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PubMed1993

Metabolic, ionic, and secretory response to D-glucose in islets from rats with acquired or inherited non-insulin-dependent diabetes.

The metabolic, ionic, and secretory response to D-glucose was investigated in islets of adult rats either injected with streptozotocin during the neonatal period (STZ rats) or presenting with inherited diabetes (GK rats). At a high concentration of D-glucose (16.7 mM), the ATP/ADP ratio was lower in islets from STZ and GK than control rats. This coincided with an impaired response of perifused islets to a rise in D-glucose concentration in terms of stimulation of insulin release, suppression of effluent radioactivity from islets prelabeled with [2-3H]adenosine, reduction in 86Rb efflux, and induction of a phosphate flush in islets prelabeled with 32P(i). The ratio in either D-[5-3H]glucose utilization or D-[2-14C]glucose oxidation at high/low hexose concentration, as well as the paired ratio between D-[2-14C]glucose oxidation and D-[5-3H]glucose utilization in islets incubated at a high concentration of the hexose, was also lower in STZ and GK rats than in control rats. Such was not the case, however, from the oxidation of [2-14C]pyruvate. Instead, the latter 2-keto acid, when tested at a 5.0 mM concentration, improved more efficiently the overall oxidative response of the islets to a rise in D-glucose concentration in STZ and GK rats than in control animals. It is proposed, therefore, that in both STZ and GK rats, the B-cell secretory defect is primarily attributable to an anomaly in oxidative glycolysis. In islets exposed to a high concentration of D-glucose, this metabolic deficiency results in impaired ATP generation, altered closing of ATP-responsive K+ channels, and, hence, diminished insulin output.

Adenosine↗
PubMedDate not supplied

Kinetics of myo-inositol transport in corneal endothelial cells: diverse effects of sugars and implications in corneal deutergensence [corrected].

Kinetics of myo-inositol (MI) uptake into primary cultures of bovine corneal endothelial cells (CEC) were studied. Confluent corneal endothelial cells accumulated 3H-MI in a time dependent and saturable process. At a narrow range of external concentrations of 3H-MI (4-50 microM), the Na(+)-dependent MI uptake followed saturation kinetics. The apparent Km value was 20 microM with a maximum velocity (Vmax) of 16 pmol/20 min/micrograms DNA. At low external 3H-MI concentrations the uptake was dependent on Na ions, but at higher levels the Na(+)-independent fraction of MI uptake significantly increased. The uptake was sensitive to removal of Ca ions and to the presence of inhibitors such as n-ethyl maleimide, phlorizin, ouabain, and amiloride (an inhibitor of Na+/H+ exchanger). The sensitivity of MI uptake toward inhibitors and ionic changes in the bathing media was reduced as external concentrations of 3H-MI increased. Citrate at 0.5 mM increased the uptake, suggesting involvement of mitochondrial oxidative metabolism in the MI uptake. Percent release of radioactivity by 2 min, after an initial 40-min incubation with 20 microM 3H-MI, was 6.6% +/- 0.8 or 35% +/- 4 when release media contained BSS alone or BSS containing 5 mM nonradioactive MI, respectively. Efflux of radioactivity from the cells also was enhanced when release media contained 40 mM glucose. Glucose and galactose as well as nonmetabolizable glucose analogues, such as 3O-methyl glucose or alpha-methyl glucose, at high concentrations (40 mM), acutely (in the incubation media) or chronically (in the growth media) inhibited MI uptake into CEC, and the extent of inhibition was inversely proportional to the external levels of 3H-MI. However, glucose at lower levels (less than or equal to 10 mM) slightly increased MI uptake. These studies indicated that the uptake of MI into corneal endothelial cells was an Na(+)-dependent active process at a narrow range of external radioactive MI concentrations. Higher levels of MI were taken up by the cells via a passive diffusion mechanism, independent of carrier protein(s). Glucose influenced the uptake of MI in a complex manner. The increased MI efflux by glucose or by MI was perhaps due to the limited capacity of CEC for accumulation or compartmentalization of this or other solutes/osmolytes, a phenomenon that may be related to the role of CEC in maintenance of corneal deutergence. High glucose-induced inhibition of Na(+)-dependent MI uptake may be in part due to glucose regulation of Na+ fluxes and cell volume.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

Insulin responsiveness in non-fa/fa and fa/fa Zucker rats: effects of adipocyte size.

Epididymal adipose tissue from non-fa/fa (lean) and fa/fa (obese) 14.5-week-old Zucker rats was used to study the influence of insulin and genotype on uptake of glucose and palmitate into adipocytes of different sizes. After incubation with radioactive substrate, adipocytes were inactivated and fixed by addition of osmium tetroxide; fixed adipocytes were isolated and separated by screening on the basis of size. Rates of substrate uptake into triacylglycerols were measured in adipocytes of each of ten size categories. Uptake rates of both glucose and palmitate increased as adipocyte size increased. Insulin had no effect on glucose uptake per adipocyte for fa/fa rats but had a highly significant (P less than 0.01) stimulatory effect on that for non-fa/fa rats. This stimulation became significantly greater with increasing adipocyte size. When insulin was included in the incubation media, glucose uptake rates were similar between similar sizes of adipocytes from non-fa/fa and fa/fa rats. Absence of insulin from the incubation media, however, resulted in lower rates of glucose uptake by adipocytes from non-fa/fa rats. Glucose uptake was maximal in adipocytes from fa/fa rats, even in the absence of insulin. Net uptake of palmitate into triacylglycerols was not influenced by insulin; a significant interaction was observed, however, between adipocyte size and genotype. Large adipocytes from fa/fa rats had greater rates of palmitate uptake than did adipocytes of similar size from non-fa/fa rats. The reverse was true for adipocytes less than 125 micron in diameter. The results of this study show that response to insulin of adipocytes of difference sizes varies with adipocyte size and with genotype.

Adipose Tissue↗
PubMed1980

Mapping stimulus-induced nervous activity in small brains by [3H]2-deoxy-D-glucose.

Nervous activity may be localized in anatomical sections of brain tissue by the autoradiographic deoxyglucose technique. The method provides sufficient structural preservation and spatial resolution for detailed functional investigation of complex but small-sized nervous systems when the original technique is modified as follows: (i) use of 3H instead of 14C as radioactive label, (ii) application of labeled deoxyglucose in concentrations close to physiological glucose levels rather than in trace amounts, (iii) stimulation for 4--9 h after deoxyglucose application instead of 20--45 min, (iv) subsequent preparation avoiding aqueous phases at all stages from fixation to autoradiography, and (v) plastic embedding of the tissue such that serial semithin sections of good structural preservation may be routinely cut. Brief aqueous fixation and dehydration at room temperature as has been described for vertebrates apparently cannot preserve stimulus-induced distribution of radioactive label in the brain of the fly Drosophila melanogaster. Aspects of the results that illustrate the potential and some limitations of the present technique are discussed.

Animals↗
PubMed1984

Liver and adipose tissue contributions to newly formed fatty acids in an ascites tumor.

We determined the contribution from host hepatic and extrahepatic tissues to newly synthesized fatty acids (FA) in the Ehrlich ascites tumor (EAT). We administered 3H2O (subcutaneously) and [14C]glucose (in a test meal) and measured the appearance of radioactivity in plasma triglyceride fatty acids (TGFA) and free fatty acids (FFA) and in tumor total lipid fatty acids (TLFA). Using [14 C]FFA, we selectively labeled epididymal fat TGFA to estimate the FA transport rate from intraperitoneal adipose tissue directly to the tumor. Contributions of four major pathways to newly synthesized FA in EAT were determined by multicompartmental analysis. De novo FA synthesis by EAT accounted for more than 93% of the TLFA radioactivity found in the tumor. Contributions from liver TGFA via plasma TGFA (less than 0.5%), adipose tissue TGFA via plasma FFA (less than 6%), and adipose tissue TGFA via direct intraperitoneal transport of FFA (less than 1%) accounted for less than 7% of all TLFA radioactivity measured in the EAT. Thus the present study establishes that practically all labeled esterified FA in the EAT is derived from de novo synthesis by tumor cells.

Adipose Tissue↗
PubMed1980

Perinatal changes in amino acid metabolism of rat brain, especially alanine and glutamic acid.

The changes in both the levels of some free amino acids and their metabolism in the rat brain during the first 24 hr of postnatal life were studied. The content of glutamic acid decreased for the first 2 hr; it remained at the lowest level for the next 4 hr, when it began to increase. The content of alanine decreased for the first 6 hr and approached the adult level. Oxygen consumption, glucose oxidation, and pyruvate formation in the cerebral slices of the 24-hr-old rats were as much as 150% of that of the 19-day-old fetus. The distribution profile of radioactivity incorporated into the cerebral amino acids from the subarachnoid-injected [U-14C]glucose was also changed. In the 2- and 6-hr-old rats, 50% of the total radioactivity recovered in the free amino acids was in alanine. Its rate decreased to 30% in the 24-hr-old and was 2% in the adult, while the radioactivity incorporated into glutamic acid increased. Alanine aminotransferase activity started to increase at birth and had the highest level at 24 hr after birth. It then decreased and finally reached the same level as shown at birth. However, aspartate aminotransferase increased during the first 6 hr after birth and did not change until the end of the first day of life.

Aging↗
PubMed1977

Incorporation of [U-14C]glucose into neutral lipids and sn-glycerol-3-phosphate in muscle from Duchenne muscular dystrophy and control patients.

Muscle biopsy tissue from patients suffering from a wide range of neuromuscular disorders, normal muscle removed during regular orthopaedic procedures, and muscle from aborted human fetuses of 12--20 weeks gestation was dissected into fibre-bundles and incubated at 37 degrees C with [U-14C]glucose at a final concentration of 4 mM. Extracts made: (i) after 30 min were used to determine content and radioactivity of sn-glycerol-3-phosphate, and (ii) after 160 min for separation of lipid species. Dystrophic muscle from 8 patients with typical Duchenne dystrophy converted significantly more glucose than normal muscle (11 samples) into neutral lipids, but less into polar lipids. Of total lipid radioactivity a mean value of 66% was found in neutral lipid in the Duchenne group, while in various control groups this proportion was about 40%. The disparity observed was the result of greater incorporation of glucose label into triglycerides, chiefly in the glycerol moiety of the Duchenne group lipids, and was some 3 times greater than normal. Other muscle disease groups also showed slightly raised levels of incorporation but these were not statistically significant in the present series of results. The content and radioactivity of glycerol phosphate in muscle from 15 patients with Duchenne dystrophy were also significantly higher than in normal muscle. These findings are discussed in relation to glucose metabolism in muscle and the energy cost of transforming glucose into triglyceride.

Child↗
PubMed1977

Chemical measurement of steady-state levels of ten aminoacyl-transfer ribonucleic acid synthetases in Escherichia coli.

Polypeptide chains of 10 aminoacyl-transfer ribonucleic acid synthetases (those for arginine, glutamine, glutamic acid, glycine, isoleucine, leucine, lysine, phenylalanine, threonine, and valine) have been identified in lysates of Escherichia coli resolved by the O'Farrell two-dimensional gel system. By labeling cells uniformly with [14C]glucose and by measuring the total amounts of these polypeptides by their radioactivity, estimations of the steady-state, molecular amounts of these enzymes were made and compared to the number of ribosomes and elongation factors in these cells. Portions of a reference culture grown on glucose and labeled with [14C]leucine or [35S]sulfate were mixed with four cultures grown in widely different media containing [3H]leucine or [3H]leucine plus [3H]isoleucine. From the isotope ratios of the total protein and of the spots containing the synthetase chains, the chemical amount of each synthetase relative to that of the reference culture was determined. The results, where comparable, show reasonable agreement with enzyme activity measurements. In general, these synthetases each exhibit a positive correlation with growth rate in unrestricted media, indicating a strong tendency for the levels of transfer ribonucleic acid, synthetases, elongation factors, and ribosomes to remain approximately, though not exactly, in balance at different growth rates.

Alanine-tRNA Ligase↗
PubMed1990

Ketone bodies and islet function: 45Ca handling, insulin synthesis, and release.

D-(-)-beta-Hydroxybutyrate and acetoacetate cause a rapid, sustained, and rapidly reversible stimulation of insulin release from rat pancreatic islets incubated in the presence, but not absence, of D-glucose. This coincides with stimulation of both proinsulin biosynthesis and 45Ca net uptake. The ketone bodies also decrease 45Ca outflow from prelabeled islets perifused in the absence of Ca2+ and, in contrast, enhance effluent radioactivity in the presence of Ca2+. In the presence of D-glucose, the secretory response to D-(-)-beta-hydroxybutyrate is concentration related in the 2.5-20 mM range, abolished in the absence of Ca2+ or presence of KCN, and enhanced by theophylline and forskolin. It corresponds grossly to a shift to the left of the sigmoidal curve relating insulin output to the ambient concentration of D-glucose. The secretory, biosynthetic, and cationic response to acetoacetate is less marked than that evoked by an equimolar concentration of D-(-)-beta-hydroxybutyrate. These features are compatible with the view that the insulinotropic action of ketone bodies would be causally linked to their metabolism in islet cells.

3-Hydroxybutyric Acid↗
PubMed1995

Techniques for the measurement of white adipose tissue metabolism: a practical guide.

A number of old and new techniques to study various aspects of white adipose tissue metabolism in vivo and in vitro are discussed. It is possible to determine lipolysis rates in vivo with tracer techniques using glycerol or fatty acids labelled with stable or radioactive isotopes. These methods allow the determination of whole body lipolysis rates but are not valuable for the investigations of regional variations in lipolysis. When combined they permit a calculation of the rate of re-esterification of free fatty acids. The vein draining abdominal subcutaneous adipose tissue can be cannulated in humans. By this method substrate turnover can be determined in vivo over the cannulated adipose region. With microdialysis it is possible to study local metabolism in vivo in different adipose tissue regions. At the same time it is possible to locally manipulate the tissue with metabolically active pharmacological substances. A number of in vitro methods to determine glucose transport in isolated fat cells are developed. The most accurate one uses 3-O-methyl glucose as tracer. These methods can be combined with studies of the further metabolism of glucose to lipids, lactate and carbon dioxide using simple (usually radioactive) methods. Lipolysis as well as release and re-esterification of free fatty acids can be investigated in detailed in vitro with sensitive techniques based on luminescence. Finally, triglyceride turnover and partial metabolism of acylglycerols can be investigated in vitro with a double isotope technique.

Adipose Tissue↗
PubMed1981

Chloride uptake by brush border membrane vesicles isolated from rabbit renal cortex. Coupling to proton gradients and K+ diffusion potentials.

Brush border membrane vesicles were isolated from rabbit renal cortex by Mg(++)-precipitation and differential centrifugation. (36)Cl(-) and [(3)H]glucose uptakes were simultaneously determined by a rapid filtration technique. Lysis of the vesicles with distilled water abolished 90-95% of the radioactivity on the filters, suggesting that nearly all of the (36)Cl(-) and [(3)H]glucose counts represented uptake into an osmotically reactive intravesicular space. Inwardly directed K(+) gradients plus valinomycin stimulated (36)Cl(-) uptake, demonstrating a conductive pathway for chloride uptake into brush-border membrane vesicles. (36)Cl(-) uptake could also be stimulated by inwardly directed proton gradients (pH(outside) < pH(inside)). This effect was seen in the absence of sodium, as well as in the presence of valinomycin when the vesicles had equal K(+) concentrations inside and out. An "overshoot" phenomenon was observed when external (36)Cl(-) was 2 mM and the external pH was lowered from 7.5 to 6.0 or to 4.5. The effect of the proton gradient was presumed to be different from the conductive mechanism because (a) the stimulation of (36)Cl(-) uptake by inwardly directed K(+) diffusion potentials was additive to the proton gradient effect, and (b) competition studies revealed statistically significant effects of thiocyanate on the conductive pathway, but not on the proton-driven pathway.HCl cotransport or anion exchange are electrically neutral mechanisms which could couple (36)Cl(-) uptake to inwardly-directed proton gradients in a brush border membrane vesicle. If both electrically neutral and conductive path ways for chloride transport are present in the luminal membrane of the proximal tubule, then the mechanism as well as the direction of net chloride transport will be influenced by the nature of the accompanying cation transport process.

Animals↗
PubMed1993

Renal micropuncture study of the effects of D-glucose tubular calcium handling in the anaesthetized rat.

Tracer microinjection studies were used to grossly locate the nephron site of altered calcium transport following D-glucose loading. Superficial proximal and distal nephrons were injected with a solution containing 45Ca and either D-glucose or L-glucose (as control). Tubule calcium transport was assessed from the recovery of radioactivity in the final urine. 45Ca recoveries from early proximal microinjections were comparable in the two groups (2.63 +/- 0.68 vs. 3.70 +/- 1.19%). However, 45Ca recoveries from late proximal microinjections were significantly higher than D-glucose was included in the injectate (7.39 +/- 1.34 vs. 2.83 +/- 0.67%, P < 0.05). 45Ca recoveries from distal microinjections were also significantly higher in the D-glucose group (87.6 +/- 2.43 vs. 71.0 +/- 2.92%, P < 0.001). These data suggest that the effects of D-glucose on renal calcium handling are mediated at the level of the distal tubule, thick ascending limb of the loop of Henle and/or pars recta. The precise mechanisms involved are not known.

Animals↗
PubMedDate not supplied

Metabolism and pharmacokinetics of [14C]-deoxyfructosylserotonin creatinine sulfate administered orally and intravenously to rats and mice.

Deoxyfructosylserotonin (DFS) has been shown in in vitro tests to inhibit L-DOPA-oxidase and also to suppress the multiplication of Mycobacterium leprae. The possible therapeutic use of DFS makes necessary the study of its metabolic fate in animal models. Labelled [14C]-DFS was synthesized by condensation of serotonin and [14C]-glucose and administered per os or intravenously to rats and mice. After oral administration, some of the radioactivity transited through the intestinal tract to be excreted in feces (20-60% of the dose) and some was destroyed in the pH conditions of the intestine and further metabolized by the flora, producing 14CO2 in the expired air (10-40% of the dose). Radioactivity excreted in the urine amounted to 8-15% after 24 h. After intravenous administration, 60-90% of the dose had already been excreted in the urine after 8 h. Feces and CO2 accounted for 5-10% each. In the urine, for both routes of administration, beside DFS, half of the radioactivity corresponded to the glucuronide conjugate, while in the feces all the radioactivity found was unchanged DFS. Whole animal body autoradiography showed the presence of radioactivity in all the organs (1-2% of the dose) mainly resulting from the incorporation of labelled carbon from glucose and CO2. These results, obtained in healthy rats, demonstrate poor intestinal absorption of DFS (10% of the dose) and when it is absorbed, rapid urinary excretion. For its possible therapeutic use as an anti-leprosy drug in humans, derivatives with higher bioavailability must be attained.

Administration, Oral↗
PubMed1967

Studies on brain-cortex slices. The influence of various inhibitors on the retention of potassium ions and amino acids with glucose or pyruvate as substrate.

1. The K(+) content of brain-cortex slices incubated in a Krebs-Ringer phosphate medium was the same whether the substrate was glucose (11mm) or pyruvate (11mm). 2. 2,4-Dinitrophenol (30mum) and ouabain (83mum) lowered the K(+) content of brain-cortex slices by approx. 40% with either glucose or pyruvate as the substrate. Triethyltin (1.3mum) lowered the K(+) content by 73% with glucose as substrate, but by only 18% when the substrate was pyruvate. 3. Quantitative estimations of six individual amino acids were made on brain-cortex slices and on the incubation medium after 75min. at 37 degrees . Some differences were found depending on whether the substrate was glucose or pyruvate. The effects of 2,4-dinitrophenol, oligomycin, triethyltin and ouabain were studied. Triethyltin caused a marked increase in the medium of each amino acid when the substrate was glucose, but not when it was pyruvate. Ouabain caused a similar increase with either glucose or pyruvate as substrate. 4. The uptake of triethyltin by brain-cortex slices was measured by using radioactive triethyl[(113)Sn]tin chloride. Various experimental conditions did not alter the uptake; these included adding glucose or pyruvate as a substrate. 5. From the author's own results and those of others it is suggested that pyruvate is an unsuitable substrate to use for the study of regulatory factors of endogenous pyruvate metabolism in brain-cortex slices. Glucose is preferable.

Alanine↗
PubMed1983

Cerebral metabolism in hypoglycemic and hyperglycemic fetal lambs.

To investigate the mechanism whereby glucose affects fetal breathing movements (FBM), cerebral metabolism was studied in 12 unanesthetized fetal lambs, during fasting-induced hypoglycemia and after a subsequent fetal infusion of glucose. Preductal arterial and sagittal sinus blood samples were analyzed for glucose and oxygen concentrations and for blood gases and pH. Measurements of regional brain blood flow were made with radioactive microspheres. Maternal fasting of 24- to 36-h duration resulted in a decrease in fetal blood glucose from 1.061 +/- 0.085 mmol X l-1 to 0.664 +/- 0.053 (P less than 0.001). Although cerebral glucose and O2 uptake remained unchanged, sagittal sinus CO2 partial pressure (Pco2) and [H+] were significantly decreased and may have contributed to the observed decrease in FBM. A 2-h infusion of glucose to the fetuses of fasted animals resulted in an increase in blood glucose to 2.452 +/- 0.173 mmol X l-1. Cerebral O2 consumption was again unchanged; however, cerebral glucose consumption was significantly increased as were sagittal sinus Pco2 and [H+], which may have contributed to the observed increase in FBM. These findings are consistent with the hypothesis that glucose affects the incidence of FBM in part by altering the environment of central chemoreceptors.

Animals↗
PubMed1996

Effect of glucose deprivation on rat glutamine synthetase in cultured astrocytes.

Glutamine synthetase was purified from the cerebral cortex of adult rats and characterized. Polyclonal rabbit antibodies were raised against the enzyme, purified and their specific anti-(glutamine synthetase) activity determined. A primary astroglial culture was prepared from newborn Sprague-Dawley rats. Astrocytes at different ages of development were incubated in the presence and absence of glucose. In glucose-deprived conditions the specific activity of glutamine synthetase decreased. This decrease was more pronounced in 8-day-old than in 21-day-old cultures. Kinetic analysis demonstrated that the reduction in activity was mainly related to a decrease in Vmax. By immunoprecipitation, it was shown that the number of enzyme molecules in astrocytes was decreased in glucose-deprived conditions. On addition of glucose, a total recovery of glutamine synthetase was obtained after 36 h in 8-day-old culture. Rates of degradation and synthesis were investigated. When compared with an incubation in the presence of glucose, glucose deprivation increased enzyme turnover, as estimated from the first-order disappearance of radioactivity from glutamine synthetase. Synthesis rate was estimated from the incorporation of [35S]methionine during a 2 h incubation period and was decreased in glucose-deprived conditions. Trichloroacetate-precipitable proteins changed only slightly in the experimental conditions, and total protein did not vary significantly during the experimental period. A mathematical model is presented which attempts to integrate degradation and synthesis in our experimental model.

Age Factors↗
PubMed2000

A simple method for non-radioactive PCR-SSCP using MDE gel solution and a midi gel format: application for the detection of variants in the GLUT1 and CTLA-4 genes.

The need to identify disease-causing mutations and DNA polymorphisms has increased with the continuing identification of new candidate genes. PCR single-strand conformation polymorphism (PCR-SSCP) is one of the techniques most widely used to identify a mutant sequence or a polymorphism in a known gene. However, the original SSCP protocols using the incorporation of radioactive label and polyacrylamide gel electrophoresis on sequencing gels for detection were labour intensive and time-consuming. Here we describe a simple SSCP protocol using MDE gel solution and a midi gel format to detect SSCP variations in the glucose transporter gene GLUT1, that we have previously analysed with the standard radioactive SSCP protocol, and we have also tested this method on the previously described point mutation (A/G transition in exon 1) of the CTLA-4 (cytotoxic T lymphocyte associated-4) gene. All known variants were detected. Based on the results, this technique appears to be simple, with no use of radioactive labels and with easy handling of the gel. Furthermore, it needs little optimisation, is relatively rapid and highly sensitive. We propose this method for the first screening for candidate gene variants.

Abatacept↗