groups for the synthesis of ACh in mammalian brian; they do not support (but do not disprove) the view that citrate is the main carrier of acetyl groups from the intramitochondrial acetyl-CoA to the extramitochondrial space in cerebral cholinergic neurons.     

The operation of the γ-aminobutyrate bypath of the tricarboxylic acid cycle in brain tissue in vitro

1. Cerebral-cortex slices prelabelled with gamma-amino[1-(14)C]butyrate (GABA) were incubated in a glucose-saline medium. After the initial rapid uptake there was no appreciable re-entry of (14)C into the GABA pool, either from the medium or from labelled metabolites formed in the tissue. The kinetic constants of GABA metabolism were determined by computer simulation of the experimental results by using mathematical procedures. The GABA flux was estimated to be 0.03mumol per min/g, or about 8% of the total flux through the tricarboxylic acid cycle. It was found that the assumption of compartmentation did not greatly affect the estimates of the GABA flux. 2. The time-course of incorporation of (14)C into amino acids associated with the tricarboxylic acid cycle was followed with [1-(14)C]GABA and [U-(14)C]-glucose as labelled substrates. The results were consistent with the utilization of GABA via succinate. This was confirmed by determining the position of (14)C in the carbon skeletons of aspartate and glutamate formed after the oxidation of [1-(14)C]GABA. These results also indicated that under the experimental conditions the reversal of reactions catalysed by alpha-oxoglutarate dehydrogenase and glutamate decarboxylase respectively was negligible. The conversion of [(14)C]GABA into gamma-hydroxybutyrate was probably also of minor importance, but decarboxylation of oxaloacetate did occur at a relatively slow rate. 3. When [1-(14)C]GABA was the labelled substrate there was evidence of a metabolic compartmentation of glutamate since, even before the peak of the incorporation of (14)C into glutamate had been reached, the glutamine/glutamate specific-radioactivity ratio was greater than unity. When [U-(14)C]glucose was oxidized this ratio was less than unity. The heterogeneity of the glutamate pool was indicated also by the relatively high specific radioactivity of GABA, which was comparable with that of aspartate during the whole incubation time (40min). The rates of equilibration of labelled amino acids between slice and medium gave evidence that the permeability properties of the glutamate compartments labelled as a result of oxidation of [1-(14)C]GABA were different from those labelled by the metabolism of [(14)C]glucose. The results showed therefore that in brain tissue incubated under the conditions used, the organization underlying metabolic compartmentation was preserved. The observed concentration ratios of amino acids between tissue and medium were also similar to those obtaining in vivo. These ratios decreased in the order: GABA>acidic acids>neutral amino acids>glutamine. 4. The approximate pool sizes of the amino acids in the different metabolic compartments were calculated. The glutamate content of the pool responsible for most of the labelling of glutamine during oxidation of [1-(14)C]GABA was estimated to be not more than 30% of the total tissue glutamate. The GABA content of the ;transmitter pool' was estimated to be 25-30% of the total GABA in the tissue. The structural correlates of metabolic compartmentation were considered.