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PubMed1995

A new method of assessing rates of the futile cycle during glycolytic and gluconeogenic metabolism.

A method for assessing rates of the futile cycle is presented, and it is illustrated in vitro. Glycolytic- and gluconeogenic-type futile cycles are simulated for the reactions catalyzed by phosphofructokinase (EC 2.7.1.11) and fructose-1,6-bisphosphatase (EC 3.1.3.11) in assays systems in which the cycle rates in either direction can be varied and determined. While either system is sustaining a net flux of carbons in a given direction, the flux of radioactively labeled carbons in the opposite direction is determined. Different cycle rates are obtained by varying phosphofructokinase activity while keeping fructose-1,6-bisphosphatase activity constant in the "gluconeogenic" simulation and varying fructose-1,6-bisphosphatase while keeping phosphofructokinase activity constant in the "glycolytic" simulation. A direct, linear relationship was found between the cycle rate and the radioactive labeling of fructose 1,6-bisphosphate from [U-14C]glucose 6-phosphate during net gluconeogenic carbon flux. Also, a direct, linear relationship was found between cycle rate and radioactive labeling of fructose-6-P from [U-14C]fructose-1,6-bisP during net glycolytic carbon flux. The applicability, advantages, and problems of the method are discussed.

Animals↗

PubMed1999

In vivo operation of the pentose phosphate pathway in frog oocytes is limited by NADP+ availability.

Evolution of CO2 from labelled glucose microinjected into frog oocytes in vivo may be ascribed to the pentose-P pathway, as measured by radioactive CO2 production from [1-(14)C] and [6-(14)C]glucose. Coinjection of NADP+ and [14C]glucose significantly stimulated 14CO2 production. The effect depends on the amount of NADP+ injected, half maximal stimulation being obtained at 0.13 mM. The increase in CO2 production was also observed with microinjected glucose-1-P, glucose-6-P or fructose-6-P used as substrates. Phenazine methosulfate, mimicked the effects of NADP+. A high NADPH/NADP+ ratio of 4.3 was found in the cells, the intracellular concentration of NADP+ being 19 microM.

Animals↗

PubMed1998

Gluconeogenesis in isolated rat hepatocytes evaluated by gas chromatography/mass spectrometry using deuterated water.

Tritiated water and radioactive tracers have been used to monitor glucose production by primary cultures of hepatocytes. More recently, 3H2O has been replaced for by 2H2O in 'in vivo' studies addressed at the evaluation of the relative contribution of gluconeogenesis to total glucose production. In this work, the possibility of using 2H2O to determine the ratio between the glucogenic flux and the overall flux through glucose 6-phosphate in isolated liver cells in vitro was evaluated. For this purpose, hepatocytes from either fasted or fed rats were incubated with a medium containing 6, 12 and 25% of 2H2O in the presence of either 2 or 20 mM pyruvate. Isotopomer analysis of six different mass clusters (m/z 328, 314, 242, 212, 187 and 145) was carried out by gas chromatography/mass spectrometry (GC/MS) of glucose aldonitrile pentaacetate. For each cluster, ions at m/z +1, +2, +3 and +4 were monitored. From the combination of different clusters the enrichment at C-6 and C-2 of glucose was computed and the C-6/C-2 ratio was considered to represent the contribution of gluconeogenesis to total glucose production, as suggested previously. Based on the results obtained, conditions selected to be optimum for the use of the method in studies on the modulation of gluconeogenesis were as follows: incubation of hepatocytes with 20 mM pyruvate in 12% 2H2O followed GC/electron ionization MS analysis of the clusters of ions at m/z 328, 314 and 187 of the glucose derivative to calculate enrichment at the C-2 and C-6 positions of glucose.

Animals↗

PubMed1980

Uptake of the components of phenylalanylphenylalanine and maltose by intestinal epithelium.

The observed rate of phenylalanine absorption into rat intestinal rings with 0.5 or 5.0 mM phenylalanine is greater than that for absorption of phenylalanine from 0.25 or 2.5 mM Phe-Phe, respectively. With the amino acid phenylalanine, V for absorption is the same whether Na+ is present (149 mM) or absent, but the concentration at which the half-maximal transport rate occurred (Kt) is greater in the absence of Na+. For Phe-Phe, the V decreases in the absence of Na+ whilst Kt is not influenced by the Na+ concentration. The different effect of Na+ on Phe and Phe-Phe transport indicates that the absorptive mechanism for Phe-Phe is different from that for phenylalanine. Absorption of a mixture of [U-14C]Phe-[he and Phe-[G-3H]Phe showed identical rates of uptake of the carboxyl and amino terminal amino acids. Studies of transport of radioactive maltose showed that the rates of uptake of the reducing and non-reducing glucosyl moieties are identical. Radioactive maltose absorption is not inhibited by glucose oxidase. These results provide evidence that in intestinal epithelium, hydrolysis of Phe-Phe and maltose does not occur on the cell surface with release of the hydrolyzed products to the medium. Rather, hydrolysis and release of the reaction products occur at a point on the cytosol side of a diffusion barrier located in the brush border membrane.

Animals↗

PubMed1993

Regulation of synthesis of lactosylceramide in normal and tumor proximal tubular cells.

We have measured the binding and degradation of low-density lipoprotein (LDL) and LDL-mediated effects on cholesteryl ester (CE) synthesis in cultured normal and tumor proximal tubular (PT) cells. The effects of LDL on the regulation of glycosphingolipid metabolism in cells was pursued employing radioactive precursors, e.g., [3H]serine, and [3H]glucose and by measuring the activity of UDP-galactose: glucosylceramide: B1-4 galactosyltransferase (GalT-2). In normal PT cells, there was a saturable and displaceable binding and degradation of 125I-LDL and a LDL mediated 14-fold stimulation of cholesteryl ester (CE) synthesis. This was accompanied by a suppression (70-80%) of incorporation of [3H]glucose and [3H]serine into GlcCer, LacCer, GbOse3Cer and GbOse4Cer and suppression (70-80%) of GalT-2 activity. In tumor PT cells, displaceable binding and degradation of 125I-LDL was not observed and LDL failed to stimulate CE synthesis. In such cells, LDL exerted a concentration-dependent stimulation of [3H]glucose and [3H]serine incorporation into GSL. Maximum stimulation (250%) of GalT-2 activity in tumor PT cells occurred with 25 micrograms LDL/ml medium. We conclude that LDL taken up via receptor mediated pathway decreases GaIT-2 activity in normal PT cells. In contrast, LDL not taken up via the LDL receptor pathway in tumor PT cells failed to suppress the incorporation of [3H]glucose and [3H]serine into glycosphingolipids and GalT-2 activity leading to a stimulation of lactosylceramide synthesis.

Antigens, CD↗

PubMed1977

Molecular and metabolic heterogeneity of liver glycogen.

On refeeding after starvation, the resynthesis of rabbit-liver glycogen proceeds inhomogeneously and over-produces material of low molecular weight. The fate of radioactivity incorporated into glycogen from D-glucose-14C can be explained if glycogen of high molecular weight is synthesised on a protein backbone. Confirmation of this view is given by the effect upon glycogen of reagents that break disulphide bonds; these cause loss of the polysaccharide of high molecular weight. Buoyant densities of glycogens are found to be independent of molecular weight and even of extensive degradation. It is concluded that glycogen synthesis proceeds by two routes; one results in the production of polysaccharide of high molecular weight which has a protein backbone capable of forming disulphide bonds, and another results in the production of polysaccharide of low molecular weight which has either no protein backbone or a protein backbone that is incapable of forming disulphide bridges. Apart from size, the two species are physicochemically indistinguishable.

Animals↗

PubMed1988

The dolichol pathway in the retina: oligosaccharide-lipid biosynthesis.

The formation of the oligosaccharide-lipid intermediates of the dolichol pathway by the bovine retina was investigated. Intact retinas were incubated in vitro for various periods of time in the presence of a variety of radioactive sugars (2-[3H]mannose, 6-[3H]glucose, 1-[3H]galactose, 1-[14C]glucosamine) using incubation conditions which have been shown previously to support the glycosylation of rhodopsin. The oligosaccharide-lipids were isolated and partially purified by DEAE cellulose chromatography. After mild acid hydrolysis and reduction, the oligosaccharides were analysed by HPLC. Further identification was obtained by chemical means and after digestion of the oligosaccharides with alpha-mannosidase and endohexosaminidase H. The full array of oligosaccharide-lipids which have been observed in other tissues were detected in the bovine retina, although some striking differences were seen in their relative distribution. Although short-term incubations (up to 15 min) indicated that the major species was the fully glucosylated oligosaccharide-lipid (Glc3Man9GlcNAc2), with longer incubation times the non-glucose-containing intermediate, Man9GlcNAc2, became the predominant species. Since glycerol was the carbon source for these incubations, the possibility was investigated that glucose starvation may have been the basis for this phenomenon, as has been reported in other tissues. It was established that this was not the case. Experiments carried out in the presence of castanospermine and bromoconduritol indicated that alpha-glucosidase activity in the retina may have resulted in the accumulation of the unglucosylated oligosaccharide-lipids. The formation of oligosaccharide-lipid intermediates by cells of the retinal pigment epithelium from the embryonic chick, maintained in cell culture, was also examined. In contrast to the bovine retina, the major species present were the glucose-containing intermediates, similar to other tissues.

Animals↗

PubMed1981

Effect of temperature on kinetics and symmetries of the hexose transporter of Novikoff rat hepatoma cells.

We have used rapid kinetic techniques to measure the accumulation of radioactively labeled 3-O-methyl-D-glucose to transmembrane equilibrium in Novikoff cell suspensions as a function of temperature. Arrhenius plots of the maximum velocities of isotopic exchange and zero-trans entry were continuous between 6 and 39 degrees C (Ea = 15-19 kcal/mol). Equilibrium exchange and zero-trans entry of 3-O-methyl-glucose at six concentrations (1-30 mM) at 16, 27, and 35 degrees C conformed to appropriate integrated rate equations derived for a single transporter. The hexose transporter exhibited directional symmetry, but the loaded carrier moved about 2 times faster than the empty carrier at all three temperatures investigated. Thus, the differential mobilities of loaded and empty carrier are not affected by temperature in this range. The Michaelis--Menten constant for equilibrium exchange increased about 2-fold with increase in temperature between 16 and 35 degrees C.

Animals↗

PubMed2003

Microbial participation in iodine volatilization from soils.

The roles of microorganisms in iodine volatilization from soils were studied. Soils were incubated with iodide ion (I-), and volatile organic iodine species were determined with a gas chromatograph. Iodine was emitted mainly as methyl iodide (CH3I), and CH3I emission was sometimes enhanced by the addition of glucose. Soils were then incubated with a radioactive iodine tracer (125I), and radioiodine emitted from soils was determined. The emission of iodine was enhanced in the presence of yeast extract but was inhibited by autoclaving of soils. The addition of streptomycin and tetracycline, antibiotics that inhibit bacterial growth, strongly inhibited iodine emission, while a fungal inhibitor cycloheximide caused little effect. Forty bacterial strains were randomly isolated from soils, and their capacities for volatilizing iodine were determined. Among these, 14 strains volatilized significant amounts of iodine when they were cultivated with iodide ion. Phylogenetic analysis based on 16S ribosomal DNA sequences showed thatthese bacteria are widely distributed through the bacterial domain. Our results suggest that iodine in soils is methylated and volatilized as CH3I by the action of soil bacteria and that iodine-volatilizing bacteria are ubiquitous in soil environments. The pathway of iodine volatilization by soil bacteria should be important for understanding the biogeochemical cycling of iodine as well as for the assessment of long-lived radioactive iodine (129I) in the environment.

Bacteria↗

PubMed2005

Supraphysiological doses of levothyroxine alter regional cerebral metabolism and improve mood in bipolar depression.

Supplementation of standard treatment with high-dose levothyroxine (L-T(4)) is a novel approach for treatment-refractory bipolar disorders. This study tested for effects on brain function associated with mood alterations in bipolar depressed patients receiving high-dose L-T(4) treatment adjunctive to ongoing medication (antidepressants and mood stabilizers). Regional activity and whole-brain analyses were assessed with positron emission tomography and [(18)F]fluorodeoxyglucose in 10 euthyroid depressed women with bipolar disorder, before and after 7 weeks of open-label adjunctive treatment with supraphysiological doses of L-T(4) (mean dose 320 microg/day). Corresponding measurements were acquired in an age-matched comparison group of 10 healthy women without L-T(4) treatment. The primary biological measures were relative regional activity (with relative brain radioactivity taken as a surrogate index of glucose metabolism) in preselected brain regions and neuroendocrine markers of thyroid function. Treatment-associated changes in regional activity (relative to global activity) were tested against clinical response. Before L-T(4) treatment, the patients exhibited significantly higher activity in the right subgenual cingulate cortex, left thalamus, medial temporal lobe (right amygdala, right hippocampus), right ventral striatum, and cerebellar vermis; and had lower relative activity in the middle frontal gyri bilaterally. Significant behavioral and cerebral metabolic effects accompanied changes in thyroid hormone status. L-T(4) improved mood (remission in seven patients; partial response in three); and decreased relative activity in the right subgenual cingulate cortex, left thalamus, right amygdala, right hippocampus, right dorsal and ventral striatum, and cerebellar vermis. The decrease in relative activity of the left thalamus, left amygdala, left hippocampus, and left ventral striatum was significantly correlated with reduction in depression scores. Results of the whole-brain analyses were generally consistent with the volume of interest results. We conclude that bipolar depressed patients have abnormal function in prefrontal and limbic brain areas. L-T(4) may improve mood by affecting circuits involving these areas, which have been previously implicated in affective disorders.

Adult↗

PubMed1998

Comparison between the effects of D-mannoheptulose and its hexaacetate ester upon D-glucose metabolism in rat erythrocytes.

At a 2.0 mM concentration, D-mannoheptulose hexaacetate, but not unesterified D-mannoheptulose, inhibited the generation of radioactive acidic metabolites by rat erythrocytes exposed to D-[U-14C]glucose (8.3 mM). It is proposed that D-mannoheptulose hexaacetate represents a valuable tool to interfere with the phosphorylation of D-glucose in cell types otherwise resistant to the heptose.

Acetates↗

PubMed1980

Kinetics of C Distribution during Photosynthesis by Chloroplast Preparations Isolated from the Siphonous Alga Caulerpa simpliciuscula.

The kinetics of (14)C-labeling of compounds produced during photosynthesis by chloroplast preparations isolated from the green alga Caulerpa simpliciuscula were studied. After 10 minutes photosynthesis sucrose contained more (14)C than any other product, and continued to accumulate radioactivity during the whole hour of incubation. Glucose-6-phosphate and alanine also behaved as end products and continued to accumulate label during the period. In these organelles, glucose-6-phosphate replaced triose phosphate as the main compound exported from the chloroplast during shorter periods of photosynthesis. When either glucose-6-phosphate or 3-phosphoglycerate was supplied to the isolated chloroplasts, they were metabolized, but were not converted to either sucrose or alanine. It is proposed that many of the differences in metabolism which distinguish these algal chloroplasts from those isolated from higher plants are due to their isolation in the form of cytoplasts, i.e. chloroplasts surrounded by a thin layer of extrachloroplastic material which is membrane-bound. The restriction of diffusion of intermediates from the chloroplast by this cytoplast membrane appears to be at least as important as the rather small amount of cytoplasm present in determining the properties observed.

Journal Article↗

PubMed1984

Fat metabolism following an intravenous bolus dose of a fat emulsion and carnitine.

Intravenous fat tolerance tests were performed with (carboxyl-14C)-triolein labelled Intralipid in four normal subjects with and without L-carnitine administration, 20 and 25 mg/kg body weight. The pharmacokinetics of L-carnitine was studied simultaneously with measurements of variables reflecting fat metabolism during 4 h. 3-OH-butyrate concentration in plasma was higher in all subjects when carnitine was given. No effect of carnitine was found in elimination of the exogenous triglycerides, the 14CO2 activity in expired air, concentration and specific radioactivity of non- esterified fatty acids or glucose in plasma. The data suggest that carnitine may slightly increase fatty acid oxidation in normal subjects provided that increase of 3-OH-butyrate concentration in plasma is the most sensitive variable reflecting fatty acid oxidation of the variables applied in this study.

Adult↗

PubMed1979

Effects of glucocorticoids on microsomal membrane synthesis in hepatocytes from adrenalectomized rats.

Biochemical and morphological studies were performed on livers from normal, adrenalectomized (ADX), and ADX and dexamethasone (DEX)-treated rats to investigate the effects of glucocorticoids on microsomal membrane synthesis. Overnight fasted normal, ADX and ADX rats treated 2 or 4 h with DEX received [3H]leucine and [14C]glycerol. Livers were removed, and tissue specimens were prepared for electron microscopy and tissue fractionation. Liver microsomal subfractions were prepared and subsequently washed to produce rough and smooth microsomal membranes. Radioactivity and membrane composition were determined, and glucose-6-phosphatase activity was measured in washed microsomal membranes. Adrenalectomy caused decreased microsomal membrane synthesis. Two and 4 h of DEX administration restored microsomal membrane synthesis to normal levels. ADX also caused an alteration in composition of the microsomal membranes (reflected in decreased phospholipid-protein ratios), which was restored to normal levels by 4 h after DEX administration. The earliest effects of the hormone on membrane synthesis were observed in smooth microsomes as part of a smooth endoplasmic reticulum (SER) proliferation. These findings were supported by observations made with the electron microscope. The proliferating SER was enriched in at least one component: glucose-6-phosphatase. Although the specific relationship of SER to glucocorticoid action remains unclear, the interpretation is offered that SER proliferation and alteration in glucose-6-phosphatase distribution are component parts of the total response of the hepatocyte to glucocorticoids.

Adrenalectomy↗

PubMed1988

Effects of L-leucine, its 2-keto acid metabolite and its non-metabolized analogue on rat tumoral islet cell function.

L-Leucine and 2-ketoisocaproate stimulated insulin release from perifused rat tumoral islet cells (RINm5F line). The secretory response coincided with an increase in the intracellular ATP/ADP ratio, a stimulation of 45Ca outflow from cells perifused in the presence of extracellular Ca2+, and an increase in 32P efflux from cells prelabelled with radioactive orthophosphate. In contrast to D-glucose, however, L-leucine or 2-ketoisocaproate failed to decrease 86Rb outflow, to inhibit 45Ca outflow from cells perifused in the absence of Ca2+ and to enhance the labelling of inositol-containing phospholipids in cells exposed to myo-[2-3H]inositol. These findings suggest that D-glucose, L-leucine and 2-ketoisocaproate exert dissimilar effects on the subcellular distribution of adenine nucleotides and/or 86Rb. The non-metabolized analogue of L-leucine, 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH), also caused an initial stimulation of insulin release and 32P efflux, but this was soon followed by a severe and irreversible inhibition of insulin output, associated with a permanent enhancement of 86Rb outflow. The dual ionic and secretory response to BCH is interpreted in the light of its dual effect on the catabolism of endogenous amino and fatty acids, and raises the view that BCH could be used to interfere with the function of insulinoma cells.

Adenine Nucleotides↗

PubMed1988

Radiolabelling patterns in alginate of Pseudomonas aeruginosa synthesized from specifically-labelled 14C-monosaccharide precursors.

Incorporations of radioactivity in alginate from specifically-radiolabelled glucose molecules by a parental strain and a gluconate-negative mutant of Pseudomonas aeruginosa were compared. The gluconate-negative mutant, which was deficient in the Entner Doudoroff pathway enzymes, produced less alginate than did the parental strain. The ratio of incorporation of radiolabel from [6-14C] versus [1-14C] glucose was 9.7:1 in the parental strain, implying that the carbon skeleton is catabolised via the Entner Doudoroff pathway. In comparison, a ratio of 1.5:1 was obtained for the mutant. Analysis of the alginate produced after growth on [6-14C] glucose showed that in both strains less radiolabel was present in carbon-6 of the constituent uronic acids than would be predicted (parental strain 8.4% and mutant 38.3% of the expected values). These results imply that TCA cycle intermediates are probably the major precursors of alginate in P. aeruginosa.

Alginates↗

PubMed1986

Preparation of "tissue-isolated" rat tumors for perfusion: a new surgical technique that preserves continuous blood flow.

A surgical technique for preparing "tissue-isolated" rat tumors for perfusion was developed that ensures continuous blood flow to the tissue. The tumor may be perfused in situ or ex vivo. Tumor venous blood flow, tumor glucose and lactic acid metabolism, as well as host animal respiration and core temperature, were unchanged during the procedure. Following addition of 14C-D-Glucose to the tumor afferent blood, all radioactivity appeared in the tumor venous blood. None appeared in the host systemic blood, indicating complete separation of tumor and host vasculature during in situ perfusion.

Animals↗

PubMed1983

Turnover of excess hemoglobin alpha chains in beta-thalassemic cells is ATP-dependent.

Blood erythroid cells from five beta-thalassemic donors were incubated with [3H]leucine at 37 degrees C to label the pool of excess, free hemoglobin alpha chains. The 3H-labeled cells were split and reincubated in nonradioactive media that either supported ATP production (with 5 mM glucose) or inhibited ATP production (without glucose but with 20 mM 2-deoxyglucose and 0.20 mM 2,4-dinitrophenol). During the 6-h incubation in the glucose medium, the total cellular protein 3H radioactivity decreased about 30%, while the ATP levels remained constant. Chromatographic separation of the alpha- and non-alpha-globin chains of the crude (stroma included) lysates and electrophoretic separation of the free alpha chains and tetrameric hemoglobins of the stroma-free soluble phases both showed that degradation of the alpha chains was responsible for the decrease in protein 3H radioactivity. Conversely, in the energy-deprived cells, the ATP levels dropped to less than 10% of that of the energy-supported cells, and the turnover of alpha-globin 3H radioactivity of the crude lysates was only 5-10%. These results indicate that proteolysis of excess, newly synthesized alpha chains in beta-thalassemic cells is ATP-dependent. The accumulation, mostly in the stromal fraction, of intact 3H-alpha chains in the ATP-deprived cells suggests that an ATP-dependent step occurs early in the biochemical pathway of alpha chain proteolysis. Denaturation resulting in insolubility of the free alpha chains may be a recognition signal for activation of this proteolysis.

Erythrocytes↗