Effects of intra-peritoneal injections of lithium chloride on the entry of radioactive carbon atoms of glucose and amino acids into mouse brain and other tissues.
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One hundred grams of glucose with 50 microCi U-14C-glucose were given orally to 17 women with widely varying amounts of body fat. Radioactivity and glucose metabolism in vitro were then measured in adipose tissue obtained by needle biopsies in the abdominal and femoral regions after four hours. Radioactivity in triglycerides was then measured in repeated biopsies 1 day, 1 week, and monthly up to 7 months after glucose administration. Glucose label in triglycerides after four hours was higher in abdominal than femoral adipocytes in obese women. It increased slightly during the following week, and then decreased exponentially with a half-life of 12 months in the abdominal region and 19 months in the femoral region. Uptake of glucose carbon in total body fat was estimated from the triglyceride label measured and determinations of body fat mass, and found to be in the order of less than 4% of given glucose. The studies in vitro suggested that much of the glucose taken up in adipose tissue is converted to lactate. If this is the case in vivo, then glucose uptake in adipose tissue might well be of significance for total body glucose homeostasis, particularly in obese subjects, amounting to maximally perhaps one third to one half of the oral glucose given. The majority of this glucose uptake would then, however, leave adipose tissue again as lactate. The shorter half-life of label in abdominal adipocytes is in agreement with findings of increased lipolysis in these adipocytes in vitro.
Cells of proso millet (Panicum miliaceum L. cv Abarr) in liquid culture and leaves of maize seedlings (Zea mays L. cv LH51 x LH1131) readily incorporated d-[U-(14)C]glucose and l-[U-(14)C]arabinose into soluble and cell wall polymers. Radioactivity from arabinose accumulated selectively in polymers containing arabinose or xylose because a salvage pathway and C-4 epimerase yield both nucleotide-pentoses. On the other hand, radioactivity from glucose was found in all sugars and polymers. Pulse-chase experiments with proso millet cells in liquid culture demonstrated turnover of buffer soluble polymers within minutes and accumulation of radioactive polymers in the cell wall. In leaves of maize seedlings, radioactive polymers accumulated quickly and peaked 30 hours after the pulse then decreased slowly for the remaining time course. During further growth of the seedlings, radioactive polymers became more tenaciously bound in the cell wall. Sugars were constantly recycled from turnover of polysaccharides of the cell wall. Arabinose, hydrolyzed from glucuronoarabinoxylans, and glucose, hydrolyzed from mixed-linkage (1-->3, 1-->4)beta-d-glucans, constituted most of the sugar participating in turnover. Arabinogalactans were a large portion of the buffer soluble (cytoplasmic) polymers of both proso millet cells and maize seedlings, and these polymers also exhibited turnover. Our results indicate that the primary cell wall is not simply a sink for various polysaccharide components, but rather a dynamic compartment exhibiting long-term reorganization by turnover and alteration of specific polymers during development.
Glucose is the principal fuel for brain metabolism and its movement across the blood-brain barrier depends on Glut1. Impaired glucose transport to the brain may have deleterious consequences. For example, Glut1 deficiency syndrome (Glut1DS) is the result of heterozygous loss of function Glut1 mutation leading to energy failure of the brain and subsequently, epileptic encephalopathy. To preserve the integrity of the energy supply to the brain in patients with compromised glucose transport function, consumption of compounds with glucose transport inhibiting properties should be avoided. Phenytoin is a widely used anticonvulsant that affects carbohydrate metabolism. In this study, the hypothesis that phenytoin and its metabolite 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH) affect cellular glucose transport was tested. With a focus on Glut1, the effects of phenytoin and HPPH on cellular glucose transport were studied. Glucose uptake assay measuring the zero-trans influx of radioactive-labeled glucose analogues showed that phenytoin and HPPH did not exert immediate effects on erythrocyte Glut1 activity or glucose transport in Hs68 control fibroblasts, Glut1DS primary fibroblasts isolated from two patients, or in rat primary astrocytes. Prolonged exposure to the two compounds could stimulate glucose transport by up to 30-60% over the control level (p <0.05) in Hs68 and Glut1DS fibroblasts as well as in rat astrocytes. The stimulation of glucose transport by HPPH was dose-dependent and accompanied by an up-regulation of GLUT1 mRNA expression (p <0.05). In conclusion, phenytoin and HPPH do not compromise cellular glucose transport. Prolonged exposure to these compounds can modify carbohydrate homeostasis by up-regulating glucose transport in both normal and Glut1DS conditions in vitro.
Measurement of glucose turnover under non-steady-state conditions has proven problematic. When the mass of the glucose pool is not changing (i.e., glucose concentrations are constant) non-steady-state error can be minimized if all glucose entering the circulation has the same specific activity as plasma [radioactive infused glucose (hot-GINF) method]. Alternatively, a second tracer can be used to measure the effective volume of glucose [variable-pV method of Issekutz (T. Issekutz, R. Issekutz, and D. Elahi. (Can. J. Physiol. 52:215-224, 1974)]. To determine whether these techniques provide concordant assessments of insulin action under non-steady-state conditions, glucose turnover was measured in six subjects. After initiation of insulin (0.6 mU.kg-1 x min-1), both methods indicated similar rates of suppression of hepatic glucose release, which was complete by approximately 100-120 min. In contrast, the traditional fixed-pV method of Steele (R. Steele, J. Wall, R. DeBodo, and N. Altszuler. Am. J. Physiol. 187:15-24 1956) underestimated turnover (P < 0.01) resulting in apparent complete suppression of glucose release within approximately 40 min (P < 0.01 vs. other methods). The hot-GINF and variable-pV methods also yielded similar estimates of turnover after discontinuation of insulin. Both indicated that resumption of hepatic glucose release was slower (P < 0.01) and fall of glucose uptake faster (P < 0.01) than suggested by the fixed-pV method. Thus both the hot-GINF and variable-pV methods avoid non-steady-state error introduced by the fixed-pV method and provide concordant assessments of the rate of onset and offset of insulin action.
Calf pancreas microsomes incorporated radioactively labeled D-glucose from UDP-D-glucose into products extracted with chloroform/methanol (2:1, v/v), chloroform/methanol/water (10:102.5, v/v), and into the residual precipitate, with a pH optimum in Tris/maleate buffer of about 5.3. The chloroform/methanol extract contained a single 14C-labeled acidic product, which was identified as dolichyl beta-D-glucosyl phosphate. It was stable to mild alkali, yielded D-[14C]glucose upon mild acid hydrolysis, and a 14C-labeled compound with the chromatographic mobility of 1,6-anhydro-beta-D-glucopyranosyl upon hot alkali treatment. The [14C]glucolipid had the same chromatographic mobility as dolichyl beta-D-[14C]mannosyl phosphate, and its formation was stimulated by exogenous dolichyl phosphate. The chloroform/methanol/water extract contained radioactive lipid-bound oligosaccharides which were retained on DEAE-cellulose more strongly than dolichyl D-[14C]glucosyl phosphate. They were stable to mild alkali, but labile to acid and hot alkali. Acid treatment yielded a D-glucose-labeled oligosaccharide fraction which was shown by gel filtration to be slightly larger than most of the D-mannose-labeled oligosaccharides. About 80% of the radioactive D-glucose residues could be removed with alpha-glucosidase, but not with beta-glucosidase. Pancreatic dolichyl beta-D-[14C]glucosyl phosphate incubated with calf pancreas microsomes served as direct donor of D-glucosyl residues to lipid-bound oligosaccharides and to the precipitate. These oligosaccharides had the same size as those labeled from UDP-D-[14C]glucose, and the D-[14C]glucose residues could also be removed with alpha-glucosidase.
1. Glucose kinetics were assessed in seven normal adult male subjects by an intravenous bolus technique with the use of a non-radioactive isotopically labelled preparation, [6,6-2H]glucose, as tracer. Tracer enrichment in plasma was assessed by gas chromatography-mass spectrometry. For comparison five subjects also received a simultaneous intravenous bolus of [6-3H]glucose and kinetics were assessed by conventional means. 2. Administration of [6,6-2H]glucose did not alter circulating glucose or insulin concentrations. 3. Glucose turnover, assessed by the use of [6,6-2H]glucose, was 11.4 (+/- 0.9) micromol min-1 kg-1 and 11.6 (+/- 0.5) micromol min-1 kg-1 with rate of glucose was 2.3 (+/- 0.3) ml min-1 kg-1 with both isotopically labelled tracers. Estimates of mean residence time, glucose pool and glucose space were also similar by each technique. 4. [6,6-2H]Glucose is therefore an effective tracer and allows investigation of glucose kinetics without administration of a radioactive label.
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Glycogen synthesis in isolated perfused livers or livers of anesthesized rats (in situ), was studied using radioactively labelled fructose, lactate, and inositol as substrates. The specific radioactivity of glucose and glycogen was measured at various times and compared with that of some intermediates. The results suggest that liver glycogen is formed from the pool of free glucose which in turn is fed by the so-called "direct and indirect pathway" of glycogen synthesis. This points to an important role of glucose-6-phosphatase, an enzyme complex subject to regulation by glucocorticoids, well known promoters of hepatic glycogen synthesis.
OBJECTIVE: The 2-[(18)F]-fluoro-2-deoxy-D-glucose-positron emission tomography (FDG-PET) technique provides information on uptake and metabolism of glucose in various tissues. Compared with resting cells, activated lymphocytes take up radioactively labeled glucose analog at a higher rate, which makes it possible to identify lymphoid organs with higher concentrations of activated lymphocytes. This study was undertaken to compare the pattern of PET images and quantitative FDG uptake in lymphoid organs of patients with active systemic lupus erythematosus (SLE) versus patients with inactive SLE and to correlate these findings with peripheral blood lymphocyte phenotypes. METHODS: Ten patients with active SLE and 9 patients with inactive SLE were studied. FDG-PET images were obtained from the inguinal region to above the ear, starting at 60 minutes after injection of FDG. Standardized uptake values using lean body mass were determined over areas of interest. RESULTS: Both patients with active lupus and those with inactive lupus had increased FDG uptake in lymph nodes when compared with healthy volunteers, and there was no statistically significant difference between the 2 groups of lupus patients. Thymic uptake was demonstrated in 5 of 10 patients with active lupus compared with 0 of 9 patients with inactive disease. Three of the 5 patients with active SLE who were over 29 years of age had thymic uptake. Of the activation markers tested, only the CD3/CD71 population of cells was significantly different between the patient groups, with an increased percentage in the active disease group (P = 0.0247). CONCLUSION: Increased FDG uptake in lymph nodes of both patients with active SLE and patients with inactive SLE suggests that metabolic, and probably immunologic, activity is enhanced not only in active, but also in clinically quiescent, disease. The increased thymic uptake observed only in patients with active disease suggests that the thymus plays an important role during periods of disease activity.
The effects of glucose starvation on glycogen synthase (GS) activity and protein expression were investigated. Fibroblasts were cultured in medium supplemented with either glucose or pyruvate. Pyruvate-cultured cells exhibited UDP-glucose contents that amounted to approximately 10% of those in cells cultured with glucose. GS activity, protein and mRNA amounts in pyruvate-cultured cells were decreased to approximately 35, 60, and 60%, respectively, of values in glucose-cultured cells. Incubation of extracts from glucose-cultured cells with radioactive UDP-glucose resulted in substantial binding of ligand to immunoprecipitated GS. However, binding in immunoprecipitates from pyruvate-cultured cells was decreased to approximately 25% of values in glucose-cultured cells. These data indicate that glucose starvation and the subsequent depletion of UDP-glucose result in: (1) inactivation of GS, owing to a decrease in its ability to bind UDP-glucose, and (2) decreased amount of GS protein, owing to a decrease in the levels of GS mRNA.
1. To determine the effect of diet and level of energy intake on glucose metabolism in sheep, four dietary treatments consisting of feeding a low-roughage (LR) and a high-roughage (HR) diet at each of two intake levels estimated to provide 586 and 1172 kJ (140 and 280 kcal) digestible energy (DE)/kg body-weight-0.75 per d were given to each of eight yearling rams in four different time periods each of 4 weeks duration. Both diets contained 140 g crude protein/kg using ground maize, mixed hay and soya-bean meal and were given in two meals/d. Estimated DE values of food were verified during the study and actual intakes of DE were within 9.5% of the estimated values. 2. To study glucose metabolism, a single intravenous injection of ]2-3H]glucose and subsequent withdrawal of nine venous blood samples with 3 h were made in each experiment. Two experiments were conducted on consecutive days for each sheep on each dietary treatment. 3. Coefficients of determination (r2) for linear regressions to measure the effect of time after a single injection of [2-3H)glucose on log specific radioactivity of plasma glucose were calculated for fifty-eight experiments. In fifty-six of the experiments, r2 values exceeding 0-95 were obtained. 4. Compared to the HR diet, the LR diet increased (P smaller than 0-05) the pool size and decreased (P smaller than 0-05) the half-life of glucose. At both intake levels, the LR diet increased (P smaller than 0-05) the plasma concentration and the entry rate of glucose compound to the HR diet but interaction (P smaller than 0-05) between diet and intake level was attributed to a greater difference obtained between diets at the higher compared to the lower level of food intake. Increasing the level of intake caused a greater (P smaller than 0-05) pool size and space, and a shorter (P smaller than 0-05) half-life of glucose. 5. It was concluded that substitution of roughage by concentrate in a ruminant's diet may increase the rate of glucose entry during a short time period after eating.
The contribution of the kidneys to postabsorptive endogenous glucose production is a matter of controversy. To assess whether this could relate to the use of various isotopical methods with different analytical performance capabilities, we measured glucose kinetics in 12 healthy subjects. Blood samples were taken from the femoral artery and the renal vein after 4 h of [6,6-2H2]glucose infusion (for gas chromatography [GC]/mass spectrometry [MS] analysis), and renal plasma flow was determined with paraaminohippurate. In addition, six subjects received uniformly labeled [13C]glucose (for GC/combustion/isotope ratio MS [IRMS]) and [3-3H]glucose (for counting of radioactive disintegrations). Arterial glucose concentrations (means +/- SD) were 4.2+/-0.1 mmol/l, and endogenous glucose production rates using [2H2]glucose were 2.2+/-0.1 mg x kg(-1) x min(-1) or 818+/-50 micromol/min. Dilution of [2H2]glucose across the kidney was 0.79+/-1.32%, and renal glucose production (RGP) rates were 27+/-72 micromol/min. In the six subjects receiving additional tracers, dilutions across the kidney were 2.83+/-0.72 and 0.54+/-1.20 (for [U-13C]glucose and [3-3H]glucose, respectively, the dilution with [U-13C] being higher than that with [2H2] (P = 0.007). Corresponding RGP values were 144+/-39 and 43+/-76 micromol/min for [U-13C] and [3-3H], respectively. In conclusion, we found that the highly sensitive [U-13C] GC/Combustion/IRMS technique showed consistent dilution of label across the kidney, whereas the less sensitive techniques gave some negative values and smaller RGP rates. Thus, depending on which technique is being used, a fivefold difference in calculated RGP values may be encountered. The methodological variability of our data suggests that extrapolation from regional renal measurements to the whole-body level should be perfumed with caution.
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An oral gavage of either 3, 1 or 0.1 mmoles of 14C-labelled glucose was given to rats under standard feeding conditions or food deprived for 24 hr. The fate of the glucose label was determined at 10, 15, 30 and 60 min after gavage; at 60 min 40% of the glucose was absorbed in fed rats (60% in food deprived). The portal vein blood flows were determined and the levels of glucose, lactate, alanine and pyruvate, and their radioactivity, as well as that of CO2 were measured in both portal and arterial blood. The net computed glucose and 3-carbon carriers (lactate, alanine and pyruvate) actually released into the portal system by the intestine was lower than the amount of glucose taken up from the intestinal lumen in one hour. Oxidation to 14CO2 accounted for a 12-15% of the absorbed glucose. The size of the gavage deeply affected the proportion of glucose released into the portal blood (c. 50% with a 3 mmoles gavage and practically nil with a 0.1 mmoles gavage), but it affected much less the generation of lactate and other 3 C carriers. In fed rats, the net intestinal balance of non-radioactive glucose was negative, and that of lactate positive; when radioactive glucose was considered, the pattern was inverted. In starved rats, both glucose and lactate were released in large proportions by the intestine, but alanine efflux was lower. It can be concluded that the intestine consumes a considerable proportion of glucose in the fed state. Glucose handling by the intestine is compartmentalized in two functional circuits: glucose is taken up from the arterial blood and used for intestinal metabolism and lactate production, luminal glucose is absorbed mainly unaltered and transferred to the portal blood. Thus, the generation of lactate is mainly related to the availability of arterial glucose. In addition to the release of the ingested glucose as 3 C carriers or glucose, an extraportal pathway for glucose transfer into the bloodstream is postulated.
Unsporulated oocysts of Eimeria tenella contain large quantities of carbohydrates, namely amylopectin, mannitol and glucose. Analysis of carbohydrate content of sporulating oocysts revealed that mannitol content increased markedly during early stages of sporogony (first 4-6h) but slowly diminished during the next 40h of sporulation. Accumulation of mannitol was accompanied by a rapid decrease in amylopectin and free glucose, suggesting that mannitol might be synthesized from glucose released from amylopectin. Mannitol was also detected in sporozoite and merozoite extracts. All four mannitol cycle enzymes were detected in oocysts. Sporozoites excysted in vitro had lower activities of all four enzymes. Mannitol-1-phosphatase and mannitol dehydrogenase activity was also detected in merozoites obtained from the second stage schizonts. Sporozoites incubated with 14C-glucose accumulated radioactively labelled precursor continuously for over 12h and some of the 14C-glucose was converted into 14C-mannitol. These results indicate that mannitol plays an important role in the metabolism and development of the intracellular stages of the parasite.
Omega-Cyclohexyl undecanoic acid and omega-cyclohexyl tridecanoic acid were found in 10 strains of acido-thermophilic bacteria isolated from different Japanese hot springs. These unusual fatty acids were found in the esterified form in glyceride type complex lipids and constituted 74 to 93% of the total fatty acids in the bacteria. The fatty acids other than omega-cyclohexyl fatty acids found were 14-methyl hexadecanoic acid (3 to 15%) and 15-methyl hexadecanoic acid (1 to 6%), and trace amounts of straight chain and methyl-branched tetra- and penta-decanoic acids. Biosynthesis of omega-cyclohexyl fatty acids increased with increase in the concentration of glucose in the culture medium. The metabolism of omega-cyclohexyl fatty acids was studied using deuterium-labeled precursors by mass fragmentation analysis. The deuterium of [2-D]glucose was specifically incorporated into position 2 of the cyclohexyl ring of the fatty acids, indicating that the ring was synthesized from the glucose molecule. Radioactivity was efficiently incorporated into the omega-cyclohexyl fatty acids from labeled glucose, shikimate, and cyclohexyl carboxylate. These findings indicate that omega-cyclohexyl fatty acids are synthesized with glucose through shikimic acid and probably cyclohexyl carboxylyl-CoA derivative as the intermediates.
1. Measurable incorporation of radioactive carbon from [U-14C]pyruvate, [U-14C]-glutamate and [14C]bicarbonate into the glycogen synthesized by brain slices in vitro was demonstrated. 2. The fructose diphosphatase activity of guinea-pig brain was determined and found to be about 0.03 mumol of substrate degraded/min per g of fresh tissue. 3. The specific radioactivity of the glucose carbon from glycogen relative to that of the precursor added to the incubation medium gave approximate values of 0.195 for glucose, 0.006 for pyruvate, 0.039 for glutamate and 0.001 for bicarbonate.