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Glucose consumption and utilization of amino acids, lipids and proteins was measured in the rat brain under normoxia and hypoxia (7%O2:93%N2). After 2 h of hypoxia the brain glucose consumption increased by over 60% of control value. In spite of this increase, the radioactivity of amino acid fraction did not increase or parallel changes of glucose radioactivity in the blood. This strongly suggested that glucose flux into amino acids remained unchanged in hypoxia. Incorporation of 14C from glucose into macromolecules was found to decrease. The above changes demonstrated that the metabolic steps which precede synthesis of amino and tricarboxylic acids were inhibited. In some experiments, the incorporation of 14C from [2-14C]-acetate into a macromolecular fraction was also measured. The amounts of radioactivity found in these fractions were unchanged under hypoxic conditions. Possible differences in the influence of hypoxia on macromolecular synthesis in different metabolic compartments of the brain are discussed.
The treatment of rats for 4 h with 6-aminonicotinamide (60 mg kg-1) resulted in an 180-fold increase in the concentration of 6-phosphogluconate in their brains; glucose increased 2.6-fold and glucose 6-phosphate, 1.7-fold. Moreover, lactate decreased by 20%, glutamate by 8% and gamma-aminobutyrate by 12%, and aspartate increased by 10%. No significant changes were found in glutamine and citrate. In blood, 6-phosphogluconate increased 5-fold; glucose, 1.4-fold and glucose 6-phosphate, 1.8-fold. The metabolism of glucose in the rat brain, via both the Embden-Meyerhof pathway and the hexose monophosphate shunt, was investigated by injecting [U-14C]glucose or [2-14C]glucose, and that via the hexose monophosphate shunt alone by injecting [3,4-14C]glucose. The total radioactive yield of amino acids in the rat brain was 5.63 mumol at 20 min after injection of [U-14C]glucose, or 5.82 mumol after injection of [2-14C]glucose; by contrast, it was 0.62 mumol after injection of [3,4-14C]glucose. The treatment of rats with 6-aminonicotinamide showed significant decreases in these values, owing to decreases in the radioactive yields of glutamate, glutamine, aspartate, gamma-aminobutyrate, and alanine+glycine+serine. Glutamate isolated from the brain contained approximately 43% of its radioactivity in carbon 1 after injection of [3,4-14C]glucose, in contrast to 13% and 18% after injection of [U-14C]glucose and [2-14C]glucose, respectively, in both the control and treated rats. The calculations based on these findings showed that approximately 69% of the 14C-labelled glutamate was formed from [14C]acetyl coenzyme A (acetyl CoA) and the residual 31% by 14CO2 fixation of pyruvate after injection of [3,4-14C]glucose in both control and treated rats. The results gave direct evidence that glutamate and gamma-aminobutyrate in the brain were formed by metabolism of glucose via the hexose monophosphate shunt as well as via the Embden-Meyerhof pathway. From the radioactive yields of glutamate formed via [14C]acetyl CoA it was estimated that approximately 7.8% of the total glucose utilized was channelled via the hexose monophosphate shunt. Assuming that [14C]glutamate formed by carbon-dioxide fixation of pyruvate was also dependent on the metabolism of glucose through the hexose monophosphate shunt, the estimated value was approximately 9.5% of the total glucose converted into glutamate. The results of the present investigation, taken in conjunction with other findings, suggest that the utilization of glucose via the hexose monophosphate shunt is functionally important in the rat brain.
Squash (Cucurbita pepo L. var. melopepo torticalis, Bailey) leaves were supplied with (14)C-sucrose, then specific radioactivities of the glucose and galactose moieties of translocated stachyose were determined. In every case, the specific radioactivity of the galactose moiety was greater than that of the glucose moiety. It is concluded that the stachyose was not synthesized at either the phloem-loading site or subsequent to phloem loading, but rather in cells that were not a part of the translocation system, possibly the mesophyll cells.
The incorporation of [1-14C]mannose into hamster liver glycolipids and glycoproteins was studied in normal and vitamin A-depleted hamsters. Severly (25% weight loss) and mildly (no weight loss) deficient animals were compared to vitamin A-fed controls. The incorporation of [14C]mannose into glycolipids and glycoproteins decreased in mild and severe vitamin A deficiency by 63-90% compared to vitamin A-fed animals. These results were essentially the same whether expressed per g of wet liver, per DNA or per protein. The size of the pools of mannose, glucose and galactose and their specific radioactivity in liver were determined by gas-liquid chromatography of the boronates of the hexitols (Eisenberg, Jr, F. (1972) Methods Enzymol. XXVIIIB, 168-178) in normal and vitamin A-deficient conditions. It was found that the amount of free hexoses per g of liver was very similar in normal and vitamin A-deficient conditions. The specific radioactivities for mannose and glucose were greater in vitamin A deficiency, thus excluding the possibility that the observed severe decrease in glycopeptide and glycolipid synthesis is a reflection of a similar decrease in the specific radioactivity of the precursor pools. Quantitation of mannose in glycoprotein showed a 79% decrease in vitamin A deficiency. Specific radioactivity of mannose in glycoproteins, 20 min after injection of the label, was 187 dpm/mug of mannose in the normal and 48 kpm/mug of mannose in the vitamin A-deficient livers. It is concluded that vitamin A is necessary for the biosynthesis of liver mannose-containing glycoproteins and glycolipids.
1. Chronically cannulated sheep foetuses and suckling lambs were injected with 14C-labelled glucose, fructose or lactate, and sequential blood samples taken under conditions of minimal stress and without anaesthesia. 2. Gluconeogenesis from lactate was not detectable in foetal sheep, but the pathway was active in suckling lambs. 3. Fructose utilization rates were low in foetal sheep, with no measurable conversion into glucose or lactate. 4. The high rates of irreversible loss of both glucose and lactate in the foetus were decreased in suckling lambs. Radioactivity from labelled glucose entered both the lactate and fructose pools in foetal sheep, and entered the lactate pool in suckling lambs. 5. A model is proposed in which carbon flow between glucose, fructose and lactate has been quantified in foetal sheep.
OBJECTIVE: To evaluate the effects of acidity, glucose degradation products (GDP), and different solution buffer systems on solute and fluid transport during acute peritoneal dialysis (PD) in rats. DESIGN: Dialysis fluid (16 mL) containing 2.5% glucose as the osmotic agent was instilled intraperitoneally in Wistar rats (280 g) via a thin catheter in dwells lasting 4 hours. Blood and dialysis fluid samples (25 microL) were taken for measurement of glucose, sodium, and radioactive markers. The mass transfer area coefficient (MTAC or PS) for glucose and for 51Cr-EDTA (given as an intravenous infusion) and the peritoneal clearance (Cl) of 125I albumin (RISA), as well as the clearance of RISA to plasma (Cl --> P) were assessed for a commercial, heat-sterilized, acidic PD solution (2.5% glucose, pH 5.5; Gambrosol, Gambro, Lund, Sweden), containing GDP, and for four filter-sterilized solutions containing either lactate (40 mmol/L, pH 5.5 or 7.2), bicarbonate (38 mmol/L, pH 7.2), or pyruvate (40 mmol/L, pH 7.2) as buffers and being devoid of GDP. RESULTS: The initial pH of the acidic solutions increased rapidly, and attained physiological levels within 40 minutes. The initial drop of sodium, which is expected during the first part of the dwell, was significantly more pronounced with neutral than with acidic lactate. The PS for glucose and 51Cr-EDTA were slightly, but significantly, higher with the acidic and heat-sterilized solution (Gambrosol) than with the neutral, sterile-filtered lactate-buffered solution (p < 0.01), especially early during the dwell. Such an increase may be due to initial vasodilatation, and hence, recruitment of capillaries by the combination of acidity and GDP. However, there were no significant differences with respect to small solute PS values among sterile-filtered solutions, regardless of the presence of acidity or of buffer choice. CONCLUSION: There were no major differences in fluid and solute transport among sterile-filtered PD solutions having differing buffer systems and pH. Neither were there any effects of GDP alone. However, the combination of a low pH and the presence of GDP in the PD solutions seemed to cause significant increases in peritoneal small solute transport.
Crossbred barrows were used for in vivo studies investigating hormonal regulation of lipogenesis. The first experiment examined an in vivo method for determining rates of lipogenesis. Three barrows were infused with [U-14C]glucose and incorporation of radioactivity into triglycerides was determined in up to five biopsies of subcutaneous adipose tissue obtained over 7 h. Incorporation was linear after blood glucose specific radioactivity had reached a plateau and was constant over the entire infusion. For the second experiment, eight pigs (71 +/- 2.5 kg) were allocated to one of two treatments involving daily injections of excipient (control) or porcine somatotropin (pST; 120 micrograms/kg of BW). On d 10, beginning 15 h after injection, glucose incorporation into adipose tissue lipid was determined under both basal and hyperinsulinemic/euglycemic conditions. Basal glucose incorporation into lipid, particularly fatty acids, was markedly reduced (greater than 90%) during pST treatment. Although glucose incorporation was increased to a similar extent in both groups by hyperinsulinemia, the pST-treated pigs still exhibited markedly lower rates. Based on kinetic data, the decrease in lipid accretion of pST-treated pigs was primarily the result of a decrease in the rate of de novo synthesis. Furthermore, the reductions in glucose incorporation into fatty acids, glucose irreversible loss rate, and feed intake that occur with pST treatment were quantitatively similar.
The metabolism of, and retention of radioactivity from, radiolabelled glucose, glutamine and pyruvate were measured in individual cattle embryos produced in vitro from the 2-cell to hatched blastocyst stage. Uptake was defined as the numeric sum of metabolism and retention of radiolabel. Glucose metabolism increased significantly between the 8- and 16-cell stages, but was accompanied by a much larger increase in glucose uptake. Consequently, the proportion of glucose uptake that was metabolized through the pentose-phosphate and Embden-Meyerhof pathways reached a minimum at those stages. From the compacted morula stage onward, the calculated uptake of [14C]glucose was only 25 to 33% of that calculated for [5-3H]glucose. This suggests that 66 to 75% of glucose carbon leaves the embryo, after metabolism to phosphoenolpyruvate, in some form other than CO2. Little or no glucose metabolism by the Krebs cycle could be detected at any stage. Both glutamine and pyruvate metabolism were relatively high at the 2- and 4-cell stages, declined to a minimum at the compacted morula stage and then increased with blastulation. Glutamine metabolism continued to increase with expansion and hatching of the blastocyst, but pyruvate metabolism did not. This suggest that, relative to the activity of the pathway from pyruvate to 2-oxoglutarate, the activity of the 2-oxoglutarate-to-oxaloacetate segment of the Krebs cycle is of increasing significance during expansion and hatching of the cattle blastocyst.
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Cell membranes from etiolated Pisum sativum (pea) tissues were separated by ultracentrifugation on linear sucrose density gradients and assayed for membrane marker and glycosyltransferase activity. Membrane fractions were shown to incorporate glucose from UDP-D-[14C]glucose into polysaccharides with glycosyl linkages consistent with synthesis of xyloglucan. A combined assay using g.c., radiogas proportional counting and m.s. was employed to determine the identities of 14C-labelled glycosyl residues and the glycosyl linkages between them. In glucan synthase I assays, membrane fractions enriched for Golgi membranes showed 14C incorporation into 4- and 4,6-glucose residues, with minor incorporation into 3-glucose residues. In glucan synthase II assays, all 14C incorporation was into 3- and 3,4-glucose. There was a shift in glycosyl linkage of 14C incorporation from predominantly 4-glucose at low UDP-glucose concentration to predominantly 3- and 3,4-glucose at high UDP-glucose concentrations. Mn2+ stimulated incorporation of radioactivity into 4,6-glucose residues characteristic of xyloglucan polysaccharides. Addition of exogenous UDP-xylose to assay mixtures stimulated incorporation into 4,6-glucose, with a maximum at 15 microM UDP-xylose.
This study describes the contribution of de novo glucose synthesis by the kidney to blood glucose homeostasis in rats. The net glucose release by the kidney in vivo was measured by an isotope-dilution method, which calculated the extent of dilution of injected [(14)C]glucose by glucose newly synthesized in the kidney. The extent of dilution was determined from the difference between the decrease of the actual blood glucose concentration and that of the radioactivity of [(14)C]glucose, after injecting [(14)C]glucose into functionally hepatectomized rats. The results indicate that the net glucose release by the kidney in vivo in normal fed rats was 0.75+/-0.13 mg/dl per min, and that its contribution to blood glucose was 25.9+/-5.0%. When unilateral nephrectomy was performed, under the same conditions, renal net glucose release was one-half of that in rats with two intact kidneys, which indicates the quantitative accuracy of the isotope-dilution method employed in this study. In rats starved for 24 h, the renal net glucose release increased to 0.99+/-0.08 mg/dl per min. Diabetic rats showed a remarkably higher renal net glucose of 2.28+/-0.33 mg/dl per min, which was 360% of the normal level. Treatment of diabetic rats with insulin, restored the renal net glucose release to the normal level. In acidotic rats, renal net glucose release was as great as 1.03+/-0.15 mg/dl per min, which suggests that the acid-base balance participates in control of renal glucose output. Measurements every 6 h throughout the day showed that glucose was supplied from the kidney at a constant rate without any circadian rhythm. These data suggest that renal gluconeogenesis is of physiological importance in the maintenance of homeostasis of blood glucose.
Under conditions of sustained hyperglycemia, glycogen accumulates in pancreatic islets, but not so in acinar pancreatic cells. We investigated whether advantage could be taken of such a situation in the perspective of the noninvasive imaging of the endocrine pancreas. Control rats or animals injected with streptozotocin (STZ) were infused with solutions of D-glucose mixed with a tracer amount of D-[U-14C]glucose, and the radioactive glycogen content of both liver and pancreas was then measured. After 48 h of infusion, the radioactive glycogen content of the pancreas was 30 times lower in STZ rats than in control animals, coinciding with a 50 times lower insulin content. In the control rats, a sizable labeling of pancreatic glycogen was also recorded when D-[U-14C]glucose was infused for only the last 4 h of unlabeled D-glucose infusion; such a labeling was not decreased when the animals were further infused for 1 h with only the unlabeled hexose. Moreover, a pronounced difference in the pancreatic gland and blood radioactive content of control rats was still observed when the hyperglycemic animals were killed only 40 min after the i.v. injection of D-[U-14C]glucose. In STZ rats transplanted with islets and later infused with D-[U-14C]glucose, the total radioactive content and radioactive glycogen content were both much higher in the transplanted islets than in the pancreatic gland. These results allow one to define the conditions under which the administration of either 2-deoxy-2-[18F]fluoro-D-glucose or 11C-labeled D-glucose could conceivably be used to favor the selective labeling of the endocrine, as distinct from exocrine, pancreas.
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1. A trace amount of glucose labelled with 14C uniformly and with 3H at position 2, 3 or 6 was injected intravenously into starved rats to measure the turnover rate of blood glucose. 2. Reliable estimates were made based on the semilogarithmic plot of specific radioactivity of the glucose contained in whole blood samples taken from the tail vein. 3. Glucose turned over more rapidly in hyperthyroid and more slowly in hypothyroid than in euthyroid rats. The percentage contribution of glucose recycling (determined from the difference in replacement rates between [U-14C]glucose and [6-3H]glucose) to the glucose utilization increased on induction of hyperthyroidism. 4. Futile cycles between glucose and glucose 6-phosphate (determined from the difference between replacement rates of [2-3H]glucose and [6-3H]glucose) were activated and inactivated by induction of hyperthyroid and hypothyroid states respectively. 5. The hepatic content of glycogen was much lower in hyper- and hypo-thyroid than in euthyroid rats. The enhanced glucose production in hyperthyroid rats resulted from not only activationof hepatic gluconeogenesis but also diversion of the final product of gluconeogenesis from liver glycogen to blood glucose. In hypothyroidism, the inhibition of gluconeogensis led to suppression of both glucose production and glycogenesis in the liver.
The effect of a low- and high-fat diet on the transport of 3-hydroxybutyrate and glucose across the blood brain barrier has been measured in two strains of animals that have a marked difference in the degree of obesity that develops when they eat a high fat diet. The S 5B/Pl rats are resistant to dietary obesity whereas the Osborne-Mendel rats readily develop obesity when eating a high-fat diet. The transport of 3-hydroxybutyrate and glucose across the blood-brain barrier was measured as the ratio of radioactive compound (3-hydroxybutyrate or glucose) to radioactively labeled water by the technique of Oldendorf. The uptake of 3-hydroxybutyrate was significantly higher in the S 5B/Pl rats than in the Osborne-Mendel rats when they were eating either the low-fat diet or the high-fat diet. In addition, there was a significant increase in the transport of 3-hydroxybutyrate in the animals of both strains when eating the high-fat diet as compared to the low-fat diet. However, there was no difference in the transport of glucose between the two strains of rats whether they ate a low-fat or high-fat diet. These data are consistent with the hypothesis that resistance to dietary obesity is associated with increased transport of 3-hydroxybutyrate across the blood-brain barrier.
We report an improvement of a published procedure using Escherichia coli to synthesize 14C-labeled trehalose from [14C]glucose (B. Brand and W. Boos, Appl. Environ. Microbiol. 55:2414-2415, 1989). Instead of inducing the expression of the trehalose-synthesizing enzymes encoded by the chromosomal genes otsAB by high osmolarity, we now induce their expression from a plasmid under normal growth conditions by the addition of IPTG (isopropyl-beta-D-thiogalactopyranoside). Instead of using a pgi zwf double mutant to prevent glucose utilization, we use a pgi::Tn10 insertion only. In addition to being defective in treA, which encodes a periplasmic trehalase, the strain is now also defective in treF, which encodes a newly discovered cytoplasmic trehalase. This strain is genetically stable; it has no growth defects; and after induction with IPTG, it will transform [14C]glucose to [14C]trehalose in minimal medium without any carbon source under aerobic conditions at a rate of 3 nmol/min/10(9) cells. With the improved method, the overall yield of trehalose from glucose is about 80% and the process takes place without dilution of the specific radioactivity of the glucose residues. The accumulated trehalose is extracted from the bacteria by 70% hot ethanol and can easily be purified radiochemically by chromatographic techniques.
A mathematical model is designed for the metabolism of D-glucose in erythrocytes under conditions in which the flux through the pentose phosphate pathway accounts for either 5% or 75% of the rate of D-glucose phosphorylation, as indeed observed in the absence or presence of menadione. This model allows to compare the fate of D-[1-1H]glucose and D-[1-2H]glucose, taking into account the isotopic discrimination towards the deuterated hexose in the reactions catalyzed by phosphoglucoisomerase and glucose-6-phosphate dehydrogenase. The study of this model is extended to the fate of tracer amounts of either D-[1-14C]glucose, D-[U-14C]glucose or D-[1-3H]glucose mixed with non-radioactive D-[1-1H]glucose or D-[1-2H]glucose. The fates of D-[1-14C, 1-2H]glucose and D-[U-14C, 1-2H]glucose in this model are also examined. A fair agreement between the data derived from the mathematical model and prior experimental findings is observed, at least as far as the fate of 14C-labelled D-glucose is concerned. The present study illustrates, therefore, the mechanism by which unequal isotopic discrimination in different enzymatic reactions may cause severe misjudgment of metabolic flow when using deuterated and/or tritiated D-glucose as substitute and/or tracer for the protonated hexose.