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Continuous biosynthesis of glycogen was demonstrated by perfusion of the skin of dogs with uniformly labeled C(14)-glucose. If the skin is irradiated in vivo with an erythemal dose of x-rays 24 hours prior to perfusion, the uptake of radioactive glucose into glycogen is significantly increased. The irradiation effect appears to be due to enzymatic dysbalance.
Inducible binding proteins for C4-dicarboxylic acids (DBP) and glucose (GBP) were isolated from Pseudomonas aeruginosa by extraction of exponential-phase cells with 0.2 M MgC12 (pH 8.5) and by an osmotic shock procedure without affecting cell viability. DBP synthesis was induced by growth on aspartate, alpha-ketoglutarate, succinate, fumarate, malate, and malonate but not by growth on acetate, citrate, pyruvate, or glucose. Binding of succinate by DBP was competitively inhibited by 10-fold concentrations of fumarate and malate but not by a variety of related substances. GBP synthesis and transport of methyl alpha-glucoside by whole cells were induced by growth on glucose or pyruvate plus galactose, 2-deoxyglucose, or methyl alpha-glucoside but not by growth on gluconate, succinate, acetate, or pyruvate. The binding of radioactive glucose by GBP was significantly inhibited by 10-fold concentrations of glucose, galactose, and glucose-1-phosphate but not by the other carbohydrates tested. The binding of glucose by GBP or succinate by DBP did not result in any chemical alteration of the substrates.
Larval stages of Oesophagostomum radiatum grown in vitro and adults grown in vivo were incubated in complex media or in a simple salt solution containing radioactive glucose. Glucose disappearance and end product accumulation of third-stage larvae in a simple salt solution indicated that they excreted CO2 and acetic, propionic, and lactic acids. Larvae in third molt, fourth stage, and adults all excreted CO2, acetic, propionic, and lactic acids at twice the rate of third-stage larvae plus an additional product, methylbutyric acid. Carbon dioxide arose primarily from the 3 or 4 carbons of glucose. An anaerobic atmosphere (95% N2:5% CO2) had no apparent effect on metabolism. When incubation was done in complex media, isobutyric and 3-methylbutyric acids were seen as major excretion products (10 and 24%, respectively). However, these acids were quantitatively minor when incubations took place in simple salts-glucose medium (1 and 0-3%, respectively).
Strain NP 315 of Escherichia coli possesses a thermolabile fructose-1, 6-diphosphate (FDP) aldolase; its growth on carbohydrate substrates is inhibited probably as a consequence of the accumulation of high intracellular levels of FDP. Studies of one class of phenotypic revertants of strain NP 315 which have regained their ability to grow on C(6) substrates at 40 C showed that in these strains the buildup of the inhibitory FDP pool is prevented by additional mutations in enzymes catalyzing the conversion of the substrate offered in the medium to FDP. For example, mutations affecting 6-phosphogluconate dehydrogenase activity (gnd(-)) may be selected in great number without any mutagenesis and enrichment simply by isolating revertants of strain NP 315 able to grow on gluconate at 40 C. Similarly, an additional mutation in phosphoglucose isomerase (pgi(-)) restores the ability of these fda(-)gnd(-) strains to grow on glucose at 40 C. Glucose metabolism of these fda(-)gnd(-)pgi(-) strains was investigated. The enzymes of the Entner-Doudoroff pathway are induced to an appreciable extent upon growth of these mutants on glucose medium; further evidence for glucose degradation via this route (which normally is induced only in the presence of gluconate) was provided by following the fate of the C1 label of radioactive glucose in l-alanine. Predominant labeling of the carboxyl-carbon of l-alanine was observed, inciating a major contribution of the Entner-Doudoroff path to pyruvate formation from glucose. Chromatographic analysis of the intermediates of glucose metabolism showed further that glucose apparently is at least partly metabolized via a bypass consisting of the accumulation of extracellular gluconic acid which arises by dephosphorylation of 6-phosphogluconolactone and possibly of 6-phosphogluconate. This extracellular gluconate is then taken up and metabolized in the normal manner via the Entner-Doudoroff enzymes.
The rate of glucose oxidation of fresh corneas of guinea pigs at different ages has been determined. Full-thickness corneal discs, 4 mm in diameter, were incubated in radioactive glucose solution. The quantity of the released CO2 was expressed in terms of the corneal discs, their dry weights, and DNA and protein contents. The rate of glucose metabolized to CO2 per hour was about 300 pmol/microgram DNA for guinea pigs weighing approximately 800 g. Glucose oxidation decreased as the age of the animals increased. Our results compared well with those obtained in other species. We therefore feel that the guinea pig should be a suitable model for research on the metabolism of corneas.
In fasting-refeeding experiments glucagon was found to inhibit the induction of rat liver glucose-6-P dehydrogenase. The kinetics of induction in the presence of glucagon indicated that the hormone decreased the rate of enzyme synthesis without altering the rate of enzyme degradation. Immunochemical titration of the amount of glucose-6-P dehydrogenase protein present in control and glucagon treated rats suggests that glucagon does not produce an inhibited species of the enzyme. Isolation of radioactive glucose-6-P dehydrogenase by precipitation with a specific antiserum provided direct evidence that glucagon inhibited the synthesis of the enzyme. Under identical conditions glucagon had no effect on the levels of 6-phosphogluconate dehydrogenase.
Human platelet membrane proteins (PMP), incubated in vitro in the presence of various concentrations of glucose, undergo nonenzymatic glycation, as evidenced by incorporation of [3-3H]glucose radioactivity into the acid-precipitable fraction. The time course of the reaction is linear for the first hours, and the rate of glycation depends on the glucose concentration in the medium: at a glucose concentration of 80 mmol/L, up to 60 nmol of glucose is bound per milligram of PMP. The ketoaminic nature of the glucose/protein linkages was demonstrated by the finding of 5-hydroxymethylfurfuraldehyde by liquid-chromatographic analysis of acid hydrolysates of PMP. We analyzed PMP from 13 subjects with type I poorly controlled diabetes and from 10 nondiabetics. Nonenzymatic glycation, evaluated as nanomoles of the aldehyde per milligram of protein, was much greater in diabetic patients than in nondiabetics: 1.58 +/- 0.70 vs 0.37 +/- 0.18 (mean +/- SD).
The mechanism (both at the whole body and cellular level) by which metformin improves insulin sensitivity has yet to be defined. In the present study, we examined in vivo insulin-mediated whole-body glucose disposal, glycogen synthesis, hepatic glucose production, and insulin secretion, as well as in vitro muscle insulin receptor tyrosine kinase activity in eight control, eight neonatal streptozotocin diabetic rats, and eight diabetic rats before and after treatment with metformin. Ten weeks after birth diabetic rats had higher fasting (132 + 5 v 101 + 2 mg/dL) and postmeal (231 + 10 v 133 + 3) plasma glucose levels compared with controls (P less than .001). Metformin treatment was followed by a significant decrease in the growth rate and normalized glucose tolerance without enhancing the deficient insulin response. Insulin-mediated glucose uptake in diabetic versus control rats was reduced (P less than .01) during the high-dose (15.4 + 0.6 v 18.3 + 1.0 mg/kg.min) insulin clamp study and was increased to values greater (P less than .05) than controls following metformin treatment. Muscle glycogen synthetic rate in vivo, measured by incorporation of 3H-3-glucose radioactivity, was diminished by 25% (P less than .01) in diabetic rats, restored to normal values with metformin, and correlated closely (r = .82, P less than .002) with total-body glucose uptake during the insulin clamp in all three groups. Insulin receptor tyrosine kinase activity, measured in partially purified insulin receptors, was reduced in diabetic rats and increased to supernormal levels after metformin. The decrease in muscle tyrosine kinase activity in diabetic versus control animals was entirely accounted for by a reduction in maximal velocity (Vmax) (32 v 45 pmol/mg.min, P less than .01) and increased to supernormal levels following metformin (91 pmol/mg.min, P less than .001) without any change in affinity (Km). Muscle tyrosine kinase activity was closely correlated with both the muscle glycogen synthetic rate (r = .82, P less than .002) and total-body insulin-mediated glucose disposal (r = .64, P less than .01) in vivo. The close correlation between in vivo insulin action, muscle glycogen synthesis, and muscle insulin receptor tyrosine kinase activity is consistent with an important role of the enzyme in the insulin resistance of diabetes and its improvement following metformin treatment.
OBJECTIVE: We assessed glucose utilization and insulin sensitivity in normal reproductive-age women after administration of micronized estradiol, micronized progesterone, or the combination of micronized estradiol and progesterone. METHODS: Hyperglycemic and euglycemic, hyperinsulinemic clamp studies were performed in normal, regularly cycling women both before and after a short course of either micronized estradiol (n = 8), micronized progesterone (n = 8), or micronized estradiol and progesterone (n = 7). All studies were performed after an overnight fast. Glucose and insulin were determined in the control period and then every 2-10 minutes in the hyperglycemic clamp studies and every 5-10 minutes in the euglycemic clamp studies. In the hyperglycemic clamp studies, hyperglycemia was maintained by a variable glucose infusion. In the euglycemic clamp studies, a primed 3-3H glucose infusion (25 microCi) followed by a 0.25-microCi continuous infusion was begun 120 minutes before initiation of the insulin infusion and variable glucose infusion. Samples for glucose radioactivity were measured during the control period and during the last 40 minutes of each step of the euglycemic clamp after establishment of a steady state. RESULTS: No differences in baseline glucose or insulin levels were detected in any of the study groups as compared with control. Glucose utilization as assessed by the hyperglycemic clamp model was not significantly different from control in the estradiol group, the progesterone group, or the group treated with the combination. In all of the treatment groups, no significant differences were noted under euglycemic, hyperinsulinemic conditions in glucose utilization, hepatic glucose production, or insulin sensitivity. CONCLUSIONS: Women of reproductive age do not demonstrate significant differences in basal levels of glucose or insulin when given a short course of micronized estradiol and progesterone, either alone or in combination. Under conditions of the hyperglycemic, hyperinsulinemic clamp, the pancreatic beta-cell response to hyperglycemia and glucose utilization is not significantly altered by exogenous administration of hormones. Conditions of the euglycemic, hyperinsulinemic clamp failed to elicit significant differences in glucose utilization and insulin sensitivity in any of the three treatment groups. These findings demonstrate that glucose homeostasis is not altered by the exogenous administration of natural hormones in reproductive-age women.
Hypothalamic sites in the monkey were labelled by micro-injections of 3H-NE and successive push-pull perfusions were carried out at a rate of 25 mul/min. When the monkey was fed, 3H-NE within the perifornical region increased. When 2-deoxy-D-glucose (2-DG) was added to the perfusate, 3H-NE release was also enhanced, whereas insulin perfused at the same rate caused a delated increase in catecholamine levels as reflected by increased radioactivity. Glucose supressed the release of 3H-NE, suggesting overall that the noradrenergic feeding system in the hypothalamus of the monkey is modulated by the regional level of glucose as well as the local concentration of insulin.
The uptake of D-[(14)C]mannose by adult rat kidney cortex slices has been examined. The process involved appeared to be saturable and oxygen dependent and to be inhibited by glucose, galactose, chi-methyl-D-glucoside, and phlorizin. Extensive metabolism of the sugar occurred. (14)CO(2) was produced to an extent comparable to that from D-glucose. Radioactive compounds accumulated in the tissue, approximately 50% of which were in the form of phosphorylated or other polar intermediates. The remaining neutral fraction consisted of mannose with a small amount of label in glucose and an unidentified compound which behaves like an alditol. Mannose concentration in the intracellular fluid was never greater than that of the medium.
Earlier investigations have revealed that polyethylene glycol-B1-insulin (PEG insulin) causes lower utilization of glucose in fat cells than does the unaltered hormone, even though the blood glucose-lowering activity of both preparations in intact animals is identical. The present study was aimed to establish whether or not PEG insulin in regard to adipose tissues of intact animals has similar functions. Radioactive glucose was used to examine the influence of both native pork insulin and its PEG derivative on the incorporation of tracers into lipids. Male Wistar rats and male domestic rabbits received 14C- and/or 3H-labeled glucose intravenously, while the insulin preparations were administered by either the subcutaneous (s.c.) or the intravenous (i.v.) route. Under the influence of PEG insulin, diminished incorporation of 14C and/or 3H into adipose tissues was observed in all cases, yet both natural insulin and the derivative lowered the blood glucose to the same extent. These observations allow the assumption that, by using certain modified insulins, it may also be possible to manipulate the extent of glucose metabolism by lipid tissues in diabetic patients.
We studied the measurement of hepatic glucose output (HGO) with prolonged [3-3H]glucose infusion in 14 patients with non-insulin-dependent diabetes mellitus (NIDDM). Over the course of 10.5 h, plasma glucose concentration fell with fasting by one-third, from 234 +/- 21 to 152 +/- 12 mg/dl, and HGO fell from 2.35 +/- 0.18 to 1.36 +/- 0.07 mg . kg-1 . min-1 (P less than .001). In the basal state, HGO and glucose were significantly correlated (r = 0.68, P = .03), and in individual patients, HGO and glucose were closely correlated as both fell with fasting (mean r = 0.79, P less than .01). Plasma [3-3H]glucose radioactivity approached a steady state only 5-6 h after initiation of the primed continuous infusion, and a 20% overestimate of HGO was demonstrated by not allowing sufficient time for tracer labeling of the glucose pool. Assumption of steady-state instead of non-steady-state kinetics in using Steele's equations to calculate glucose turnover resulted in a 9-24% overestimate of HGO. Stimulation of glycogenolysis by glucagon injection demonstrated no incorporation of [3-3H]glucose in hepatic glycogen during the prolonged tracer infusion. In a separate study, plasma glucose was maintained at fasting levels (207 +/- 17 mg/dl) for 8 h with the glucose-clamp technique. Total glucose turnover rates remained constant during this prolonged tracer infusion. However, HGO fell to 30% of the basal value simply by maintaining fasting hyperglycemia in the presence of basal insulin levels.(ABSTRACT TRUNCATED AT 250 WORDS)
Calorimetry was used to assess the importance of endogenous metabolism towards total ATP synthesis in bovine sperm in the presence of extracellular glucose. Sperm were incubated in the calorimeter with D-[U-14C]glucose without or with electron transport inhibitors, rotenone and antimycin A. Steady-state heat production during the incubations was measured for 30 min, the incubations were terminated, and the cell suspensions removed for analysis of radioactive glucose and its metabolic end-products. Heat production (mean +/- S.E.) associated with the metabolism of glucose was calculated, from enthalpies of formation of glucose and its end-products, as -412 +/- 34 mJ/h/10(8) cells in control incubations and -263 +/- 18 mJ/h/10(8) cells in the incubations with electron transport inhibitors. Measured heat production was -455 +/- 36 and =263 +/- 17 mJ/h/10(8) cells, respectively. Thus, heat production by endogenous pathways, the difference between measured total heat production and calculated exogenous heat production, was -43 +/- 14 mJ/h/10(8) cells for control cells and about -6 mJ/h/10(8) cells for inhibited cells. The ratio of heat produced per mol of ATP synthesized is similar for all ATP-producing pathways. Therefore, about 10% of total ATP synthesis in control cells and less than 2% in inhibited cells is provided by endogenous pathways when extracellular glucose is present.
1. d-Glucose and l-arabinose serve as precursors of the pectic polysaccharides of sycamore suspension-callus tissue. 2. The rates and characteristics of the incorporation of radioactive sucrose, glucose and mesoinositol by sycamore callus tissue have been compared and shown to be different. 3. The time-course of the incorporation of radioactive glucose into the major fractions within the cells has been determined. Approx. 7-10% of the radioactivity incorporated is present in the whole pectin of the cells. 4. A study of the continuous incorporation of radioactive glucose showed that the neutral arabinan-galactan fraction of the pectin quickly became saturated with the radioactive label. During the incorporation of radioactivity from a pulse of radioactive glucose the neutral fraction became progressively less labelled, with a corresponding increase in the radioactivity of the weakly acidic pectinic acid, which is known to contain neutral sugars. 5. When the cells were exposed to a pulse of radioactive l-arabinose, the label accumulated first in the neutral fraction and then after 4hr. it passed to the weakly acidic pectinic acid with a corresponding decrease in the radioactivity of the neutral fraction. 6. The product that was initially labelled during the first hour of exposure of the cells in the stationary phase to radioactive glucose was identified as an incompletely methylated galacturonan in which the radioactivity was present in the anhydrogalacturonide residues. This polysaccharide probably acts as the precursor of the polyuronide portions of both the strongly acidic and weakly acidic pectinic acids. 7. The observations are discussed in relation to the structure of the pectic substances and their function in cell growth and development. A tentative model for their metabolic relationship is put forward.
1. A study of wall synthesis has been made by following the incorporation of radioactive glucose and threonine into the cytoplasm and wall of yeast. 2. Both glucose and threonine are incorporated into a mannan glycopeptide. The glucose is also synthesized into a structural glucan of the wall. 3. The mannan glycopeptide contains high-molecular-weight mannan and low-molecular-weight mannose and oligosaccharide units composed of mannose. Both types of carbohydrate are attached to the peptide. The extent of radioactive incorporation into these different carbohydrate constituents of the glycopeptide remained constant during a pulse-chase experiment. No evidence of a sequential synthesis of oligosaccharides and high-molecular-weight mannan was obtained. 4. Cycloheximide inhibits the incorporation of threonine into the wall but only partially inhibits the incorporation of glucose. Thus not all the polysaccharide deposited into the wall is dependent on a simultaneous peptide synthesis and incorporation. 5. Protoplasts grown in an iso-osmotic medium secreted a mannan polymer that was probably a glycopeptide.
After 20 min infusion of L-[U-14C]alanine through the left uterine artery in 21-day-pregnant rats, the radioactivity in the plasma of fetuses from the left uterine horn was much higher than in their mothers and was composed of approximately equal parts of [14C]alanine and [14C]lactate, with a minor percentage of [14C]glucose. Radioactivity in fetal plasma was much lower when the mothers were infused with alpha-amino[14C]isobutyric acid. The simultaneous infusion of aminooxyacetate decreased materno-fetal transfer of radioactivity from [14C]alanine but not from alpha-amino[14C]isobutyric acid, and this effect corresponded to a complete disappearance of the [14C]lactate in fetal plasma without affecting [14C]alanine levels or alanine concentration in the fetuses. Placenta slices in vitro metabolized L-[U-14C]alanine into [14C]lactate and 14CO2 at the rate of 7 nmol/g per min, and this process was inhibited by the presence of 1 mM aminooxyacetate in the medium. Placental uptake of alpha-amino[14C]isobutyric acid was half that of [U-14C]alanine, and aminooxyacetate did not affect this parameter with either of the labelled compounds. Results indicate that the lower transfer to the rat fetus of the 14C atoms from alpha-amino[14C]isobutyric acid as compared to that from [14C]alanine is due not only to the diminished placental carrier system of the former but also to its non-metabolyzable condition. It is proposed that the capacity of the placenta to metabolyze L-alanine to lactate and the subsequent release of lactate to the fetus constitute important factors for the fetal metabolic economy.
A method for quantitative estimation of total radioactivity present in the free amino acid fraction of tissue samples has been described. Samples deproteinized with cold acetone were extracted, in acidic medium, with ethyl ether (peroxide free); after centrifugation, the aqueous phase was used for amino acid derivatization at 40 degrees C for 15 h with 1-fluoro-2,4-dinitrobenzene in bicarbonate-buffered medium. Aliquots of the derivatized samples were acidified and extracted twice again with ethyl ether. The combined organic phases were placed in glass scintillation vials, dried, and used for the determination of its radioactivity, corresponding to the radioactivity present in the free amino acid fraction of the sample. Deproteinized samples of rat blood plasma, as well as hen egg white and yolk were tested after addition of known quantities of 14C-labelled amino acids or glucose, for validation of the method. No glucose radioactivity was found in any of the extracted samples. All radioactivity added to the samples in the form of 14C-labelled alanine, glutamic acid, leucine and phenylalanine was quantitatively recovered in the derivatized fraction; only a fraction of arginine radioactivity was recovered.