Gluconeogenesis in chick embryo isolated hepatocytes

1. The effectiveness of gluconeogenic precursors in hepatocytes isolated from 18 day old chick embryos is:Lactate much much greater than pyruvate greater than alanine = glutamine greater than glycerol and other amino acids. This result is qualitatively and quantitatively similar to hepatocytes isolated after hatching. 2. In the presence of endogenous glycogenolysis, conversion of [U-14C]lactate to glucose was used to estimate gluconeogenic flux and its control by hormones. 3. Glucagon failed to stimulate lactate gluconeogenesis although simultaneously increasing glycogenolysis. Insulin had no effects on gluconeogenesis.     

Use of 3H and 14C doubly labeled glucose and amino acids in the study of hormonal regulation of gluconeogenesis in rats.

Double isotope procedures (3H and 14C) were used in vivo to investigate a) slow long-term gluconeogenic actions of adrenal glucocorticoids, and b) rapid stimulation of gluconeogenesis by glucagon. [U-14C,6-3H]Glucose was administered to normal and adrenalectomized rats. No effect was observed on the [6-3H]glucose half-life suggesting the dicarboxylic acid shuttle is unaffected by adrenalectomy; the Cori cycle is also not influenced. Loads of [14C]aspartate, [14C]glutamate, or [14C]alanine were given to normal and adrenalectomized rats. Simultaneously, in vivo transaminase activity was studied by measuring the appearance of 3H2O in body water after administration of [2-3H]aspartate, [2-3H]glutamate, or [2-3H]alanine, Adrenalectomy has no influence on the incorporation of glutamate or aspartate into glucose or on their in vivo transaminases. Diminution of incorporation of [14C]alanine into glucose and alanine transaminase activities occurs only when rats are given unphysiological loads. These studies support the contention that glucocorticoid rate-limiting actions occur in extrahepatic tissues to produce an increased flow of glucose precursors to the liver. [U-14C,3-3H]Glucose was used to investigate the effect of glucagon on the hepatic fructose-6-phosphate (F-6-P) cycle. Glucagon administration resulted in a rapid drop in the 3H/14C ratio of circulating glucose, suggesting an increase in F-6-P recycling caused by activation of FDPase with little or no decrease in phosphofructokinase. Such a change would direct substrate flux toward gluconeogenesis.     

Simultaneous determination of glucose turnover, alanine turnover, and gluconeogenesis in human using a double stable-isotope-labeled tracer infusion and gas chromatography-mass spectrometry analysis

We have developed and validated a new method to measure simultaneously glucose turnover, alanine turnover, and gluconeogenesis in human, in steady and non-steady states, using a double stable-isotope-labeled tracer infusion and GC-MS analysis. The method is based on the concomitant infusion and dilution of D-[2,3,4,6,6-2H5]glucose and L-[1,2,3-13C3]alanine. The choice of the tracers was done on the basis of a minimal overlap between the ions of interest and those arising from natural isotopic abundances. Alanine was chosen as the gluconeogenic substrate because it is the major gluconeogenic amino acid extracted by the liver and, with lactate, constitutes the bulk of the gluconeogenic precursors. The method was validated by comparing the results obtained during simultaneous infusion of trace amounts of both stable isotope labeled compounds with the radioactive tracers (D-[3-3H]glucose and L-[1,2,3-14C3]alanine) in a normal and a diabetic subject; the radiolabeled tracers were used as the accepted reference procedure. A slight overestimation of glucose turnover (7.3 versus 6.8 in normal and 10.8 versus 9.2 mumol/kg min in diabetic subject) was noticed when the stable isotope-labeled tracers were used. For the basal turnover rate of alanine, similar values were obtained with both methods (6.2 mumol/kg min). For gluconeogenesis, higher values were observed in the basal state with the stable isotopes (0.42 versus 0.21 mumol/kg min); however, these differences disappeared in the postprandial period after the ingestion of a mixed meal. Despite those minor differences, the overall correlation with the reference method was excellent for glucose turnover (r = 0.87) and gluconeogenesis (r = 0.86).(ABSTRACT TRUNCATED AT 250 WORDS)     

Glutamine gluconeogenesis in the small intestine of 72 h-fasted adult rats is undetectable

Recent reports have indicated that 48-72 h of fasting, Type 1 diabetes and high-protein feeding induce gluconeogenesis in the small intestine of adult rats in vivo. Since this would (i) represent a dramatic revision of the prevailing view that only the liver and the kidneys are gluconeogenic and (ii) have major consequences in the metabolism, nutrition and diabetes fields, we have thoroughly re-examined this question in the situation reported to induce the highest rate of gluconeogenesis. For this, metabolically viable small intestinal segments from 72 h-fasted adult rats were incubated with [3-13C]glutamine as substrate. After incubation, substrate utilization and product accumulation were measured by enzymatic and NMR spectroscopic methods. Although the segments utilized [13C]glutamine at high rates and accumulated 13C-labelled products linearly for 30 min in vitro, no substantial glucose synthesis could be detected. This was not due to the re-utilization of [13C]glucose initially synthesized from [13C]glutamine. Arteriovenous metabolite concentration difference measurements across the portal vein-drained viscera of 72 h-fasted Wistar and Sprague-Dawley rats clearly indicated that glutamine, the main if not the only gluconeogenic precursor taken up, could not give rise to detectable glucose production in vivo. Therefore we challenge the view that the small intestine of the adult rat is a gluconeogenic organ.     

Parasitism enhances the induction of glucogenesis by the insect, Manduca sexta L

Metabolic alterations that accompany parasitism of invertebrate animals can play an important role in parasite development. Employing 13C NMR, this study examined pyruvate cycling from (2-(13)C)pyruvate in the lepidopteran insect Manduca sexta, and the effects of parasitism by the hymenopteran Cotesia congregata on the gluconeogenic formation of trehalose, the haemolymph or blood sugar of insects. Larvae were maintained on a semi-synthetic sucrose-free diet, or on the same diet with sucrose at 8.5 g/l. Pyruvate cycling was evident from the 13C enrichment in C3 of alanine, derived following carboxylation to oxaloacetate, and was similar in parasitized and normal insects regardless of diet. Trehalose was formed following de novo synthesis of glucose, and net synthesis was estimated from the 13C distribution in trehalose and alanine. The 13C-enrichment ratio [2trehalose C6/alanine C3] is an indicator of the level of gluconeogenesis relative to glycolysis, both enrichments were derived from (2-(13)C)pyruvate in the same manner. The ratio was greater than unity in all insects, regardless of diet, but was significantly greater in parasitized larvae, demonstrating an enhanced level of gluconeogenesis. This was confirmed by analysis of the 13C distribution in trehalose and glutamine derived from (3-(13)C)alanine. Despite enhanced de novo trehalose formation in parasitized insects, the haemolymph sugar level was similar to that of normal larvae. Because haemolymph trehalose regulates dietary carbohydrate intake, but not gluconeogenesis, the results suggest that accelerated induction of gluconeogenesis is an adaptive response to parasitism that provides increased carbohydrate for parasite growth and simultaneously maintains nutrient intake.     

The metabolic effects of sodium dichloroacetate in the starved rat

1. Sodium dichloroacetate (300mg/kg body wt. per h) was infused in 24h-starved rats for 4h. 2. Blood glucose decreased significantly, an effect that had previously only been noted in diabetic animals 3. Plasma insulin concentration decreased by 63%; blood lactate and pyruvate concentrations decreased by 50 and 33%, whereas concentrations of 3-hydroxybutyrate and acetoacetate increased by 81 and 73% respectively. 4. Livers were freeze-clamped at the end of the 4h infusion. There were significant decreases in hepatic [glucose], [glucose 6-phosphate], [2-phosphoglycerate], the [lactate]/[pyruvate] ratio, [citrate] and [malate], and also [alanine], [glutamate] and [glutamine], suggesting a diminished supply of gluconeogenic substrates. 5. Animals subjected to a functional hepatectomy at the end of 2h infusions showed no difference in blood-glucose disappearance but a highly significant decrease in the rate of accumulation of lactate, pyruvate, glycerol and alanine, compared with control animals. Dichloroacetate decreased ketone-body clearance. 6. After functional hepatectomy an increase in glutamine accumulation appeared to compensate for the decrease in alanine accumulation. 7. It is concluded that dichloroacetate causes hypoglycaemia by decreasing the net release of gluconeogenic precursors from extrahepatic tissues while inhibiting peripheral ketone-body uptake. 8. These findings are consistent with the activation of pyruvate dehydrogenase (EC 1.2.4.1) in rat muscle by dichloroacetate previously described by Whitehouse & Randle (1973).     

13C NMR study of hepatic pyruvate carboxylase activity in tumor rats

Alanine and lactate, as major gluconeogenic substrates, must be converted into oxaloacetate by way of pyruvate carboxylase before their entry into gluconeogenesis. Although it is well known that hepatic gluconeogenesis from these substrates is increased in tumor hosts, the involvement of pyruvate carboxylase has not been demonstrated. In the present study, we examined pyruvate carboxylase activity in the perfused livers of tumor rats using 13C NMR spectroscopy with [3-13C]-alanine as the gluconeogenic substrate. A substantial increase in hepatic [3-13C]-aspartate production was found in the tumor rats. Since aspartate accumulation directly reflects fluxes of alanine through pyruvate carboxylase, the observed increase in hepatic production of [3-13C]-aspartate in tumor rats indicates that pyruvate carboxylase activity is significantly enhanced.     

Caveolae and the organization of carbohydrate metabolism in vascular smooth muscle

We have previously found that glycolysis and gluconeogenesis occur in separate "compartments" of the VSM cell. These compartments may result from spatial separation of glycolytic and gluconeogenic enzymes (Lloyd and Hardin [1999] Am J Physiol Cell Physiol. 277:C1250-C1262). We have also found that an intact plasma membrane is essential for compartmentation to exist (Lloyd and Hardin [2000] Am J Physiol Cell Physiol. 278:C803-C811), suggesting that glycolysis and gluconeogenesis may be associated with distinct plasma membrane microdomains. Caveolae are one such microdomain, in which proteins of related function colocalize. Thus, we hypothesized that membrane-associated glycolysis occurs in association with caveolae, while gluconeogenesis is localized to non-caveolae domains. To test this hypothesis, we disrupted caveolae in vascular smooth muscle (VSM) of pig cerebral microvessels (PCMV) with beta methyl-cyclodextrin (CD) and examined the metabolism of [2-(13)C]glucose (a glycolytic substrate) and [1-(13)C]fructose 1,6-bisphosphate (FBP, a gluconeogenic substrate in PCMV) using (13)C nuclear magnetic resonance spectroscopy. Caveolar disruption reduced flux of [2-(13)C]glucose to [2-(13)C]lactate, suggesting that caveolar disruption partially disrupted the glycolytic pathway. Caveolae disruption may also have resulted in a breakdown of compartmentation, since conversion of [1-(13)C]FBP to [3-(13)C]lactate was increased by CD treatment. Alternatively, the increased [3-(13)C]lactate production may reflect changes in FBP uptake, since conversion of [1-(13)C]FBP to [3-(13)C]glucose was also elevated in CD-treated cells. Thus, a link between caveolar organization and metabolic organization may exist.     

In vivo 13C-NMR studies on the metabolism of the lugworm Arenicola marina

13C-NMR natural-abundance spectra of specimens of Arenicola marina obtained, showed seasonal changes in the concentration of some metabolites, with the osmolite alanine as well as triacylglyceride storage compounds present at high concentrations. Glycogen was sometimes only barely detectable due to the low natural abundance level of 13C. Glycogenic metabolism of the lugworm A. marina was studied in vivo by 13C-NMR spectroscopy using 13C-labelled glucose. During recovery from a hypoxic period [1-13C]glucose was incorporated into glycogen. [1-13C]Glucose was injected 5 h after the end of hypoxia to guarantee sufficient and reliable 13C labelling of glycogen. An earlier injection of [1-13C]glucose led to considerably diminished incorporation of 13C-labelled glucosyl units into glycogen, probably due to the consumption of the available glucose as fuel for ATP production. No scrambling of 13C into the C6 position of glycogen was observed, indicating a lack of gluconeogenic activity. 13C was also incorporated into the C3 positions of alanine and alanopine. To assign correctly this last 13C-NMR resonance, the compound was synthesized biochemically. No labelling of glycogen was observed when [3-13C]alanine was injected into the coelomic cavity with similar incubation conditions being used. The 13C of [1-13C]glucose, incorporated into glycogen, showed a very low turnover rate in normoxic lugworms as shown by two 13C(1H)-NMR spectra, one obtained 48 h after the other. On the other hand, in hypoxia lugworms the signal due to 13C-labelled glycogen decreased very rapidly proving a high turnover rate. The disappearance of 13C from glycogen during the first 24 h of hypoxia indicates that the last glycosyl units to be synthesized are the first to be utilized. Lugworms were quite sensitive to the 1H-decoupling field used for obtaining the 13C(1H)-NMR spectra, especially at 11.7 T. Using bi-level composite-pulse decoupling and long relaxation delays, no tissue damage or stress-dependent phosphagen mobilization, as judged by 31P-NMR spectroscopy, was observed.     

Inhibitory effect of tumor necrosis factor α on gluconeogenesis in perfused rat liver

Tumor necrosis factor α (TNFα) is a cytokine involved in many metabolic responses in both normal and pathological states. Considering that the effects of TNFα on hepatic gluconeogenesis are inconclusive, we investigated the influence of this cytokine in gluconeogenesis from various glucose precursors. TNFα (10 μg/kg) was intravenously injected in rats; 6 h later, gluconeogenesis from alanine, lactate, glutamine, glycerol, and several related metabolic parameters were evaluated in situ perfused liver. TNFα reduced the hepatic glucose production (p < 0.001), increased the pyruvate production (p < 0.01), and had no effect on the lactate and urea production from alanine. TNFα also reduced the glucose production (p < 0.01), but had no effect on the pyruvate production from lactate. In addition, TNFα did not alter the hepatic glucose production from glutamine nor from glycerol. It can be concluded that the TNFα inhibited hepatic gluconeogenesis from alanine and lactate, which enter in gluconeogenic pathway before the pyruvate carboxylase step, but not from glutamine and glycerol, which enter in this pathway after the pyruvate carboxylase step, suggesting an important role of this metabolic step in the changes mediated by TNFα.     

Stimulation by alpha-adrenergic agonists of Ca2+ fluxes, mitochondrial oxidation and gluconeogenesis in perfused rat liver.

Glucose output from perfused livers of 48 h-starved rats was stimulated by phenylephrine (2 microM) when lactate, pyruvate, alanine, glycerol, sorbitol, dihydroxyacetone or fructose were used as gluconeogenic precursors. Phenylephrine-induced increases in glucose output were immediately preceded by a transient efflux of Ca2+ and a sustained increase in oxygen uptake. Phenylephrine decreased the perfusate [lactate]/[pyruvate] ratio when sorbitol or glycerol was present, but increased the ratio when alanine, dihydroxyacetone or fructose was present. Phenylephrine induced a rapid increase in the perfusate [beta-hydroxybutyrate]/[acetoacetate] ratio and increased total ketone-body output by 40-50% with all substrates. The oxidation of [1-14C]octanoate or 2-oxo[1-14C]glutarate to 14CO2 was increased by up to 200% by phenylephrine. All responses to phenylephrine infusion were diminished after depletion of the hepatic alpha-agonist-sensitive pool of Ca2+ and returned toward maximal responses after Ca2+ re-addition. Phenylephrine-induced increases in glucose output from lactate, sorbitol and glycerol were inhibited by the transaminase inhibitor amino-oxyacetate by 95%, 75% and 66% respectively. Data presented suggest that the mobilization of an intracellular pool of Ca2+ is involved in the activation of gluconeogenesis by alpha-adrenergic agonists in perfused rat liver. alpha-Adrenergic activation of gluconeogenesis is apparently accompanied by increases in fatty acid oxidation and tricarboxylic acid-cycle flux. An enhanced transfer of reducing equivalents from the cytoplasmic to the mitochondrial compartment may also be involved in the stimulation of glucose output from the relatively reduced substrates glycerol and sorbitol and may arise principally from an increased flux through the malate-aspartate shuttle.     

The stimulation of hepatic gluconeogenesis by acetoacetate precursors. A role for the monocarboxylate translocator

The regulation of the gluconeogenic pathway from the 3-carbon precursors pyruvate, lactate, and alanine was investigated in the isolated perfused rat liver. Using pyruvate (less than 1 mM), lactate, or alanine as the gluconeogenic precursor, infusion of the acetoacetate precursors oleate, acetate, or beta-hydroxybutyrate stimulated the rate of glucose production and, in the case of pyruvate (less than 1 mM), the rate of pyruvate decarboxylation. alpha-Cyanocinnamate, an inhibitor of the monocarboxylate transporter, prevented the stimulation of pyruvate decarboxylation and glucose production due to acetate infusion. With lactate as the gluconeogenic precursor, acetate infusion in the presence of L-carnitine stimulated the rate of gluconeogenesis (100%) and ketogenesis (60%) without altering the tissue acetyl-CoA level usually considered a requisite for the stimulation of gluconeogenesis by fatty acids. Hence, our studies suggest that gluconeogenesis from pyruvate or other substrates which are converted to pyruvate prior to glucose synthesis may be limited or controlled by the rate of entry of pyruvate into the mitochondrial compartment on the monocarboxylate translocator.     

Plasmodium berghei: gluconeogenesis in the infected mouse liver studied by 13C nuclear magnetic resonance

Using 13C nuclear magnetic resonance, we have compared the gluconeogenic activity of perfused livers isolated from normal starved mice and mice highly parasitized with Plasmodium berghei, using [2-13C]pyruvate as substrate. In both types of livers, 13C labeling of glucose carbons occurred in positions 1, 2, 5, and 6. The equal proportions of [1,6-13C]- and [2,5-13C]glucose in livers from malarial and normal mice suggests that pyruvate enters the gluconeogenic pathway directly and, to an equal extent, via the tricarboxylic acid cycle. The normalized signal heights indicated that at a given time after the addition of [2-13C]pyruvate the degree of 13C labeling in glucose carbons was reduced in livers from malarial animals, when compared to livers from normal animals. During the course of the perfusion experiment, the [2-13C]lactate resonance signal was always more intense from livers of malarial animals than from normal animals. A reduced activity of hepatic gluconeogenesis in malarial animals was further confirmed by a separate set of perfusion experiments which showed a 56% reduction of the measured rate of glucose production in livers from malarial animals, with respect to that of normal animals. A lowered NAD/NADH ratio in livers from malarial animals would explain the increased proportion of lactate observed in the spectra and be related to a decreased gluconeogenic rate. A more reduced oxidoreduction level in the hepatocytes of a malarial animal would result from a defect in the oxidative phosphorylation activity of mitochondria.     

Effects of maternal fasting on hepatic gluconeogenesis and glucose metabolism in fetal lambs.

In unstressed, normoglycaemic fetal lambs, the liver produces little glucose, and gluconeogenesis is insignificant. Indirect measurements have suggested that the fetus may produce glucose endogenously during hypoglycaemia induced by prolonged maternal starvation. In eight fetal lambs we directly measured total and radiolabelled substrate concentration differences across the liver to determine whether the fetal liver produces glucose after four days of fasting-induced hypoglycaemia. Simultaneously we measured umbilical glucose uptake and fetal glucose utilization. Glucose concentrations in ewes (1.78 +/- 0.44 mmol.-1) and fetuses (0.61 +/- 0.17 mmol.l-1) were decreased. Fetal glucose utilization rate (21.7 +/- 8.9 mumol.min-1.kg-1) was not significantly different from umbilical glucose uptake (17.2 +/- 8.9 mumol.min-1.kg-1). Hepatic glucose production (8.9 +/- 17.2 mumol.min-1.100 g-1) and gluconeogenesis (6.1 +/- 4.4 mumol.min-1.100 g-1) were present, but could account for only 13% and 8% of fetal glucose requirements, respectively. To determine whether glucose output by the fetal liver was limited by substrate availability, we infused lactate, acetate, and acetone into the umbilical veins of four fasted animals, increasing hepatic substrate delivery. Hepatic glucose output did not increase during infusion of gluconeogenic substrates, indicating that substrate availability did not limit gluconeogenesis. We conclude that the gluconeogenic pathway is intact in late-gestation fetal lambs and that the fetal liver is capable of gluconeogenesis. However, the primary change in fetal metabolism during maternal starvation is the reduction in fetal glucose utilization, obviating the need for substantial hepatic glucose production. The factors stimulating this modest increase in fetal hepatic glucose production remain to be elucidated.     

Glucose turnover and hepatocyte glucose production of starved and toxaemic pregnant sheep

Ewes bearing twins were starved for 10 days during the last month of gestation to induce ovine pregnancy toxaemia (OPT). Glucose turnover was measured by a primed continuous infusion of [U-14C]- and [6-3H]glucose at the end of 10 days of starvation (non-susceptible), or earlier when ewes became recumbent with OPT (susceptible). All ewes were slaughtered at the end of the infusion and hepatocytes were prepared in order to measure glucose production from different substrates. Many of the ewes had dead foetuses when slaughtered. Glucose production rates by hepatocytes with the substrates propionate, lactate or alanine were significantly less from the susceptible ewes than were those from non-susceptible ewes. These low rates were not stimulated by incubation with glucagon (10(-8) M), glutamine or glycerol. Rates of glucose turnover and of hepatic glucose production from all substrates were higher for ewes with dead than with live foetuses. The data support the hypothesis that pathogenesis of OPT is related to an impairment of hepatic gluconeogenesis, and further suggest that, in starved pregnant ewes, maternal glucose production may be restrained in the presence of a live foetus.     

Blood sugar formation due to abnormally elevated gluconeogenesis: aberrant regulation in a parasitized insect, Manduca sexta Linnaeus.

Alterations of carbohydrate metabolism associated with parasitism were examined in an insect, Manduca sexta L. In insect larvae maintained on a low carbohydrate diet gluconeogenesis from [3-13C]alanine was established from the fractional 13C enrichment in trehalose, a disaccharide of glucose and the blood sugar of insects and other invertebrates. After transamination of the isotopically substituted substrate to [3-13C]pyruvate, the latter was carboxylated to oxaloacetate ultimately leading to de novo glucose synthesis and trehalose formation. Trehalose was selectively enriched with 13C at C1 and C6 followed by C2 and C5. 13C enrichment of blood sugar in insects parasitized by Cotesia congregata (Say) was significantly greater than was observed in normal animals. The relative contributions of pyruvate carboxylation and decarboxylation to trehalose labeling were determined from the 13C distribution in glutamine, synthesized as a byproduct of the tricarboxylic acid cycle. The relative contribution of carboxylation was significantly greater in parasitized larvae than in normal insects providing additional evidence of elevated gluconeogenesis due to parasitism. Despite the increased gluconeogenesis in parasitized insects the level of blood sugar was the same in all animals. Because de novo glucose synthesis does not normally maintain blood sugar level in insects maintained under these dietary conditions the findings suggest an aberrant regulation over gluconeogenesis. The 13C labeling in trehalose was nearly symmetric in all insects but the mean C1/C6 13C ratio was higher in parasitized animals suggesting a lower activity of the pentose phosphate pathway that brings about a redistribution of 13C in trehalose following de novo glucose synthesis. Additional studies with insects maintained on a high carbohydrate diet and administered [1,2-13C2]glucose confirmed a decreased level of pentose cycling during parasitism consistent with a lower level of lipogenesis. It is suggested, however, that the pentose pathway may facilitate the synthesis of trehalose from dietary carbohydrate by directing hexose phosphate cycled through the pathway to the production of energy.     

Inhibition of glutamine transport depletes glutamate and GABA neurotransmitter pools: further evidence for metabolic compartmentation

The role of glutamine and alanine transport in the recycling of neurotransmitter glutamate was investigated in Guinea pig brain cortical tissue slices and prisms, and in cultured neuroblastoma and astrocyte cell lines. The ability of exogenous (2 mm) glutamine to displace 13C label supplied as [3-13C]pyruvate, [2-13C]acetate, l-[3-13C]lactate, or d-[1-13C]glucose was investigated using NMR spectroscopy. Glutamine transport was inhibited in slices under quiescent or depolarising conditions using histidine, which shares most transport routes with glutamine, or 2-(methylamino)isobutyric acid (MeAIB), a specific inhibitor of the neuronal system A. Glutamine mainly entered a large, slow turnover pool, probably located in neurons, which did not interact with the glutamate/glutamine neurotransmitter cycle. This uptake was inhibited by MeAIB. When [1-13C]glucose was used as substrate, glutamate/glutamine cycle turnover was inhibited by histidine but not MeAIB, suggesting that neuronal system A may not play a prominent role in neurotransmitter cycling. When transport was blocked by histidine under depolarising conditions, neurotransmitter pools were depleted, showing that glutamine transport is essential for maintenance of glutamate, GABA and alanine pools. Alanine labelling and release were decreased by histidine, showing that alanine was released from neurons and returned to astrocytes. The resultant implications for metabolic compartmentation and regulation of metabolism by transport processes are discussed.     

Modulation of branched-chain amino acid oxidation in rat hemidiaphragms in vitro by glucose and ketone bodies

Branched-chain amino acid metabolism in hemidiaphragms from 40 h-starved rats is influenced by the provision of glucose as co-substrate. Glucose inhibits 14CO2 production from [l-14C]valine and [U-14C]valine but stimulates 14CO2 production from [l-14C]leucine, [U-14C]leucine and [U-14C]isoleucine. In the presence of glucose, ketone bodies inhibit alanine release and 14CO2 production from [l-14C]valine, [l-14C]leucine and [U-14C]isoleucine, but inhibition is not observed in the absence of glucose as cosubstrate. Glucose-dependent inhibition by ketone bodies of branched-chain amino acid oxidation via inhibition of the branched-chain 2-oxo acid dehydrogenase complex or branched-chain amino acid aminotransferase may account in part for the reported hypoalanaemic action of ketone bodies in vivo.     

Hepatic glycogen synthesis from duodenal glucose and alanine. An in situ 13C NMR study

An in situ and in vivo surface coil 13C NMR study was performed to study hepatic glycogen synthesis from [3-13C]alanine and [1-13C]glucose administered by intraduodenal infusion in 18-h fasted male Sprague-Dawley rats. Combined, equimolar amounts of alanine and glucose were given. Hepatic appearance and disappearance of substrate and concurrent glycogen synthesis was followed over 150 min, with 5-min time resolution. Active glycogen synthesis from glucose via the direct (glucose----glycogen) and indirect (glucose----lactate----glycogen) pathways and from alanine via gluconeogenesis was observed. The indirect pathway of glycogen synthesis from [1-13C]glucose accounted for 30% (+/- 6 S.E.) of total glycogen formed from labeled glucose. This estimate does not take into account dilution of label in the hepatic oxaloacetate pool and is, therefore, somewhat uncertain. Hepatic levels of [3-13C]alanine achieved were significantly lower than levels of [1-13C]glucose in the liver, and the period of active glycogen synthesis from [3-13C]alanine was longer than from glucose. However, the overall pseudo-first-order rate constant during the period of active glycogen synthesis from [3-13C]alanine (0.075 min-1 +/- 0.026 S.E.) was almost 3 times that from [1-13C]glucose via the direct pathway (0.025 min-1 +/- 0.005 S.E.). The most likely reason for the small rate constant governing direct glycogen formation from duodenally administered glucose compared to that from duodenally administered alanine is a low level of glucose phosphorylating capacity in the liver.     

Pathways of glutamine metabolism in Spodoptera frugiperda (Sf9) insect cells: evidence for the presence of the nitrogen assimilation system, and a metabolic switch by 1H/15N NMR

1H/15N and 13C NMR were used to investigate metabolism in Spodoptera frugiperda (Sf9) cells. Labelled substrates ([2-15N]glutamine, [5-15N]glutamine, [2-15N]glutamate, 15NH4Cl, [2-15N]alanine, and [1-13C]glucose) were added to batch cultures and the concentration of labelled excreted metabolites (alanine, NH4+, glutamine, glycerol, and lactate) were quantified. Cultures with excess glucose and glutamine produce alanine as the main metabolic by-product while no ammonium ions are released. 1H/15N NMR data showed that both the amide and amine-nitrogen of glutamine was incorporated into alanine in these cultures. The amide-nitrogen of glutamine was not transferred to the amine-position in glutamate (for further transamination to alanine) via free NH4+ but directly via an azaserine inhibitable amido-transfer reaction. In glutamine-free media 15NH4+ was consumed and incorporated into alanine. 15NH4+ was also incorporated into the amide-position of glutamine synthesised by the cells. These data suggest that the nitrogen assimilation system, glutamine synthetase/glutamate synthase (NADH-GOGAT), is active in glutamine-deprived cells. In cultures devoid of glucose, ammonium is the main metabolic by-product while no alanine is formed. The ammonium ions stem both from the amide and amine-nitrogen of glutamine, most likely via glutaminase and glutamate dehydrogenase. 13C NMR revealed that the [1-13C] label from glucose appeared in glycerol, alanine, lactate, and in extracellular glutamine. Labelling data also showed that intermediates of the tricarboxylic acid cycle were recycled to glycolysis and that carbon sources, other than glucose-derived acetylCoA, entered the cycle. Furthermore, Sf9 cell cultures excreted significant amounts glycerol (1.9-3.2 mM) and ethanol (6 mM), thus highlighting the importance of sinks for reducing equivalents in maintaining the cytosolic redox balance.