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PubMed1977

Estimation of glucose turnover with stable tracer glucose-1-13C.

Glucose turnover was measured in normal and disbetic dogs by the dilution of glucose-1-13C and glucose-1-14C tracers infused simultaneously at constant rates. In order to quantify the stable isotope, an enzymatic assay for the analysis of glucose-1-13C was developed and evaluated. CO2 from C-1 glucose was evolved by coupling hexokinase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconic dehydrogenase activities. The 13C/12C ratio of the CO2 was measured with a high-precision magnetic-deflection double-collector mass spectrometer, and the radioactivity of 14CO2 was quantified by liquid scintillation. The ratio of 13C/12C was reproducible in assays of CO2 evolved from either naturally occurring or 13C-enriched glucose. Furthermore, systemic glucose production rates measured with 13C- and 14C-labeled tracers were similar over a wide range from 2 to 12 mg./kg.-min. Thus, glucose-1-13C may be employed as a tracer for glucose metabolism in human subjects without incurring the risk of radiation.

Animals↗

PubMed1989

Islet function after storage at -2 degrees C and -196 degrees C.

The radical treatment of type 1 diabetes by transplantation requires the extracorporeal storage of islets, and this has frequently been studied. Damage from ice formation, however, has prevented the development of any satisfactory method for preservation. We compared islet function after frozen storage with that after non-frozen storage. Isolated rat islets immersed in 10% dimethyl sulfoxide were kept at -2 degrees C (group A) and -196 degrees C (group B) for 7 days. After one day of culture, some of the islets were incubated in 3.3 and 16.7 mM glucose-containing Krebs--Henseleit bicarbonate buffer for 60 min. The other islets were incubated with 3H-leucine for 2 h. The radioactivity of whole-islet homogenate and the insulin extracted from it were measured. We also counted the number of islets before and after the 7-day storage. The islets thus preserved were transplanted into streptozotocin-induced diabetic rats and the fasting plasma glucose was determined weekly. Non-cooled islets were used as controls (group C). Insulin release in the presence of 16.7 mM glucose did not significantly differ between groups C and A, whereas it was lower in group B than in groups A or C. The islet uptake of 3H-leucine was lower in A and B compared with C, but the insulin synthesis was similar in all three groups. More islets were recovered from A than B. Fasting plasma glucose was lowered similarly in the diabetic rats after transplantation of islets from A and B. The relative ease of preservation at -2 degrees C, and the positive results of this experiment, favor this method of preservation.

Animals↗

PubMed1977

Glucose-stimulated and La3+-nondisplaceable Ca2+ pool in pancreatic islets.

To study intracellular pools of calcium tissue specimens from noninbred ob/ob mice were labeled with 45Ca2+ and subsequently washed with La3+. D-glucose, 20 mM, enhanced the labeling of the La3+-nondisplaceable calcium in pancreatic islets but not in pieces of exocrine pancreas or liver. The disappearance of 45Ca2+ from labeled islets was accelerated by the ionophore, X-537A, but not by dibutyryl cyclic AMP, theophylline, or pentobarbital. On fractionation of 45Ca2+-labeled islets, the greatest radioactivity per unit of protein occured in a fraction rich in insulin secretory granules. The radioactivity of this fraction was higher after the islets had been loaded with 45Ca2+ in the presence of 20 mM glucose as compared to 3 mM glucose. It is concluded that the secretory granules make up a considerable part of the glucose-sensitive calcium store in the beta-cells.

Animals↗

PubMed2000

D-mannoheptulose uptake and its metabolic and secretory effects in human pancreatic islets.

D-mannoheptulose was recently proposed to be transported into cells at the intervention of GLUT2. Since GLUT1, rather than GLUT2, represents the major carrier system for the transport of monosaccharides across the islet B-cell plasma membrane in human subjects, the uptake of D-mannoheptulose and its metabolic and secretory effects were investigated in human islets. The uptake of D-glucose reached much more rapidly a close-to-equilibrium value in isolated islets than in pieces of pancreas obtained from the same donor. The distribution space of D-[3H]mannoheptulose in the human islets largely exceeded that of [U-14C]sucrose, considered as an extracellular marker, and did not differ significantly from that of 3HOH. In the human islets, the heptose (10.0 mM) inhibited both D-[5-3H]glucose utilization and D-[U-14C] glucose oxidation, and decreased glucose-stimulated insulin release to the same extent as D-mannoheptulose hexaacetate. These findings indicate that a suitable radioactive analog of D-mannoheptulose could be used, in human like in rat islets, for preferential labelling of the endocrine moiety of the pancreatic gland.

Carbon Radioisotopes↗

PubMed1975

Effect of light stimulation on the synthesis and release of GABA in rat and frog retinae.

The effect of light stimulation on the incorporation of label from [14C]glucose and [3H]acetate into aspartate, glutamate, glutamine and GABA, both in vivo and in vitro, was studied in dark-adapted frog and rat retinae. With [14C]glucose as the precursor, but not with [3H]acetate, flashes of light appeared to increase the in vivo syntheses of both GABA and glutamine at the expense of glutamate in frog retina, while attenuating the labelling of aspartate and glutamate in isolated frog tissues. Amino acid synthesis from these radioactive substrates in rat retina was not altered under these conditions. Also, the turnover rates of amino acids derived from [14C]-glucose in rat retina in vivo were closely similar in light and darkness. Isolated retinae from both species accumulated [14C]GABA from the incubation medium. In each case the subsequent release of this compound was augmented by electric field stimulation, high K+ and by a high concentration of cold GABA in the medium, but not by a flashing light. Similarly, application of a light stimulus was unable to evoke the liberation of [14C]GABA derived from [14C]glucose in either species.

Aminobutyrates↗

PubMed1984

Phosphate flush and glucose metabolism in pancreatic islets of young and old diabetic mice (C57BL/KsJ-db/db).

Pancreatic islets from normal C57BL/KsJ-+/+-mice and diabetic C57BL/KsJ-db/db-mice were collagenase-isolated and incubated with 33P-labelled inorganic phosphate. No significant difference was observed in phosphate uptake between normal and diabetic mouse islets whether the animals were young (6-8 weeks) or old (27 +/- 9 weeks). When 33P-labelled islets were perifused with non-radioactive medium, all types of islets exhibited a brisk and transient peak of phosphate release in response to a change of glucose concentration from 2.8 to 16.7 mmol/l. Expressed in absolute terms, both the basal and peak efflux of phosphate appeared to be diminished in the diabetic mice. In relative terms (peak over basal), the glucose-stimulated phosphate efflux was not lower in diabetic than in normal mice. At both a low (3 mmol/l) and a high (20 mmol/l) glucose concentration, the production of 3H2O from D-[5-3H]glucose was reduced in old but not in young diabetic mouse islets. The percentage increase in glucose metabolism in response to a rise in glucose concentration from 3 to 20 mmol/l was about the same in all types of islets. The results add to previous observations of disturbed ionic fluxes in the pancreatic islets of diabetic KsJ-db/db-mice. These effects are probably not due to gross alterations in glycolytic metabolism but more probably reflect alterations in the function of the beta-cell plasma membrane.

Age Factors↗

PubMed1981

Positron computed tomography studies of cerebral glucose metabolism in man: theory and application in nuclear medicine.

The capability of positron computed tomography (PCT) to delineate the substructures of the brain and its facility for accurately measuring the local tissue radioactivity concentration allow the application of tracer kinetic models for the study of local cerebral function in man. This principle and an adaptation of the 14C-deoxyglucose (DG) model of Sokoloff et al. with 18F-2-fluoro-deoxy-D-glucose (FDG) is being used at UCLA. Brookhaven National Laboratory, University of Pennsylvania, NIH, and the Massachusetts General Hospital to determine the local cerebral glucose metabolic rate (LCMRGIc) in normal man at rest and during sensory activation and the changes that occur in patients with a variety of cerebral disorders. Kinetic studies with PCT have been employed to measure the rate constants of the model in different gray and white matter structures of the brain in both normal and ischemic states. The precision of the method in normals has been shown to be about +/- 5% for 1.5-2.0 sq cm regions of the brain. Studies in normals have yielded values for hemispheric CMRGIc that are in agreement with measurement using the Kety-Schmidt technique and LCMRGIc values in agreement with values in monkeys using DG autoradiography. Studies in volunteers subjected to visual and auditory stimulation are demonstrating the potential of this technique for investigating the human brain's response to different stimuli. STudies in patients with stroke show excellent correlation between the degree, extent, and particular structures involved and the clinical symptoms. The method consistently detected hypometabolism in cortical, thalamic, and striatal tissues that were dysfunctional due to deactivation or damage but which appeared normal on x-ray CT. Studies in patients with partial epilepsy have shown hypometabolic zones that highly correlated anatomically with interictal EEG spike foci and were associated with normal x-ray CT studies in 77% of the patients studied. The studies on epilepsy at UCLA have resulted in the integration of the LCMRGIc study into the clinical workup of patients with partial epilepsy that are candidates for surgical resection of their epileptogenic focus (effective June 1979). Studies on Huntington's chorea, Parkinson's disease, aphasia, dementia, schizophrenia, and tumors are in early stage of investigation but also are providing exciting new results. Further studies are needed to determine the role of the local function information obtained with the PCT-FDG method in elucidating the basic mechanism and the potential to aid in improving the approach to medical therapy.

Brain↗

PubMed2003

Lactate carbon does not enter the sugars of lipopolysaccharide when gonococci are grown in a medium containing glucose and lactate: implications in vivo.

In media containing glucose, lactate stimulates the metabolism of gonococci at concentrations that simulate conditions in vivo. Nuclear magnetic resonance (NMR) spectroscopy of (13)C-labelled lipids obtained from gonococci grown in a synthetic medium with (13)C-labelled lactate and unlabelled glucose (culture A), (13)C-labelled glucose alone (culture B) or (13)C-labelled glucose and unlabelled lactate (culture C) showed lactate carbon was not present in glycerol/ethanolamine residues of lipids from culture A. This indicated that, in the presence of glucose, lactate gluconeogenesis is shut down. Hence, the stimulation of metabolism could result from the production of extra energy because lactate is used solely for conversion to acetyl-CoA, the precursor of fatty acid synthesis and the components of the tricarboxylic acid cycle. In this paper, additional evidence for lack of gluconeogenesis has been sought using a different approach. The carbohydrate moieties of lipopolysaccharide (LPS) have been examined for lactate carbon after gonococci were grown with lactate and glucose. Two methods were used: NMR spectroscopy of (13)C-labelled lipopolysaccharide purified from the three cultures described above showed that, in the presence of glucose, lactate carbon, in contrast to glucose carbon, was not in the carbohydrate moiety. Also, (14)C-labelled lactate was added to a culture containing unlabelled glucose and lactate (culture A) and [(14)C]glucose to cultures containing unlabelled glucose without unlabelled lactate (culture B) and with unlabelled lactate (culture C). When LPS samples purified from these cultures were subjected to hydrazinolysis, the ratio of the radioactivity of water-soluble products (carbohydrate moieties) to those of chloroform-soluble products (fatty acids) was much lower when [(14)C]lactate was used in culture A, than when [(14)C]glucose was used in cultures B and C. Thus, in the presence of glucose, lactate carbon, unlike glucose carbon, is incorporated predominantly into fatty acids of LPS, not into its carbohydrate moieties. There is no doubt, therefore, that gluconeogenesis is shut off when lactate is present with glucose and there is a consequent stimulation of metabolism. This probably occurs in vivo on mucous surfaces, where gonococci are surrounded by a mixture of glucose and lactate in the secretions.

Carbohydrates↗

PubMedDate not supplied

Transport characteristics of the blood--perilymph barrier.

The inner ear must maintain a delicate homeostasis to preserve high sensitivity to acoustic and vestibular inputs. Various experimental conditions were imposed upon chinchillas to help identify these homeostatic mechanisms for the inner ear fluids. Radioactive ions (sodium, chloride, and calcium) pass into perilymph more slowly than they pass into cerebrospinal fluid or aqueous humor. The concentration of glucose in perilymph relative to that in serum was found to be quite constant (about 45 per cent) over a wide range of serum values (130-943 mg/dl) generated by continuous intravenous infusion. The transport of albumin into perilymph was very slight after injection of the radioactive protein intravenously. Osmotic agents cause an efflux of water from perilymph into a relatively hypertonic circulating blood, resulting in a secondary elevation of perilymph osmolarity. Recent findings relating to cyclic AMP metabolism and the possible role of this second messenger in the regulation of inner ear fluid metabolism are discussed. A new hypothesis that excess cyclic AMP in the inner ear may be related to endolymphatic hydrops is presented.

Animals↗

PubMed1983

The rate of substrate cycling between glucose and glucose 6-phosphate in muscle and fat-body of the hawk moth (Acherontia atropos) at rest and during flight.

1. The rate of substrate cycling between glucose and glucose 6-phosphate was measured in tissues of the hawk moth (Acherontia atropos). 2. The insect was injected with [2-3H,2-14C]glucose, and after periods of time at rest or flying the animal was freeze-clamped. Separation of glucose and hexose monophosphate from the tissues was performed by paper chromatography and t.l.c., and the 3H and 14C radioactivities in these compounds were measured. 3. On the basis of the 3H/14C ratios in these compounds and the measured rate of glycolysis, the rate of cycling was calculated. The rates of cycling were 0.03, 0.10, 0.06 and 3.9 mumol/min per g for fat-body at rest and during flight and for flight muscle at rest and during flight respectively. 4. The marked increase in the cycling rate between glucose and glucose 6-phosphate upon flight contrasts with the finding of Clark, Bloxham, Holland & Lardy [(1973) Biochem. J. 134, 589-597] in the bumble-bee, in which this condition inhibited cycling. It is suggested that the increased rate of cycling increases the sensitivity of glucose phosphorylation to changes in the concentrations of effectors of hexokinase should it be necessary to increase the rate of glycolysis in muscle, for example, to increase power output of the flight muscle for increased speed of flight.

Adipose Tissue↗

PubMed1996

The insulin status of sheep with genetic differences in glucose clearance.

Lines of sheep have been selected for Slow or Fast glucose clearance after a glucose tolerance test. The aim of this work was to establish what characteristics of the insulin status were altered by the breeding program. Six animals from each line with consistently Slow (T-half > 70 min) or Fast (T-half < 60 min) decreases in plasma glucose concentration were studied in three different experiments. After the injection of [125I]insulin, blood was sampled for 300 min. The change in radioactivity with time was used in a three-compartment series model to estimate theoretical insulin pool sizes and flow rates between pools. All three pools were significantly (P < 0.05) larger in the Slow (61, 115, and 191 mU) than in the Fast glucose clearance animals (45, 82, and 112 mU). Flow rates between the pools were not significantly different. A euglycemic clamp experiment was performed at two insulin infusion rates, each for 4 hr. A significantly higher glucose infusion rate was required to maintain blood glucose at basal levels in the Slow (3 and 9 mg of glucose/kg liveweight [lwt]0.75 per min) than in the Fast glucose clearance animals (1 and 5 mg/kg lwt0.75 per min). The increase in glucose infusion rate when the insulin infusion rate was increased from 0.63 to 3.46 mU/kg lwt0.75 per min (insulin sensitivity index) was significantly greater in Slow than in Fast glucose clearance animals (0.68 vs. 0.35 mU of insulin/kg lwt0.75 per min). There was no difference between the lines in insulin binding to membranes isolated from muscle or adipose tissue. It is concluded that selection for Slow or Fast glucose clearance has altered several aspects of insulin status, but further work is required to identify the primary difference between the lines.

Adipose Tissue↗

PubMed1984

Metabolic consequences of nutritional support of the cancer patient.

The year 1983-1984 has been a period of evaluation and examination of efficacy of nutritional support of the cancer patient. The early observation that individual patients, unable to ingest, digest, or absorb food could be nutritionally maintained by intravenous nutrition has been repeated in every cancer center. The premature application of parenteral nutrition as an adjunct to cancer care and the results of clinical trials with small numbers of patients have initiated a reevaluation of this therapy, as a routine. The most exciting advances have come from the opportunity to utilize nutritional support as a means of investigating the basic mechanisms by which the cancer patient disposes of exogenously delivered substrate. This opportunity had been initially under-appreciated and is only now receiving the full and intensive clinical and laboratory research necessary to delineate abnormalities of metabolism that occur in the cancer-bearing host. Studies have not been performed not only in intact man, but in specific organ systems in man during nutritional support. These studies, using stable and radioactive isotopes, have given us new insight into the way that the cancer patient handles glucose and amino acids. Whether these abnormalities of metabolism are due to the cancer or are due to treatment, complications of treatment, or inherent starvation accompanying progressive cancer cachexia is being examined. Such studies should indicate whether or not therapeutic interventions in limiting substrate availability for the tumor while preserving the host are possible.

Body Composition↗

PubMed1994

Preparation and properties of metabolically 3H- or 13C-labeled (1-->3)-beta-D-glucan, SSG, from Sclerotinia sclerotiorum IFO 9395.

Metabolically labeled (1-->3)-beta-D-glucan (SSG) obtained from the culture filtrate of Sclerotinia sclerotiorum IFO 9395, with the radio (3H) or the stable isotope (13C) was prepared. The specific radioactivity of 3H-SSG was increased in accordance with the amount of D-[3H]glucose added, and was 15-20 kBq/mg SSG when 222 kBq/mL of D-[3H]glucose was added. Physicochemical analyses of 3H-SSG suggested a structural similarity between SSG and 3H-SSG. 13C NMR spectra of 13C-labeled SSG (13C-SSG) revealed that a strong signal of the 13C-nucleus was observed at the C-1 or C-2 position when D-[1-13C]glucose or D-[2-13C]glucose was used, respectively, suggesting that most of the glucose residues in SSG were directly taken up from the medium. In addition, part of the 13C-nucleus was incorporated into the SSG molecule at all carbon atoms after metabolic degradation and reconstruction of the glucose molecule. Analyses of the culture filtrate and the mycelium of the fungus suggested that part of the glucose was also metabolized to trehalose and mannitol.

Ascomycota↗

PubMed1975

Wall mannan of Saccharomyces cerevisiae. Metabolic stability and release into growth medium.

Selective labelling of cell wall mannan with radioactive precursors in growing Saccharomyces cerevisiae showed that this polysaccharide is metabolically stable during exponential growth. Mannan once inserted into the wall is not subject to turnover or release into the growth medium. However, about 10% of the amount of mannan incorporated into the cell wall fraction can be recovered in the non-dialyzable material isolated from the growth medium. Therefore, the mannan escaping from the cell must be either a mannan de novo synthesized, not trapped in the growing wall structure, or a mannan with a non-structural role. Radioactivity was also retained in the wall fraction of cells pre-labelled with [14C] glucose which pointed to metabolic stability of all cell wall polysaccharides in growing S. cerevisiae.

Cell Wall↗

PubMed2004

Quantification in PET.

Quantification provides the link between the concentrations of radioactivity measured in tissue and the underlying physiologic processes occurring in the organ. It relates the rate at which radioactivity levels in the body change over time to quantitative parameters such as absolute rate glucose metabolism, regional blood flow, or concentrations of receptors or other binding sites. Absolute measurement of physiologic parameters generally requires accurate measurement of activity concentrations in arterial blood, which provides the input function to the kinetic model. Although absolute quantification can be a difficult process, simplifications of these invasive techniques, involving reference tissues or normalization approaches (eg, SUV), have been applied with some success. Any simplified model of tracer behavior must be validated against the full model to test for bias and systematic errors.

Algorithms↗

PubMed1986

Comparison and contrast of affinity chromatographic determinations of plasma glycated albumin and total glycated plasma protein.

Techniques for affinity measurement of glycated albumin and for glycated total plasma protein have been developed. The two techniques were contrasted. Both techniques are linear over a 100-fold range of sample concentrations. There appears to be a non-specific early glucose binding phase to non-albumin plasma proteins. Although this phase is detected by radioactive incorporation and thiobarbituric acid, it does not interfere with the affinity determination, which does not appear to detect the early binding species. The correlation of glycated albumin levels with glycated hemoglobin levels is much stronger than that of glycated globulin levels with glycated hemoglobin levels. Due to the large contribution of glycated albumin levels to total glycated serum protein levels, the correlation of the latter with glycated hemoglobin levels is sufficiently strong to allow the use of either technique as an adequate index of glycation.

Boronic Acids↗

PubMed1975

Production of CO2 in the living mouse immediately after injection of 1- or 2-14C acetate.

Three groups of mice, normally fed, fasted and fed after a fasting period are injected intravenously with either 1- or 2-14C acetate. The respiratory 14CO2 as well as that of the liver, the adipose tissue and the carcass are collected after 3 min and the radioactivity measured. A determination is also made of the radioactivity of the tissue fatty acids and, for two groups of mice, of the circulating glucose. A calculation is suggested by which the number of revolutions performed by the acetate C in the Krebs cycle before it is transformed into CO2 can be deduced. The results suggest that the Krebs cycle is very open, that the acetate C found in the glucose has already broken away from the cycle after one revolution and that the C which appears in the form of CO2 has performed an average of only 2.8 to 3.6 revolutions. The results are evaluated as a function of the experimental conditions chosen.

Acetates↗

PubMed1991

Localization in the Golgi apparatus of rat liver UDP-Gal:glucosylceramide beta 1----4galactosyltransferase.

The presence and subcellular localization of UDP-Gal:glucosylceramide beta 1----4galactosyltransferase (GalT-2) was investigated in rat liver. For this purpose, purified Golgi apparatus, endoplasmic reticulum, and plasma membrane fractions were prepared from the liver and used as the enzyme source for detecting GalT-2. A pure Golgi apparatus, highly enriched in many glycosyltransferases, was the only fraction where GalT-2 was measurable. The reaction product formation rate under appropriate assay conditions, which requires high detergent concentration and Mn2+, was low but comparable with that of other glycosyltransferases. The product formation was stimulated by exogenously added acceptor GlcCer, donor UDP-Gal, and Golgi protein. The reaction product was a single spot that was identified by chromatographic behavior, sensitivity to beta-galactosidase, and permethylation studies as Gal beta 1----4Glc beta 1----1'Cer (lactosylceramide). A metabolic experiment, performed by determining the glycosphingolipids which became radioactive in the above subcellular fractions prepared from the liver of animals treated with glucose-labeled glucosylceramide, further indicated that the in vivo glycosylation of glucosylceramide takes place in the Golgi apparatus.

Animals↗