The fate of labelled glucose molecules in the rat adipocyte. Dependence on glucose concentration

Isolated rat adipocytes were incubated with 15 nM [3-3H]glucose or 100 nM [U-14C]glucose with or without insulin and in the absence or presence of unlabelled glucose. Following a 2 h incubation with 15 nM [3-3H]glucose, about two thirds of the cell-associated 3H-labelled metabolic products were hydrophilic largely anionic intermediates and about one third was lipids. The equivalent values were 40 and 60%, respectively, when using 100 nM [U-14C]glucose. The only 14C-labelled metabolite escaping to the incubation medium was 14CO2, which accounted for about 15% of the rate of metabolism. Therefore, the rate of incorporation of 100 nM [U-14C]glucose into the cell-associated metabolites was quite a good measure of its net influx rate. The conversion of the two tracers to the sum of the metabolic products in cells treated with a maximally stimulating insulin concentration remained constant with glucose concentrations up to about 100 microM and then decreased progressively. The incorporation of radioactivity into the different metabolites varied markedly over the glucose concentration range 0-100 microM, presumably due to the saturation of different metabolic pools at different glucose concentrations. This variation was much less in cells not stimulated with insulin. Consequently, the maximal effect of insulin on the incorporation of the tracers into a given metabolite (e.g., labelled lipids) varied over the entire glucose concentration range. In addition, the apparent sensitivity (ED50) with respect to the incorporation into a given metabolite was also dependent on the glucose concentration.     

Stimulation by glucagon of the incorporation of U-14 C-labeled substrates into glucose by isolated hepatocytes from fed rats

The effect of glucagon on the incorporation of U-¹⁴ C-labeled lactate, pyruvate or alanine into glucose has been studied using isolated hepatocytes from livers of fed rats. Rates of incorporation into glucose were about the same as observed in perfused liver preparations provided precautions were taken to avoid depletion of certain metabolities by the preparative procedures. With each substrate, stimulation of the incorporation into glucose by a maximally effective concentration of glucagon (10 nM) was associated with about a 75% reduction in the substrate concentration required for a half-maximal rate and with about a 30% increase in maximum rate. Consequently, the hormone caused a substantial (2–4-fold) stimulation when any one of the above substrates was present at a near physiological concentration, but brought about only a relatively small stimulation (1.4-fold) when very high substrate concentrations were used. Provision of cytoplasmic reducing equivalents (by ethanol addition), or of precursor for acetyl-coenzyme A formation (by acetate addition)-stimulated incorporation of labeled alanine into glucose and their effects were additive with that of glucagon. This suggested that provision of either of these intermediates was not a means by which the hormone increased the incorporation of labeled substrate into glucose. NH₄⁺ stimulated the incorporation of 20 mM [U-¹⁴ C] lactate into glucose 2-fold, probably by promoting glutamate synthesis and thus enhancing the transamination of oxaloacetate to aspartate. Evidence was obtained to support the view that glucagon also increases glutamate production (presumably from endogenous protein). However, the stimulation of incorporatio into glucose from 20 mM [U-¹⁴ C] lactate by NH₄⁺ plus glucagon was synergistic. This suggested that glucagon also stimulates the incorporation of labeled substrate into glucose by additional means. Stimulation of the incorporation of [U-¹⁴ C] alanine into glucose by β-hydroxybutyrate plus glucagon was also synergistic. This suggested that another action of glucagon may be to provide more intramitochondrial reducing potential.     

Glucose handling by hepatocytes from obese Zucker rats

Hepatocytes isolated from obese Zucker rats showed a significantly higher rate of both [U-¹⁴C]glucose and [U-¹⁴C]lactate incorporation into [¹⁴C]lipid than those from their lean counterparts. This was associated with a marked increase in the lipogenic rate measured by the incorporation of³H₂O into the cell esterified fatty acids. Although there were no changes in the incorporation of the tracer into either [¹⁴C]glycogen or¹⁴CO₂, the [¹⁴C] total uptake was significantly higher in the obese animals. The high rate of [¹⁴C]lipid synthesis from glucose was observed both at 15 and 30 mM substrate concentrations and was linked to an enhanced uptake of the tracer into the cell as measured using the decarboxilation of [1-¹⁴C]glucose in the presence of phenazine methosulphate. The presence of insulin in the incubation medium had no effect on the uptake of glucose by the liver cells. However, the large uptake of glucose by the hepatocytes from the obese animals was not related to an enhanced rate of transport as measured using 3-O-methyl[U-¹⁴C]glucose. The activity of glucose-6-phosphate dehydrogenase together with a higher [1-¹⁴C]glucose/[U-¹⁴C]glucose descarboxylation ratio indicate a predominant very active pentose phosphate pathway which may be responsible for the enhanced glucose uptake observed in the hepatocytes from the obese animals.     

METABOLISM OF HEXOSES IN RAT CEREBRAL CORTEX SLICES

Abstract—

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

     

THE METABOLISM OF LEUCINE BY DEVELOPING RAT BRAIN: EFFECT OF LEUCINE AND 2-OXO-4-METHYLVALERATE ON LIPID SYNTHESIS FROM GLUCOSE AND KETONE BODIES

Abstract— The oxidation of l -[U-¹⁴C]leucine and l -[l-¹⁴C]leucine at varying concentrations from 0.1 to 5mM to CO₂ and the incorporation into cerebral lipids and proteins by brain slices from 1-week old rats were markedly stimulated by glucose. Although the addition of S mM-dl -3-hydroxybutyrate had no effect on the metabolism of [U-¹⁴C]leucine by brain slices from suckling rats, the stimulatory effects of glucose on the metabolism of l -[U-¹⁴C]leucine were markedly reduced in the presence of dl -3-hydroxybutyrate. The stimulatory effect of glucose on leucine oxidation was, however, not observed in adult rat brain. Furthermore, the incorporation of leucine-carbon into cerebral lipids and proteins was also very low in the adult brain. The incorporation of l -[U-¹⁴C]leucine into cerebral lipids by cortex slices was higher during the first 2 postnatal weeks, which then declined to the adult level. During this time span, the oxidation of l -[U-¹⁴C]leucine to CO₂ remained relatively unchanged. The incorporation in vivo of D-3-hydroxy[3-¹⁴C]butyrate into cerebral lipids was markedly decreased by acute hyperleucinemia induced by injecting leucine into 9-day old rats. In in vitro experiments, 5 mM-leucine had no effect on the oxidation of [U-¹⁴C]glucose to CO₂ or its incorporation into lipids by brain slices from 1-week old rats. However, 5 mM-leucine inhibited the oxidation of d -3-hydroxy-[3-¹⁴C]butyrate, [3-¹⁴C]acetoacetate and [1-¹⁴C]acetate to CO₂ by brain slices, but their incorporation into cerebral lipids was not affected by leucine. In contrast 2-oxo-4-methylvalerate, a deaminated metabolite of leucine, markedly inhibited both the oxidation to CO₂ and the incorporation into lipids of labelled glucose, ketone bodies and acetate by cortex slices from 1-week old rats. These findings suggest that the reduction in the incorporation in vivo of d -3-hydroxy[3-¹⁴C]butyrate into cerebral lipids in rats injected with leucine is most likely caused by 2-oxo-4-methylvalerate formed from leucine. Since the concentrations of leucine and 2-oxo-4-methylvalerate in plasma of untreated patients with maple-syrup urine disease are markedly elevated, our findings are compatible with the possibility that an alteration in the metabolism of glucose and ketone bodies in the brain may contribute to the pathophysiology of this disease.     

A simple method for determining total glucose utilisation by isolated adipocytes using (5-3H)-glucose

Glucose utilisation by adipocytes incubated with and without insulin and at two concentrations of extracellular glucose has been estimated by three different procedures. Glucose disappearance from the medium was calculated by using glucose oxidase to determine the glucose concentration remaining after incubation and comparing this with the glucose concentration in standard solutions made up by appropriate dilution of the original medium. [U-¹⁴C]-glucose utilisation was calculated by summing the ¹⁴C found in CO₂, triglycerides, and anions. [³H]-H₂O formation from [5-³H]-glucose was the third measure of glucose utilisation. All three methods gave similar answers, but the [5-³H]-glucose is simpler to use than [U-¹⁴C]-glucose and gives substantially more reproducible results than glucose oxidase.     

Cerebral lipid metabolism in experimental hyperphenylalaninaemia: incorporation of 14C-labelled glucose into total lipids

—1. Effects of the administration of phenylalanine to rats on incorporation in vivo or in vitro of [U-¹⁴C]glucose into cerebral lipids were studied during the first 5–10 days of postnatal development. In addition, the effects of added phenylalanine and its deaminated metabolites on incorporation of [U-¹⁴C]glucose by homogenates into lipids of developing rat brain were investigated. Hyperphenylalaninaemia reduced incorporation both in vivo and in vitro of [U-¹⁴C]glucose into cerebral lipids. 2. Phenylalanine or tyrosine added in vitro at concentrations equivalent to those in the brain of the hyperphenylalaninaemic rat (0-1 μmole/ml incubation medium) did not inhibit incorporation of [U-¹⁴C)glucose into lipids, although at much higher concentrations of phenylalanine (36 μumoles/ml incubation medium) slight inhibition (10 per cent) of incorporation of [U-¹⁴C]glucose into lipids was observed. 3. In contrast, the deaminated metabolites in general exerted greater inhibitory effects at lower concentrations. Phenyllactic acid, in comparison to phenylpyruvic and phenyl-acetic acid, was the most potent inhibitor of the incorporation in vitro of [U-¹⁴C]glucose into cerebral lipids. These results indicated that these metabolites of phenylalanine were the more potent inhibitors of cerebral lipid metabolism in immature animals.     

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

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

The action of insulin of glycogen synthesis in rat diaphragm from intracellular and extracellular glucose

Rat hemidiaphragms were loaded with [U-¹⁴C] glucose at 2°C and subsequently incubated at 37°C with non-labeled glucose or [¹⁴C] glucose in the presence or absence of insulin. The incorporation of isotope into glycogen and lactate was determined. The results showed that insulin markedly stimulated the synthesis of glycogen from extracellular glucose while it had no effect on incorporation of isotope into glycogen from intracellular glucose. Lactate formation was not influenced by insulin. It was concluded that glucose transport in muscle is linked to glycogen sythesis and that insulin preferentially directs glucose entering the cell toward the formation of glycogen.     

DEVELOPMENT OF LIPOGENESIS IN RAT BRAIN CORTEX: THE DIFFERENTIAL INCORPORATION OF GLUCOSE AND ACETATE INTO BRAIN LIPIDS IN VITRO

—The oxidation to CO₂ and the incorporation of [U-¹⁴C]glucose and [U-¹⁴C]acetate into lipids by cortex slices from rat brain during the postnatal period were investigated. The oxidation of [U-¹⁴C]glucose was low in 2-day-old rat brain, and increased by about two-fold during the 2nd and 3rd postnatal weeks. The oxidation of [U-¹⁴C]acetate was increased markedly in the second postnatal week, but decreased to rates observed in 2-day-old rat brain at the time of weaning. Both labeled substrates were readily incorporated into non-saponifiable lipids and fatty acids by brain slices from 2-day-old rat. Their rates of incorporation and the days on which maximum rates occurred were different, however, maximum incorporation of [U-¹⁴C]glucose and [U-¹⁴]acetate into lipid fractions being observed on about the 7th and 12th postanatal days, respectively. The metabolic compartmentation in the utilization of these substrates for lipogenesis is suggested. The activities of glucose-6-phosphate dehydrogenase, cytosolic NADP-malate dehydrogenase, cytosolic NADP-isocitrate dehydrogenase, ATP-citrate lyase and acetyl CoA carboxylase were measured in rat brain during the postnatal period. All enzymes followed somewhat different courses of development; the activity of acetyl CoA carboxylase was, however, the lowest among other key enzymes in the biosynthetic pathway, and its developmental pattern paralleled closely the fatty acid synthesis from [U-¹⁴C]glucose. It is suggested that acetyl CoA carboxylase is a rate-limiting step in the synthesis de novo of fatty acids in developing rat brain.     

Incorporation of carbon from glucose into cerebral amino acids, proteins and lipids, and alterations during recovery from hypoglycaemia

Abstract— [U-¹⁴C]Glucose was given to dogs by intravenous infusion to maintain a fixed level of specific radioactivity of the plasma glucose. The time course of incorporation of ¹⁴C into free amino acids, proteins and lipids of the cerebrum was observed for periods up to 2 h. Labelling of amino acids closely related to the tricarboxylic acid cycle increased progressively throughout the infusion, approaching specific radioactivities per μg-atom of carbon equal to that of the plasma glucose. No significant dilution by unlabelled carbon entering the metabolic pathways was apparent. In dogs subjected to profound insulin hypoglycaemia, [U-¹⁴C]glucose mixed with unlabelled glucose was given to bring about recovery. The incorporation of ¹⁴C into glutamate, glutamine and aspartate during a 40-min period was greater than during a comparable period in control animals, whereas the incorporation into serine was reduced. When considered in relation to alterations in amino acid levels, the data suggest that during recovery from hypoglycaemia the rates of synthesis of amino acids related to the tricarboxylic acid cycle are increased. During the period of recovery the rates of incorporation of ¹⁴C from glucose into proteins exceeded the pre-insulin rates to a degree surpassing the increased incorporation into free amino acids. The labelling of the ganglioside, cerebroside-sulphatide and cephalin fractions was also increased, The rates of incorporation into the lecithin-sphin-gomyelin and cholesterol fractions during recovery were the same as in the pre-insulin period. Hypoglycaemia decreased the cerebral content of the phospholipid fractions; the lecithin-sphingomyelin fraction returned toward normal during recovery, whereas the cephalins did not increase significantly. The electrographic patterns and the occurrence of convulsive activity are discussed, both in insulin hypoglycaemia and during recovery.     

The involvement of sucrose,glucose and other metabolites in the synthesis of triterpenes and dopa in the laticifers of Euphorbia lathyris

A quantitative triterpene analysis was made of latex stem tissue of Euphorbia lathyris. Young plants seedlings of E. lathyris were incubated with various labelled precursors. Incorporation into triterpenes was obtained from [2-¹⁴C]mevalonic acid, [1-¹⁴C]acetate, [3-¹⁴C]pyruvate, [U-¹⁴C]sucrose, [U-¹⁴C]glucose, [U-¹⁴C]xylose, [U-¹⁴C]glyoxylate, [2,3-¹⁴C]succinic acid, [1-¹⁴C]glycerol [U-¹⁴C]serine. Both sugars tyrosine appeared to be effective precursors in DOPA synthesis inside the laticifers. Exogenously supplied mevalonic acid was only involved in triterpene synthesis outside the laticifers. GC-RC of triterpenes synthesized from [U-¹⁴C]glucose revealed the origin of these compounds in the latex. The labelled triterpenes obtained after incorporation of the other mentioned labelled precursors were only partly synthesized in the laticifers. For quantitative data on latex triterpene synthesis seedlings were incubated with [U-¹⁴C]sucrose, [U-¹⁴C]glucose, [U-¹⁴C]xylose [1-¹⁴C]acetate in the presence of increasing amounts of unlabelled substrate. From the amount of ¹⁴C incorporated into the triterpenes the amount of substrate directly involved in triterpene synthesis was calculated, as was the absolute triterpene yield. Sucrose showed the highest triterpene yield, equivalent to the daily increase of the triterpene content of growing seedlings. The possible significance of the other precursors in triterpene synthesis in the laticifers is discussed.     

Effect of triperidol on carbohydrate and amino acid metabolism in rat brain-cortex slices

1. The effect of triperidol on the metabolism of glucose, pyruvate, glutamate, aspartate and glycine was studied with rat brain-cortex slices, U-¹⁴C-labelled substrates and a quantitative radiochromatographic technique. 2. Triperidol at a concentration of 0·2mm decreased the oxygen uptake and the ¹⁴CO₂ production by about 30% when glucose, pyruvate and glutamate were used as substrates, whereas no effects were observed with aspartate and glycine. 3. The drug did not alter qualitatively the metabolic pattern of the substrates. 4. Quantitatively, triperidol decreased the incorporation of ¹⁴C from [U-¹⁴C]glucose and [U¹⁴-C]-pyruvate into glutamate, glutamine and γ-aminobutyrate but not into lactate, alanine and aspartate. The overall utilization rates of glucose and pyruvate were decreased. The relative specific radioactivities of glutamate and aspartate were also decreased. 5. Triperidol increased the rate of disappearance of U-¹⁴C-labelled glutamate, aspartate and glycine from the incubation medium, and altered the distribution of their metabolites between medium and tissue. 6. No appreciable effect of triperidol on [1-¹⁴C]galactose disappearance was found.     

Studies in lipogenesis in vivo: Fatty acid and cholesterol synthesis during starvation and re-feeding

1. Lipogenesis in vivo has been studied in mice given a 250mg. meal of [U-¹⁴C]glucose (2·5μc) or given an intraperitoneal injection of 25μg. of [U-¹⁴C]glucose (2·0μc). 2. The ability to convert a [U-¹⁴C]glucose meal into fatty acid was not significantly depressed by 6–7hr. of starvation. In contrast, incorporation of ¹⁴C into fatty acid in the liver after the intraperitoneal dose of [¹⁴C]glucose was depressed by 80% and by more than 90% by 1 and 2hr. of starvation respectively. Carcass fatty acid synthesis from the [U-¹⁴C]glucose meal was not depressed by 12hr. of starvation, whereas from the tracer dose of [U-¹⁴C]glucose the depression in incorporation was 80% after 6hr. of starvation. 3. Re-feeding for 3 days, after 3 days' starvation, raised fatty acid synthesis and cholesterol synthesis in the liver fivefold and tenfold respectively above the levels in non-starved control mice. These increases were associated with an increased amount of both fatty acid and cholesterol in the liver. 4. After 18hr. of starvation incorporation of a [U-¹⁴C]glucose meal into carcass and liver glycogen were both increased threefold.     

Fluoride-Induced Inhibition of Starch Biosynthesis in Developing Potato, Solanum tuberosum L., Tubers Is Associated with Pyrophosphate Accumulation

Pretreatment of discs excised from developing tubers of potato (Solanum tuberosum L.) with 10 millimolar sodium fluoride induced a transient increase in 3-phosphoglycerate content. This was followed by increases in triose-phosphate, fructose 1,6-bisphosphate and hexose-phosphate (glucose 6-phosphate + fructose 6-phosphate + glucose 1-phosphate). The effect of fluoride is attributed to an inhibition of glycolysis and a stimulation of triose-phosphate recycling (the latter confirmed by the pattern of ¹³C-labeling [NMR] in sucrose when tissue was supplied with [2-¹³C]glucose). Fluoride inhibited the incorporation of [U-¹⁴C] glucose, [U-¹⁴C]sucrose, [U-¹⁴C]glucose 1-phosphate, and [U-¹⁴C] glycerol into starch. The incorporation of [U-¹⁴C]ADPglucose was unaffected. Inhibition of starch biosynthesis was accompanied by an almost proportional increase in the incorporation of ¹⁴C into sucrose. The inhibition of starch synthesis was accompanied by a 10-fold increase in tissue pyrophosphate (PPi) content. Although the subcellular localization of PPi was not determined, a hypothesis is presented that argues that the PPi accumulates in the amyloplast due to inhibition of alkaline inorganic pyrophosphatase by fluoride ions.     

Inhibition of glucose utilization for acetylcholine synthesis by cerebrospinal fluid.

Abstract— Replacement of bicarbonate-Locke incubation medium with feline CSF reduced [¹⁴C]ACh formation from [U-¹⁴C]glucose by rat brain mince approx 30%. CSF was obtained from a cannula leading to the cisterna magna of freely moving cats. The component of CSF responsible for inhibition was characterized as a dialyzable heat-stable organic anion. Choline acetyltransferase activity was not altered by CSF. [¹⁴C]ACh synthesis and ¹⁴CO₂ production from [U-¹⁴C]glucose but not from [2-¹⁴C]-pyruvate were inhibited by CSF, suggesting inhibition in the metabolism of glucose to pyruvate. The anionic fraction of human CSF was as potent as that from feline CSF in inhibiting ¹⁴CO₂ production from [U-¹⁴C]glucose. Brain hexokinase was inhibited by the anionic fraction of feline CSF. The inhibition was non-competitive with respect to glucose and uncompetitive with respect to ATP. It is suggested that inhibition of hexokinase by CSF was responsible at least in part for the inhibition of glucose metabolism which resulted in decreased [¹⁴C]ACh synthesis and ¹⁴CO₂ production.     

An Hg-sensitive channel mediates the diffusional component of glucose transport in olive cells

In several organisms solute transport is mediated by the simultaneous operation of saturable and non-saturable (diffusion-like) uptake, but often the nature of the diffusive component remains elusive. The present work investigates the nature of the diffusive glucose transport in Olea europaea cell cultures. In this system, glucose uptake is mediated by a glucose-repressible, H⁺-dependent active saturable transport system that is superimposed on a diffusional component. The latter represents the major mode of uptake when high external glucose concentrations are provided. In glucose-sufficient cells, initial velocities of d- and l-[U-¹⁴C]glucose uptake were equal and obeyed linear concentration dependence up to 100 mM sugar. In sugar starved cells, where glucose transport is mediated by the saturable system, countertransport of the sugar pairs 3-O-methyl-d-glucose/d-[U-¹⁴C]glucose and 3-O-methyl-d-glucose/3-O-methyl-d-[U-¹⁴C]glucose was demonstrated. This countertransport was completely absent in glucose-sufficient cells, indicating that linear glucose uptake is not mediated by a typical sugar permease. The endocytic inhibitors wortmannin-A and NH₄Cl inhibited neither the linear component of d- and l-glucose uptake nor the absorption of the nonmetabolizable glucose analog 3-O-methyl-d-[U-¹⁴C]glucose, thus excluding the involvement of endocytic mediated glucose uptake. Furthermore, the formation of endocytic vesicles assessed with the marker FM1-43 proceeded at a very slow rate. Activation energies for glucose transport in glucose sufficient cells and plasma membrane vesicles were 7 and 4 kcal mol^− 1, respectively, lower than the value estimated for diffusion of glucose through the lipid bilayer of phosphatidylethanolamine liposomes (12 kcal mol^− 1). Mercury chloride inhibited both the linear component of sugar uptake in sugar sufficient cells and plasma membrane vesicles, and the incorporation of the fluorescent glucose analog 2-NBDG, suggesting protein-mediated transport. Diffusive uptake of glucose was inhibited by a drop in cytosolic pH and stimulated by the protein kinase inhibitor staurosporine. The data demonstrate that the low-affinity, high-capacity, diffusional component of glucose uptake occurs through a channel-like structure whose transport capacity may be regulated by intracellular protonation and phosphorylation/dephosphorylation.     

Specific maltose labeling in the reducing glucose moiety.

Radioactive maltose with label in the reducing glucose moiety was prepared using a glucosyltransferase enzyme to catalyze exchange of [6-³H]glucose into unlabeled maltose. The enzyme was isolated from spinach by ammonium sulfate precipitation followed by DEAE column chromatography. A 77% yield of [6-³H]maltose was obtained after a reaction of 100 nmol of maltose with 0.0147 nmol of [6-³H]glucose was catalyzed by the most active column peak. The product was exclusively labeled in the reducing glucose moiety as indicated by the label occurring only in sorbitol following sodium borohydride reduction and sulfuric acid hydrolysis. Between 88.3 and 96.0% of the tritium in the synthesized preparation was present as [6-³H]maltose by Dowex 1-X4 chromatography. This column separates [6-³H]maltose-[U-¹⁴C]maltose mixtures and [6-³H]glucose-[U-¹⁴C]glucose mixtures apparently as a result of an isotope effect.     

Acetylcholine formation from glucose following acute choline supplementation

The effects of choline administration on acetylcholine metabolism in the central nervous system are controversial. Although choline supplementation may elevate acetylcholine (ACh) content in brain, turnover studies with labelled choline precursors suggest that systemic choline administration either has no effect or actually diminishes brain ACh synthesis. Since choline supplementation elevates brain choline levels, the apparent decreases in previous turnover studies may reflect dilution of the labelled choline precursor pool rather than altered ACh formation. Therefore, brain ACh formation from [U-¹⁴C]glucose was determined after choline supplementation. A two to three fold elevation of brain choline did not alter ACh levels or [U-¹⁴C]glucose incorporation into ACh in the cortex, hippocampus or striatum. Although atropine stimulated ACh formation from [U-¹⁴C]glucose in hippocampus, two to three fold increases in brain choline did not augment ACh synthesis or content in atropine pretreated animals. Atropine depressed brain regional glucose utilization and this effect was not reversed by choline treatment. These results suggest that shorttern elevation of brain choline does not enhance ACh formation from [U-¹⁴C]glucose, and argue against enhanced presynaptic cholinergic function after acute, systemic choline administration.Special issue dedicated to Dr. Louis Sokoloff.     

Insulin treatment can abolish changes in glucose and glutamine metabolism of lymphocytes and macrophages caused by the implantation of the walker 256 tumour

Activation of lymphocytes and macrophages by the implantation of tumour cells (10⁷ cells per rat) into the left flank of rats increased the conversion of glucose to lactate and of glutamine to glutamate and aspartate and the decarboxylation of [U-¹⁴C]-glucose and [U-¹⁴C]-glutamine in incubated cells. In addition, the amount of GLUT1 was increased in macrophages. The effect of insulin treatment on glucose and glutamine metabolism of lymphocytes and macrophages activated by Walker 256 tumour implantation was also examined. For this purpose, insulin was injected subcutaneously (4 U/100 g b.w. daily) after the fourth day of tumour implantation and the rats were killed 10 days afterwards. Insulin treatment fully reverted the changes due to tumour implantation in the metabolism of glucose and glutamine in lymphocytes and of glucose in macrophages.