In vitro sugar uptake by grapefruit (Citrus paradisi) juice-sac cells

To further our understanding of the mechanisms of sugar uptake and accumulation into grapefruit (Citrus paradisi Macf. cv. Marsh seedless), the patterns of uptake and utilisation of sucrose, glucose and fructose by Citrus juice cells was investigated. Analyses were conducted on sliced juice sacs that were incubated in radioactive [14C]-sugar solutions with unlabelled sugars, in the presence or absence of metabolic inhibitors. Both hexoses demonstrated an initial uptake peak in December and a second uptake peak in February–March. From March through April the rates of sucrose uptake increased to levels comparable to those of glucose and fructose. Sucrose and its moieties fructose and glucose entered the juice cells of Citrus juice fruit by an insaturable, and mostly by an independent, process. However, NaN3 and carbonylcyanide m-chlorophenylhydrazone (CCCP) produced slight inhibition of these processes. Cells took up hexoses at a greater rate than sucrose, with accumulation reaching a plateau by 4–8 h, and then continuing unabated, in the case of glucose, for 42 h. Uptake of all three sugars increased linearly in the range of sugar concentrations tested, which extended from 0.01 to 320 mm, denoting an insaturable system for sugar uptake. 14CO2 evolution was relatively low in all the experiments, the lowest evolution being recorded when the uptake of [14C]-sucrose was studied, while the highest 14CO2 evolution was recorded when the uptake of [14C]-glucose was studied. The data demonstrate a preferential utilisation of glucose over fructose and sucrose. In all the experiments, the two metabolic inhibitors significantly inhibited the decarboxylation of the three sugars.

Metabolism of cysteine in rat hepatocytes. Evidence for cysteinesulphinate-independent pathways

The metabolism of cysteine and cysteinesulphinate was studied in freshly isolated rat hepatocytes. Over 80% of the 14CO2 formed from [1-14C]cysteinesulphinate could be accounted for by production of hypotaurine plus taurine in incubations of rat hepatocytes with either 1 mM- or 25 mM-cysteinesulphinate. In similar incubations with 1 mM- or 25 mM-cysteine, less than 10% of 14CO2 evolution from [1-14C]cysteine could be accounted for by production of hypotaurine plus taurine. In incubations with cysteine, but not with cysteinesulphinate, the production of urea and ammonia was substantially increased above that observed in incubations without substrate. Addition of unlabelled cysteinesulphinate did not affect 14CO2 production from [1-14C]cysteine. Addition of 2-oxoglutarate resulted in a marked increase in cysteinesulphinate catabolism via the transamination pathway, but addition of neither 2-oxoglutarate nor pyruvate to the incubation system had any effect on cysteine catabolism. Inhibition of cystathionase with propargylglycine decreased 14CO2 production from [1-14C]cysteine about 50% and markedly decreased production of ammonia plus urea N; cysteinesulphinate catabolism by cysteinesulphinate-independent pathways in the rat hepatocyte and, furthermore, that cleavage of cyst(e)ine by cystathionase may be an important physiological pathway for cysteine catabolism in rat liver.

Cerebral glucose use measured with [14C]glucose labeled in the 1, 2, or 6 position

The efficacy of [14C]glucose molecules labeled in various positions as tracers of regional cerebral glucose utilization (rCMRGlc) was examined in rats. Arteriovenous differences of different [14C]-glucose species and 14CO2 were measured across brain to determine the relative rates of 14CO2 loss. As anticipated, 14CO2 evolution decreased in the order: [U-14C]glucose greater than [2-14C]glucose greater than [1-14C]glucose greater than [6-14C]glucose. Release of 14CO2 from [6-14C]glucose was undetectable at 5 min and barely detectable at 10 min, and release from [1-14C]glucose, which includes the pentose phosphate pathway, was only slightly greater. rCMRGlc was measured with [1-14C]-,[2-14C]-, or [6-14C]glucose in 5-min experiments. The results of [1-14C]- and [6-14C]glucose were indistinguishable; no difference due to the activity of the pentose phosphate pathway was found. Both [1-14C]- and [6-14C]-glucose gave values similar to, but on the whole slightly higher than, [2-14C]glucose. It was concluded that when knowledge of total rCMRGlc is required, [6-14C]glucose is the labeled substrate of choice. When the experimental objective is measurement of energy metabolism, use of [1-14C]glucose avoids inclusion of the nonenergy-yielding pentose phosphate pathway.

[U-14C]glucose, -alanine, and -leucine oxidation in rats at rest and two intensities of running

The oxidations of injected [U-14C]glucose, [U-14C]alanine, and [U-14C]leucine were investigated in laboratory rats during rest or 2 h of easy and hard treadmill running. After [U-14C]glucose injection, the rate and magnitude of 14CO2 evolution were relatively low at rest and increased as a linear function of metabolic rate (VO2). Evolution of 14CO2 after [U-14C]alanine injection was faster and larger during exercise than rest. The peak of alanine decarboxylation occurred before glucose and, therefore, did not reflect conversion of alanine to glucose prior to decarboxylation. The rate and magnitude of 14CO2 evolution after [U-14C]leucine injection were proportional to metabolic rate, but less than after glucose or alanine injection. During exercise, levels of alanine and leucine in muscle and blood were unchanged or elevated compared to rest. During exercise, alanine levels were unchanged or increased in liver. Liver leucine levels were depressed when exercise began, but increased toward control values during exercise. The metabolism of selected amino acids is joined to carbon flow sustaining exercise.

RHEUMATOID SERA AND SOLUBLE COMPLEXES: NITROBLUE TETRAZOLIUM DYE TEST AND HEXOSE MONOPHOSPHATE SHUNT ACTIVATION

An increased number of cells containing reduced dye in the nitroblue tetrazolium (NBT) test were observed when peripheral blood leukocytes of adult rheumatoid arthritis (RA) patients having rheumatoid factor (RF), as well as RF-negative pediatric RA patients were studied. Therefore, the relationship between soluble antigen -antibody complexes and NBT dye reduction following phagocytosis was investigated. After intravenous injection of 131I labeled bovine serum albumin (IBSA) into six rabbits, the mean onset of immune clearance was 8.2 days (range, 7 to 9). NBT-positive cells increased from control levels (3% to 10%) at four to six days and were maximal at six days. The mean maximal response was 35% (range, 22% ot 47%). In vitro studies of the hexose monophosphate (HMP) shunt activity of human leukocytes exposed to BSA-anti-BSA complexes demonstrated a twofold to threefold stimulation in the range of slight antigen excess. Leukocyte NBT reduction was not consistently increased above that of the controls. However, when high titer RF sera was incubated with normal human leukocytes, maximal NBT responses were observed when the RF titer was 200 to 600 units (mean, 27%; controls, 2% to 8%) and 14CO2 evolution via the HMP shunt was accelerated. RF-negative sera from children with RA elicited a slight increase in NBT-positive cells and a definite increase in 14CO2 evolution was observed. It was then concluded that soluble antigen-antibody complexes in slight antigen excess altered hexose monophosphate shunt activity and increased NBT dye reduction. The NBT-positive cell in RA may be due to phagocytosis of RF accompanied by metabolic activation.

Organ-culture studies of achondroplastic rabbit cartilage: evidence for a metabolic defect in glucose utilization

Organ-culture studies were made using cartilage from achondroplastic (dwarf) rabbits (ac/ac) and their phenotypically normal litter-mates. A significantly higher incorporation of 14C from glucose and galactose was measured in the dwarf; incorporation of 35S from sulfate and 3H from thymidine was equal in the two types of explant. Also, 14CO2 and [14C]lactate production from glucose by the dwarf cartilage was increased. No difference in the ratio of 14CO2 evolution from [1-14C]- and [6-14C]glucose was found between the two cartilage types. Explants of dwarf cartilage utilized more glucose from the medium than did the controls. Radioautographs of tissue sections from the explants showed an increased number of grains from [14C]glucose overlying the dwarf cartilage, and this difference was particularly great over the central portions of cartilage. The proportion of 14C grains from glucose was greater over the dwarf nuclei, less over the dwarf matrix and equal over the cytoplasm of the two tissues. Grain counts of sulfate and of thymidine did not differ in the two types of cartilage.