Fatty acids and glycerol or lactate are required to induce gluconeogenesis from alanine in isolated rabbit renal cortical tubules

Summary In isolated rabbit renal cortical tubules, glucose synthesis from 1 mM alanine is negligible, while the amino acid is metabolized to glutamine and glutamate. The addition of 0.5 mM octanoate plus 2 mM glycerol induces incorporation of [U-¹⁴C]Alnine into glucose and decreases glutamine synthesis, whereas oleate and palmitate in the presence of glycerol are less potent than octanoate. Gluconeogenesis is also significantly accelerated when glycerol is substituted by lactate. In view of an increase in¹⁴CO₂ fixation and elevation of both cytosolic and mitochondrial NADH/NAD⁺ ratios, the activation of glucose formation from alanine upon the addition of glycerol and octanoate is likely due to (i) stimulation of pyruvate carboxylation, (ii) increased availability of NADH for glyceraldehyde-3-phosphate dehydrogenase and (iii) elevation of mitochondrial redox state causing a diminished provision of ammonium for glutamine synthesis. The induction of gluconeogenesis in the presence of alanine, glycerol and octanoate is not related to cell volume changes. The results presented in this paper show the importance of free fatty acids and glycerol for regulation of renal gluconeogenesis from alanine. The possible physiological significance of the data is discussed.     

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.     

The synthesis of glucose and ammonia by kidney tubules isolated from suckling and early-weaned lambs

Glucose and ammonia production were examined in kidney tubules isolated from suckling and early-weaned lambs, on days 10-30 after birth, with abrupt weaning occurring at day 14. There were no differences in the rates of glucose or ammonia production for a given substrate by tubules isolated from any of the lambs, regardless of age or stage of weaning. The preferred substrates for gluconeogenesis were glycerol = lactate greater than propionate = pyruvate = fructose = proline greater than alanine greater than glutamate greater than glutamine greater than aspartate greater than glycine greater than serine, and for ammoniagenesis were glutamine much greater than alanine greater than aspartate much greater than serine greater than glycine = glutamate = proline.     

Metabolism of isolated kidney tubules. Interactions between lactate, glutamine and oleate metabolism.

Kidney-cortex tubule suspensions were prepared by collagenase treatment of kidney cortex from fed and starved rats. This preparation, consisting mainly of proximal convoluted tubules was incubated with three major renal substrates, L-lactate, glutamine and oleate to study the dose dependence of substrate uptake rates from medium substrate combinations. All three substances, when added at near physiological concentrations, modified the uptake rate and fate of the other substrates. In accordance with previous observations, oleate inhibited lactate uptake, and lactate decreased glutamine metabolism. Glutamine on the other hand led to a marked increase in lactate uptake. Both, glutamine and lactate increased oleate metabolism. Glucose was the main product of lactate and glutamine metabolism, lactate being preferentially taken up for this process. Oleate led to a net synthesis of triglycerides in the tubules, which was stimulated by the addition of lactate and glutamine. More than 75% of the oleate taken up was recovered as triglycerides. In the absence of fatty acids, triglyceride content of tubules decreased. The results indicate that oleate is taken up in preference to lactate and glutamine when all three substrates are offered to the tubule. Glucose and triglycerides are the main metabolic products of tubular substrate metabolism. Whereas glucose is released into the medium, triglycerides are stored in the tubule cell.     

Evidence that L‐glutamine is better than L‐alanine as gluconeogenic substrate in perfused liver of weaned fasted rats submitted to short‐term insulin‐induced hypoglycaemia

Gluconeogenesis in livers from overnight fasted weaned rats submitted to short‐term insulin‐induced hypoglycaemia (IIH) was investigated. For this purpose, a condition of hyperinsulinemia/hypoglycaemia was obtained with an intraperitoneal (ip) injection of regular insulin (1.0 U kg^?1). Control group (COG group) received ip saline. The studies were performed 30 min after insulin (IIH group) or saline (COG group) injection. The livers from IIH and COG rats were perfused with L‐alanine (5 mM), L‐lactate (2 mM), L‐glutamine (10 mM) or glycerol (2 mM). Hepatic glucose, L‐lactate and pyruvate production from L‐alanine was not affected by IIH. In agreement with this result, the hepatic ability in producing glucose from L‐lactate or glycerol remained unchanged (IIH group vs. COG group). However, livers from IIH rats showed higher glucose production from L‐glutamine than livers from COG rats and, in the IIH rats, the production of glucose from L‐glutamine was higher than that from L‐alanine. The higher glucose production in livers from the IIH group, when compared with the COG group was due to its entrance further on gluconeogenic pathway. Taken together, the results suggest that L‐glutamine is better than L‐alanine, as gluconeogenic substrate in livers of hypoglyceaemic weaned rats. Copyright © 2008 John Wiley & Sons, Ltd.     

A quantitative analysis of the metabolic pathways of hepatic glucose synthesis in vivo with 13C-labeled substrates

A quantitative analysis of the major metabolic pathways of hepatic glucose synthesis in fasted rats was conducted. [2-13C]Acetate was administered intraintestinally into awake fasted rats. 13C NMR and GC-MS analysis were used to quantitate the isotopic enrichments of glutamate, glutamine, lactate, alanine and the newly synthesized liver glucose. By measuring the ratio of carbon atoms in glutamate molecules derived from acetyl-CoA to carbon atoms in the glucose molecule derived from oxaloacetate and gluconeogenic substrates, such as lactate and alanine, the relative activities of the Krebs cycle and gluconeogenesis were quantified. Our results indicate that the percentage of glucose carbons originating by 'metabolic exchange' with the oxaloacetate pool, via the Krebs cycle, is less than 7%.     

Stimulation of glutamine metabolism by 3-aminopicolinate in isolated dog kidney-cortex tubules

1. The effects of 3-aminopicolinate, a known hyperglycaemic agent in the rat, on glutamine metabolism were studied in isolated dog kidney tubules. 2. 3-Aminopicolinate greatly stimulated glutamine (but not glutamate) removal and glutamate accumulation from glutamine as well as formation of ammonia, aspartate, lactate, alanine and glucose. 3. The increased accumulation of aspartate from glutamine and glutamate, and the inhibition of glucose synthesis from various non-nitrogenous gluconeogenic substrates, as well as the increased accumulation of malate from succinate, support the proposal that 3-aminopicolinate is an inhibitor rather than a stimulator of phosphoenolpyruvate carboxykinase (EC 4.1.1.32) in dog kidney tubules. 4. With glutamine as substrate, the increase in flux through glutamate dehydrogenase (EC 1.4.1.3) could not explain the large increase in glutamine removal caused by 3-aminopicolinate. 5. Inhibition by amino-oxyacetate of accumulation of aspartate and alanine from glutamine caused by 3-aminopicolinate did not prevent the acceleration of glutamine utilization. 6. These data are consistent with a direct stimulation of glutaminase (EC 3.5.1.2) by 3-aminopicolinate in dog kidney tubules.     

The metabolic response of the kidney to acute sodium lactate alkalosis

In vivo studies were performed in the dog to verify if sodium lactate had an important effect on the metabolism of glutamine by the kidney. The animals were infused with 0.6 M sodium lactate to induce acute metabolic alkalosis with plasma bicarbonate of 29.7 mM. During these experiments, it was demonstrated that the renal uptake of glutamine increased by 46%, while the renal production of ammonia was unchanged. The renal production of alanine rose from 6.0 to 16.8 mumol/min. Plasma concentration of lactate increased from 1.3 to 19.2 mM, while that of pyruvate increased from 0.075 to 0.454 mM. In the renal tissue, alpha-ketoglutarate, malate, oxaloacetate, lactate, pyruvate, citrate, and alanine increased significantly. Similar changes were found in the liver and skeletal muscle. The observed changes are best described by transamination of pyruvate and glutamate under the influence of alanine aminotransferase (GPT). It can be calculated that this reaction was responsible for 76% of the production of ammonia from glutamine, the latter being necessary to provide glutamate for the synthesis of alanine. Dogs infused with 0.3 M sodium bicarbonate instead of sodium lactate with the same degree of acute metabolic alkalosis, showed a depression of 40% in the renal uptake of glutamine with a 38% decrease in renal ammoniagenesis and a 20% fall in the production of alanine. The present studies demonstrate that the production of ammonia from glutamine is not necessarily related to changes in acid-base balance, but may be associated with biochemical alterations related to the synthesis of alanine by the kidney.     

Renal cortical intermediary metabolism in the recovery phase of postischemic acute renal failure in the dog.

Renal metabolism has been studied in eight dogs before and 48 hr after a 60-min period of renal ischemia induced by clamping the left renal artery with the simultaneous removal of the right kidney, and in 12 sham-operated animals. The study involved the measurement of renal uptake and production of lactate, glutamine, glutamate, alanine, ammonium, and oxygen, and the measurement of the tissue concentrations of ATP, glutamine, lactate, alpha-ketoglutarate, aspartate, and alanine in the renal cortex. Two days after a temporary renal ischemia, the remaining kidney showed a 22% decrease in glomerular filtration rate (GFR) and a 25% decrease in renal plasma flow. Fractional sodium and potassium excretions were similar to those of control dogs. Renal production or extraction of glutamine, glutamate, alanine, ammonium, and oxygen (all expressed by 100 ml of GFR) was not significantly different in basal conditions or 2 days after ischemia, but lactate extraction was reduced in postischemic kidneys (-101 +/- 29 vs -204 +/- 38 mumol/100 ml GFR in control dogs). The cortical concentrations of glutamine and glutamate were lower in postischemic than in control kidneys. No differences were found in cortical concentration of alpha-ketoglutarate, aspartate, lactate, pyruvate, or ATP, but total nucleotides and inorganic phosphate were decreased in postischemic kidneys. It is concluded that in the recovery phase of the ischemia, a decreased lactate uptake is the main metabolic change, and total ATP production is adapted to the decrease of GFR and sodium reabsorption.     

METABOLISM OF GLUCOSE AND GLUTAMATE BY SYNAPTOSOMES FROM MAMMALIAN CEREBRAL CORTEX

—(1) Synaptosomes incubated in high sodium, low potassium media showed high linear respiration in the presence of glucose which was converted into lactate, aspartate, glutamate, glutamine, alanine and GABA during 1 hr incubation periods. (2) Total conversion of glucose into most of these substrates over the incubation period was similar in synaptosomes and cortex slices. Half the lactate and only a small fraction of the glutamine made by slices was formed by synaptosomes. (3) Pool sizes of amino acids in cortex slices after incubation with glucose were, in general, higher than in synaptosomes, glutamate and glutamine being four-fold higher in slices. (4) Most of the amino acids made from glucose by synaptosomes were contained within their structure and not lost to the medium. (5) Glutamate was actively metabolized by synaptosomes to aspartate, glutamine, alanine and GABA. The specific radioactivities of the amino acids (except glutamine) after 1 hr incubation, approached that of the glutamate. (6) Pyridoxal phosphate added to the incubation medium increased GABA production from glutamate but not from glucose.     

Effect of the antiepileptic drug sodium valproate on glutamine and glutamate metabolism in isolated human kidney tubules

We studied the effects of sodium valproate, a widely used antiepileptic drug and a hyperammonemic agent, on L-[1-14C]glutamine and L-[1-14C]glutamate metabolism in isolated human kidney-cortex tubules. Valproate markedly stimulated glutamine removal as well as the formation of ammonia, 14CO2, pyruvate, lactate and alanine, but it inhibited glucose synthesis; the increase in ammonia formation was explained by a stimulation by valproate mainly of flux through glutaminase (EC 3.5.1.2) and to a much lesser extent of flux through glutamate dehydrogenase (EC 1.4.1.3). By contrast, valproate did not stimulate glutamate removal or ammonia formation, suggesting that the increase in flux through glutamate dehydrogenase observed with glutamine as substrate was secondary to the increase in flux through glutaminase. Accumulation of pyruvate, alanine and lactate in the presence of valproate was less from glutamate than from glutamine. Inhibition by aminooxyacetate of accumulation of alanine from glutamine caused by valproate did not prevent the acceleration of glutamine utilization and the subsequent stimulation of ammonia formation. It is concluded from these data, which are the first concerning the in vitro metabolism of glutamine and glutamate in human kidney-cortex tubules, that the stimulatory effect of valproate is primarily exerted at the level of glutaminase in human renal cortex.     

Fetal hepatic and umbilical uptakes of glucogenic substrates during a glucagon-somatostatin infusion

To test the hypothesis that fetal hepatic glutamate output diverts the products of hepatic amino acid metabolism from hepatic gluconeogenesis, ovine fetal hepatic and umbilical uptakes of glucose and glucogenic substrates were measured before and during fetal glucagon-somatostatin (GS) infusion and during the combined infusion of GS, alanine, glutamine, and arginine. Before the infusions, hepatic uptake of lactate, alanine, glutamine, arginine, and other substrates was accompanied by hepatic output of pyruvate, aspartate, serine, glutamate, and ornithine. The GS infusion induced hepatic output of 1.00 +/- 0.07 mol glucose carbon/mol O(2) uptake, an equivalent reduction in hepatic output of pyruvate and glutamate carbon, a decrease in umbilical glucose uptake and placental uptake of fetal glutamate, an increase in hepatic alanine and arginine clearances, and a decrease in umbilical alanine, glutamine, and arginine uptakes. The latter result suggests that glucagon inhibits umbilical amino acid uptake. We conclude that fetal hepatic pyruvate and glutamate output is part of an adaptation to placental function that requires the fetal liver to maintain both a high rate of catabolism of glucogenic substrates and a low rate of gluconeogenesis.     

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).     

Fructose-induced hepatic gluconeogenesis: effect of L-carnitine

High fructose feeding (60 g/100 g diet) in rodents induces alterations in both glucose and lipid metabolism. The present study was aimed to evaluate whether intraperitoneal carnitine (CA), a transporter of fatty acyl-CoA into the mitochondria, could attenuate derangements in carbohydrate metabolizing enzymes and glucose overproduction in high fructose-diet fed rats. Male Wistar rats of body weight 150-160 g were divided into 4 groups of 6 rats each. Groups 1 and 4 animals received control diet while the groups 2 and 3 rats received high fructose-diet. Groups 3 and 4 animals were treated with CA (300 mg/Kg body weight/day, i.p.) for 30 days. At the end of the experimental period, levels of carnitine, glucose, insulin, lactate, pyruvate, glycerol, triglycerides and free fatty acids in plasma were determined. The activities of carbohydrate metabolizing enzymes and glycogen content in liver and muscle were assayed. Hepatocytes isolated from liver were studied for the gluconeogenic activity in the presence of substrates such as pyruvate, lactate, glycerol, fructose and alanine. Fructose-diet fed animals showed alterations in glucose metabolizing enzymes, increased circulating levels of gluconeogenic substrates and depletion of glycogen in liver and muscle. There was increased glucose output from hepatocytes of animals fed fructose-diet alone with all the gluconeogenic substrates. The abnormalities associated with fructose feeding such as increased gluconeogenesis, reduced glycogen content and other parameters were brought back to near normal levels by CA. Hepatocytes from these animals showed significant inhibition of glucose production from pyruvate (74.3%), lactate (65.4%), glycerol (69.6%), fructose (56.2%) and alanine (63.6%) as compared to CA untreated fructose-fed animals. The benefits observed could be attributed to the effect of CA on fatty acyl-CoA transport.     

Influence of dietary energy intake and substrate addition on the in vitro metabolism of glucose and glutamine in rumen epithelial tissue

Ten Holstein steers were fed either 14.2 or 26.2 Mcal ME for 28 days prior to investigating the effect of dietary energy on epithelial metabolism. Rumen papillae were incubated in vitro with glucose (5 mM) or glutamine (1 mM) as well as additional energy substrates. Increased dietary intake increased production of 14CO2 from glucose and glutamine, increased uptake and net lactate production from glucose, and decreased net glutamate and alanine production from glutamine. At these substrate concentrations, rates of glucose oxidation to 14CO2 were sevenfold higher than glutamine.     

Calcium-ion transport by intact synaptosomes. Intrasynaptosomal compartmentation and the role of the mitochondrial membrane potential.

1. The pathways and the fate of glutamate carbon and nitrogen were investigated in isolated guinea-pig kidney-cortex tubules. 2. At low glutamate concentration (1 mM), the glutamate carbon skeleton was either completely oxidized or converted into glutamine. At high glutamate concentration (5 mM), glucose, lactate and alanine were additional products of glutamate metabolism. 3. At neither concentration of glutamate was there accumulation of ammonia. 4. Nitrogen-balance calculations and the release of 14CO2 from L-[1-14C]glutamate (which gives an estimation of the flux of glutamate carbon skeleton through alpha-oxoglutarate dehydrogenase) clearly indicated that, despite the absence of ammonia accumulation, glutamate metabolism was initiated by the action of glutamate dehydrogenase and not by transamination reactions as suggested by Klahr, Schoolwerth & Bourgoignie [(1972) Am. J. Physiol. 222, 813-820] and Preuss [(1972) Am. J. Physiol. 222, 1395-1397]. Additional evidence for this was obtained by the use of (i) amino-oxyacetate, an inhibitor of transaminases, which did not decrease glutamate removal, or (ii) L-methionine DL-sulphoximine, an inhibitor of glutamine synthetase, which caused an accumulation of ammonia from glutamate. 5. Addition of NH4Cl plus glutamate caused an increase in both glutamate removal and glutamine synthesis, demonstrating that the supply of ammonia via glutamate dehydrogenase is the rate-limiting step in glutamine formation from glutamate. NH4Cl also inhibited the flux of glutamate through glutamate dehydrogenase and the formation of glucose, alanine and lactate. 6. The activities of enzymes possibly involved in the glutamate conversion into pyruvate were measured in guinea-pig renal cortex. 7. Renal arteriovenous-difference measurements revealed that in vivo the guinea-pig kidney adds glutamine and alanine to the circulating blood.     

Effect of alloxan-diabetes on gluconeogenesis and ureogenesis in isolated rabbit liver cells.

1. Neither alloxan-diabetes nor starvation affected the rate of glucose production in hepatocytes incubated with lactate, pyruvate, propionate or fructose as substrates. In contrast, glucose synthesis with either alanine or glutamine was increased nearly 3- and 12-fold respectively, in comparison with that in fed rabbits. 2. The addition of amino-oxyacetate resulted in about a 50% decrease in glucose formation from lactate in hepatocytes isolated from fed, alloxan-diabetic and starved rats, suggesting that both mitochondrial and cytosolic forms of rabbit phosphoenolpyruvate carboxykinase function actively during gluconeogenesis. 3. Alloxan-diabetes resulted in about 2-3-fold stimulation of urea production from either amino acid studied or NH4Cl as NH3 donor, whereas starvation caused a significant increase in the rate of ureogenesis only in the presence of alanine as the source of NH3. 4. As concluded from changes in the [3-hydroxybutyrate]/[acetoacetate] ratio, in hepatocytes from diabetic animals the mitochondrial redox state was shifted toward oxidation in comparison with that observed in liver cells isolated from fed rabbits.     

Renal tissue metabolism in the rat during chronic metabolic alkalosis: importance of glycolysis

Chronic metabolic alkalosis was induced in rats drinking 0.3 M NaHCO3 and receiving 1 mg furosemide/100 g body weight per day intraperitoneally. Another group of animals received a potassium supplement in the form of 0.3 M KHCO3. In this group, hypokalemia did not develop and muscle potassium fell by only 18% versus 50% in those not receiving potassium. In vitro renal production of ammonia and uptake of glutamine fell by 40% with a decrease in the activity of glutaminase I and glutamate dehydrogenase. Activity of phosphofructokinase, a major enzyme of glycolysis, rose only in the kidney of animals receiving a potassium supplement. Fructose-1,6-diphosphatase fell as well as phosphoenolpyruvate carboxykinase. Malate dehydrogenase also fell. The activity of phosphofructokinase also rose in the liver, heart, and leg muscle. The major biochemical changes in the renal cortex were the following: glutamate, alpha-ketoglutarate, malate, lactate, pyruvate, alanine, aspartate, and citrate rose as well as calculated oxaloacetate. The concentration of intermediates like 2-phosphoglycerate, 3-phosphoglycerate, and glucose-6-phosphate fell. The cytosolic redox potential (NAD+/NADH) decreased. In addition to the fall in ammoniagenesis, it could be demonstrated in vitro that the renal tubules incubated with glutamine showed decreased glucose production and increased production of lactate and pyruvate. The concentration of lactate was elevated in all tissues examined including liver, heart, and leg muscle. This study confirms in the rat that decreased renal ammoniagenesis takes place following decreased uptake of glutamine in metabolic alkalosis. All other changes are accounted for by the process of increased glycolysis, which appears to take place in all tissues in metabolic alkalosis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of gluconeogenesis in hepatocytes isolated from the American eel,Anguilla rostrata LeSueur,

Summary Isolated hepatocyte preparations from fed immature American eels,Anguilla rostrata Le Sueur, were used to study gluconeogenic, lipogenic, glycogenic and oxidative rates of radioactively labelled lactate, glycerol, alanine and aspartate. Eel hepatocytes maintain membrane integrity and energy charge during a 2 h incubation period and are considered a viable preparation for studying fish liver metabolism.Incubating eel hepatocytes with 10 mM substrates, the following results were obtained: glycerol, alanine and lactate, in that order, were effective gluconeogenic substrates; these three substrates reduced glucose release from glycogen stores, while aspartate had no such effect; lactate, alanine and aspartate led to high rates of glycerol production, with subsequent incorporation into lipid; incorporation into glycogen was low from all substrates; and, alanine oxidation was seven times higher than that observed with other substrates.When eel hepatocytes were incubated with low or physiological substrate concentrations gluconeogenic rates from lactate were twice those from alanine; rates from aspartate were very low. Glucagon stimulated lactate gluconeogenesis, but not amino acid gluconeogenesis, and had no significant effect on glycogenolysis. Cortisol increased gluconeogenic rates from 1 mM lactate.Thus, in the presence of adequate substrate, eel liver gluconeogenesis is preferentially stimulated relative to glycogenolysis to produce plasma glucose. These data support three important roles for gluconeogenesis: the recycling of muscle lactate, the synthesis of glucose from dietary amino acids to supplement glucose levels, and the production of glycerol for lipogenesis.This work was supported from operating grants to TWM from the National Research Council of Canada (A6944)     

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

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