Autoradiographic studies on glucose utilization in individual zone of the rat thymus

Summary Wistar rats were injected intraperitoneally with 2-(¹⁴C)deoxyglucose and their thymuses were processed for thaw-mount autoradiography after 5, 10 or 35 min. Highest levels of radioactivity were demonstrated in the thymic medulla (5-fold higher than in the cortex). Scanning of autoradiograms for regional differences in grain densities indicated particularly intense glucose utilization in the cortico-medullary zone. Differences in glucose utilization between individual thymic zones seem to reflect differences in cellular composition, i. e., ratio of stroma cells to thymocytes.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Glucose utilization rates are linked to the internal free glucose gradient in the rat tapeworm

Hymenolepis diminuta is able to acquire plasma-borne glucose 3-O-[14C]methylglucose in vivo. Free glucose concentrations estimated for this helminth in vivo are comparable to that of the host intestine. Both in vivo and in vitro examinations indicate that the scolex-neck regions (first quartile) of this tapeworm have the highest glucose content, and an anterior-posterior gradient along the second, third, and fourth quartiles was observed. Substrate concentration was rate affecting for glucose utilization rates (measured as substrate depletion from the medium in vitro). Glucose utilization per minute exceeds glucose content by a factor of more than 5. The half-life of glucose was about 10 sec, emphasizing that sugar metabolism is a very rapid process. In addition, utilization was highest in the first quartile and decreased in succession in the second, third, and fourth quartiles. It is concluded that while the exogenous glucose concentration remains stable, regional differences in glucose utilization rates are linked (R = 0.98; P less than 0.01) to free glucose content in H. diminuta.     

Abnormal glucose metabolism accompanies failure of glucose to stimulate insulin release from a rat pancreatic cell line (RINm5F).

Glucose metabolism and insulin release were studied in isolated rat islets and in an insulin-producing rat cell-line (RINm5F). Intact islets displayed two components of glucose utilization, with glucose stimulation of insulin release being associated with the high-Km component (reflecting glucokinase-like activity). Glucose failed to stimulate insulin release from RINm5F cells, which only displayed a single low-Km component of glucose utilization. Only low-Km (hexokinase-like) glucose-phosphorylating activity was found for disrupted RINm5F cells. These changes in glucose metabolism may contribute towards the failure of glucose to stimulate insulin release from RINm5F cells.     

Heterogeneity in glucose sensitivity among pancreatic beta-cells is correlated to differences in glucose phosphorylation rather than glucose transport.

Rat beta-cells differ in their individual rates of glucose-induced insulin biosynthesis and release. This functional heterogeneity has been correlated with intercellular differences in metabolic redox responsiveness to glucose. The present study compares glucose metabolism in two beta-cell subpopulations that have been separated on the basis of the presence (high responsive) or absence (low responsive) of a metabolic redox shift at 7.5 mM glucose. Mean rates of glucose utilization and glucose oxidation in high responsive beta-cells were 2- to 4-fold higher than in low responsive beta-cells, whereas their leucine and glutamine oxidation was only 10-50% higher. This heterogeneity in glucose metabolism cannot be attributed to differences in GLUT2 mRNA levels or in glucose transport. In both cell subpopulations, the rates of glucose transport (13-19 pmol/min/10(3) beta-cells) were at least 50-fold higher than corresponding rates of glucose utilization. On the other hand, rates of glucose phosphorylation (0.3-0.7 pmol/min/10(3) beta-cells) ranged within those of total glucose utilization (0.2-0.4 pmol/min/10(3) beta-cells). High responsive beta-cells exhibited a 60% higher glucokinase activity than low responsive beta-cells and their glucokinase mRNA level was 100% higher. Furthermore, glucose phosphorylation via low Km hexokinase was detected only in the high responsive beta-cell subpopulation. Heterogeneity in glucose sensitivity among pancreatic beta-cells can therefore be explained by intercellular differences in glucose phosphorylation rather than in glucose transport.     

Metabolism of Escherichia coli injured by copper

Escherichia coli injured by copper in carbonate buffer simulating the drinking water environment showed decreased oxygen utilization. Oxygraph measurements revealed that copper-injured bacteria had a rate of oxygen utilization that was less than 25% of that of control cells. Respirometry experiments measured rates over a longer period of time and showed similar trends. Nuclear magnetic resonance spectroscopy (13C nmr) and gas chromatography were used to identify differences in metabolism between healthy and injured populations of E. coli. The rate of glucose utilization by injured cells under anaerobic conditions was 64% of that of healthy cells. The rates of lactate and ethanol accumulation were 88 and 50% of the control, respectively. The 13C nmr studies of oxygenated cultures revealed differences in the accumulation of acetate and glutamine. Aerobic utilization of glucose and succinate by injured cells were 87 and 21% of the rate of the controls, respectively. Additional studies revealed injured cells had a decreased ability to reduce 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT) with a variety of carbohydrate substrates. Injured cells reduced greater quantities of INT than healthy cells when NADH was used as a substrate. A comparison of metabolic end products suggested that injured cells also had considerable differences in carbon flow compared with healthy cells.     

Depth-Related Differences in Organic Substrate Utilization by Major Microbial Groups in Intertidal Marine Sediment

Stable isotope probing of magnetic-bead-captured rRNA (Mag-SIP) indicated clear differences in in situ organic substrate utilization by major microbial groups between the more oxidized (0 to 2 cm) and sulfate-reducing (2 to 5 cm) horizons of marine intertidal sediment. We also showed that cyanobacteria and diatoms may survive by glucose utilization under dark anoxic conditions.     

Insulin resistance of hind-limb tissues in vivo in lactating sheep.

1. The effects of varying the plasma insulin concentration by infusion while maintaining euglycaemia by infusion of glucose on nutrient arterio-venous differences across the hind-limb and mammary gland in lactating and non-lactating sheep were investigated. 2. Insulin infusion increased the glucose arterio-venous difference across the hind-limb; this effect of insulin was decreased by lactation, suggesting that lactation induces insulin resistance in skeletal muscle. 3. Lactation increased but insulin infusion decreased the plasma concentrations of acetate, beta-hydroxybutyrate and non-esterified fatty acids. 4. Insulin infusion decreased the arterio-venous differences of acetate and hydroxybutyrate across the hind-limb; this effect of insulin is probably indirect, resulting from the decrease in plasma concentrations of these metabolites. 5. Infusion of insulin had no effect on the glucose arterio-venous difference across the mammary gland, but did decrease the oxygen arterio-venous difference. 6. The results suggest that lactation results in insulin resistance in skeletal muscle, at least with respect to glucose utilization; this should facilitate the preferential utilization of glucose by the mammary gland.     

Effects of fatty acids, ketone bodies, lactate and pyruvate on glucose utilization by guinea-pig cerebral cortex slices

1. Starvation did not affect the rates of glucose utilization or lactate formation by guinea-pig cerebral cortex slices. 2. Palmitate (1mm), butyrate (5mm) or acetoacetate (5mm) did not affect glucose utilization or lactate formation by cerebral cortex slices from guinea pigs starved for 48hr. 3. dl-beta-Hydroxybutyrate (10mm) increased the formation of lactate without affecting glucose utilization by cerebral cortex slices from guinea pigs starved for 48hr. This implies that beta-hydroxybutyrate decreased the rate of glucose oxidation. 4. Metabolism of added ketone bodies can account for 20-40% of observed rates of oxygen consumption. 5. Lactate or pyruvate (5mm) decreased the rates of glucose utilization by guinea-pig cerebral cortex slices.     

Alteration of glucose transport and diauxic growth in 5-thio-D-glucose-resistant mutants of Azotobacter vinelandii.

Spontaneous mutants of Azotobacter vinelandii defective for glucose utilization were selected as resistant to 5-thio-D-glucose. Mutant strains AM2, AM38, and AM39 exhibited longer generation times than the wild type when grown on glucose. Mutant strain AM2 also exhibited an altered Km and Vmax for glucose uptake. During acetate-glucose diauxie, glucose utilization in the 5-thio-D-glucose-resistant mutants was subject to severe inhibition by acetate. These mutants did not exhibit the normal glucose phase of diauxie. Transport studies during diauxie indicated that glucose uptake was not induced in mutant strain AM2. However, increasing the glucose concentration from 25 to 200 mM relieved the severe acetate inhibition, and under these conditions the mutant strain AM2 exhibited normal diauxie. Revertants of mutant strain AM2 exhibited normal glucose and diauxie growth. The results are discussed in terms of a model for acetate regulation of glucose utilization in A. vinelandii.     

The pentose cycle and insulin release in mouse pancreatic islets

1. Rates of insulin release, glucose utilization (measured as [(3)H]water formation from [5-(3)H]glucose) and glucose oxidation (measured as (14)CO(2) formation from [1-(14)C]- or [6-(14)C]-glucose) were determined in mouse pancreatic islets incubated in vitro, and were used to estimate the rate of oxidation of glucose by the pentose cycle pathway under various conditions. Rates of oxidation of [U-(14)C]ribose and [U-(14)C]xylitol were also measured. 2. Insulin secretion was stimulated fivefold when the medium glucose concentration was raised from 3.3 to 16.7mm in the absence of caffeine; in the presence of caffeine (5mm) a similar increase in glucose concentration evoked a much larger (30-fold) increase in insulin release. Glucose utilization was also increased severalfold as the intracellular glucose concentration was raised over this range, particularly between 5 and 11mm, but the rate of oxidation of glucose via the pentose cycle was not increased. 3. Glucosamine (20mm) inhibited glucose-stimulated insulin release and glucose utilization but not glucose metabolism via the pentose cycle. No evidence was obtained for any selective effect on the metabolism of glucose via the pentose cycle of tolbutamide, glibenclamide, dibutyryl 3':5'-cyclic AMP, glucagon, caffeine, theophylline, ouabain, adrenaline, colchicine, mannoheptulose or iodoacetamide. Phenazine methosulphate (5mum) increased pentose-cycle flux but inhibited glucose-stimulated insulin release. 4. No formation of (14)CO(2) from [U-(14)C]ribose could be detected: [U-(14)C]xylitol gave rise to small amounts of (14)CO(2). Ribose and xylitol had no effect on the rate of oxidation of glucose; ribitol and xylitol had no effect on the rate of glucose utilization. Ribose, ribitol and xylitol did not stimulate insulin release under conditions in which glucose produced a large stimulation. 5. It is concluded that in normal mouse islets glucose metabolism via the pentose cycle does not play a primary role in insulin-secretory responses.     

Alpha- and beta-anomeric preference of glucose-induced insulin secretion at physiological and higher glucose concentrations, respectively

We determined the anomeric preference of glucose phosphorylation by islet glucokinase, glucose utilization by pancreatic islets, and insulin secretion induced by glucose over a wide range of glucose concentrations. alpha-D-Glucose was phosphorylated faster than beta-D-glucose by islet glucokinase at lower glucose concentrations (5 and 10 mM), whereas the opposite anomeric preference was observed at higher glucose concentrations (40 and 60 mM). At 20 mM, there was no significant difference in phosphorylation rate between the two anomers. Similar patterns of anomeric preference were observed both in islet glucose utilization and in glucose-induced insulin secretion. The present study affords strong evidence that glucokinase is responsible for the anomeric preference of glucose-stimulated insulin secretion through anomeric discrimination in islet glucose utilization.     

Hypoglycaemic effect of metformin in genetically obese (fa/fa) rats results from an increased utilization of blood glucose by intestine.

The insulin-resistant obese fa/fa rat is a convenient model in which to study a potential effect of metformin, a biguanide used in the treatment of non-insulin-dependent diabetes, on insulin-mediated glucose utilization. Female fa/fa rats were given metformin orally for 8 days. Studies were performed on anaesthetized post-absorptive rats 5 h after the last dose of metformin. Glucose production and utilization were enhanced 1.5-fold in metformin-treated rats. The enhanced glucose production was almost entirely due to increased glucose recycling. The digestive tract was the only tissue responsible for the enhanced glucose utilization.     

Utilization of energy-providing substrates in the isolated working rat heart.

1. An improved perfusion system for the isolated rat heart is described. It is based on the isolated working heart of Neely, Liebermeister, Battersby & Morgan (1967) (Am. J. Physiol. 212, 804-814) and allows the measurement of metabolic rates and cardiac performance at a near-physiological workload. The main improvements concern better oxygenation of the perfusion medium and greater versatility of the apparatus. Near-physiological performance (cardiac output and aortic pressure) was maintained for nearly 2 h as compared with 30 min or less in the preparations of earlier work. 2. The rates of energy release (O2 uptake and substrate utilization) were 40-100% higher than those obtained by previous investigators, who used hearts at subphysiological workloads. 3. Values are given for the rates of utilization of glucose, lactate, oleate, acetate and ketone bodies, for O2 consumption and for the relative contributions of various fuels to the energy supply of the heart. Glucose can be replaced to a large extent by lactate, oleate or acetate, but not by ketone bodies. 4. Apart from quantitative differences there were also major qualitative differences between the present and previous preparations. Thus insulin was not required for maximal rates of glucose consumption at near-physiological, in contrast with subphysiological, workloads when glucose was the sole added substrate. When glucose oxidation was suppressed by the addition of other oxidizable substrates (lactate, acetate or acetoacetate), insulin increased the contribution of glucose as fuel for cardiac energy production at high workload. 5. In view of the major effects of workload on cardiac metabolism, experimentation on hearts performing subphysiologically or unphysiologically is of limited value to the situation in vivo.     

Chromium (III) Propionate and dietary fructans supplementation stimulate erythrocyte glucose uptake and beta-oxidation in lymphocytes of rats

The study describes the effects of 10-wk dietary supplementation with fructans (inulin and oligofructose, 5% and 10%, respectively) as well as the biomimetic Cr(III) propionate complex (0.5 and 5 mg Cr/kg diet) on blood glucose, insulin, glucose transmembrane transport, and β-oxidation of fatty acids in healthy male rats. No significant differences in blood serum glucose concentrations were found. Rats fed diets supplemented with the biomimetic complex (5 mg Cr/kg diet) had markedly decreased serum insulin level by 15%, whereas the red blood cells (RBCs) glucose transmembrane transport and β-oxidation of fatty acids in white blood cells (WBCs) were elevated by 9% and 77%, respectively. These effects were accompanied by a slight decrease of the insulin-resistance index. Oligofructose and the high-fructan diet (10%) were more effective in increasing the RBCs glucose transmembrane transport vs inulin and lowfructan diet (5%). Also, β-oxidation of fatty acids in WBCs was increased by 37.5% in groups fed the high-fructan diet (10%). The results suggest that dietary fructans and the biomimetic Cr(III) complex exerted beneficial effects on glucose and lipid metabolism, increasing the efficiency of their utilization.     

Insulin resistance in soleus muscle from obese Zucker rats. Involvement of several defective sites

1. The effect of insulin upon glucose transport and metabolism in soleus muscles of genetically obese (fa/fa) and heterozygote lean Zucker rats was investigated at 5–6 weeks and 10–11 weeks of age. Weight-standardized strips of soleus muscles were used rather than the intact muscle in order to circumvent problems of diffusion of substrates. 2. In younger obese rats (5–6 weeks), plasma concentrations of immunoreactive insulin were twice those of controls, whereas their circulating triacylglycerol concentrations were normal. Insulin effects upon 2-deoxyglucose uptake and glucose metabolism by soleus muscles of these rats were characterized by both a decreased sensitivity and a decrease in the maximal response of this tissue to the hormone. 3. In older obese rats (10–11 weeks), circulating concentrations of insulin and triacylglycerols were both abnormally elevated. A decrease of 25–35% in insulin-binding capacity to muscles of obese rats was observed. The soleus muscles from the older obese animals also displayed decreased sensitivity and maximal response to insulin. However, at a low insulin concentration (0.1m-i.u./ml), 2-deoxyglucose uptake by muscles of older obese rats was stimulated, but such a concentration was ineffective in stimulating glucose incorporation into glycogen, and glucose metabolism by glycolysis. 4. Endogenous lipid utilization by muscle was calculated from the measurements of O₂ consumption, and glucose oxidation to CO₂. The rate of utilization of fatty acids was normal in muscles of younger obese animals, but increased in those of the older obese rats. Increased basal concentrations of citrate, glucose 6-phosphate and glycogen were found in muscles of older obese rats and may reflect intracellular inhibition of glucose metabolism as a result of increased lipid utilization. 5. Thus several abnormalities are responsible for insulin resistance of muscles from obese Zucker rats among which we have observed decreased insulin binding, decreased glucose transport and increased utilization of endogenous fatty acid which could inhibit glucose utilization.     

Kinetics of utilization of organic substrates by Mycoplasma mycoides subsp. mycoides in a salts solution: a flow-microcalorimetric study

The metabolism of various organic substrates by suspensions of Mycoplasma mycoides subsp. mycoides in a salts solution was followed by microcalorimetry. Enthalpy changes associated with metabolism were in good agreement with theoretical values. Substrate utilization showed Michaelis kinetics, allowing saturation constants (Km) and maximum specific rates of substrate utilization (Vmax) to be determined. In cells grown on a complex medium containing glucose, Km values were: glucose, fructose, N-acetylglucosamine, glycerol and pyruvate, less than 5 microM; lactate, 20 microM; glucosamine, 130 microns, and mannose, 1 mM. Values of Vmax for glycerol, pyruvate and lactate were similar and approximately twice those for glucose, mannose, glucosamine and N-acetylglucosamine; Vmax for fructose was one-quarter of that for glucose. In cells grown on complex medium in which glucose was replaced by mannose, glucosamine or N-acetylglucosamine, Vmax and Km for the respective growth sugars and for glucose were not significantly affected. However, in cells grown in the presence of fructose, Vmax for fructose increased to the value observed for glucose. It is suggested that M. mycoides is adapted to, and is constitutive for, the utilization of a single sugar (glucose), and a single amino sugar (N-acetylglucosamine), but that in the presence of fructose a fructose-utilizing pathway is induced.     

Peripheral CD4+ lymphocytes derived from fetal versus adult thymic precursors differ phenotypically and functionally

There is growing evidence that the differentiation processes in the fetal and adult thymus are not identical. However, there is little information on whether these developmental differences influence the properties of mature cells that exit the thymus and seed peripheral lymphoid organs. We have addressed this issue by comparing the development of Ag-specific Th1/Th2 function by fetal vs adult thymic derived CD4(+) cells in the same adoptive adult hosts. Host mice were irradiated and transplanted with 14- to 15-day fetal thymic lobes from Thy-1 congenic mice. Ag (keyhole limpet hemocyanin)-specific Th1/Th2 responses of fetal-derived (donor) or adult-derived (host) CD4(+) cells were analyzed by ELISA following primary or secondary immunization. Fetal-derived cells produced up to 10-fold more of both Th1 (IFN-gamma) and Th2 (IL-4) cytokines than did adult-derived cells. Comparisons of the IL-4:IFN-gamma ratios showed that the responses of fetal-derived cells were Th2-skewed in an Ag dose-dependent manner. At low doses of Ag, the fetal-derived ratio was approximately 5 times higher than the adult-derived ratio. As the Ag dose was increased, the differences between the ratios of the fetal- and adult-derived responses were minimized. These relative responses were established initially during the primary effector phase but were maintained for weeks, into the memory phase of the immune response. Importantly, fetal-derived CD4(+) cells showed these properties whether the fetal thymic precursors matured within the fetal or adult thymic microenvironment. These results demonstrate that cells arising from fetal thymic precursors are functionally different both qualitatively and quantitatively from adult-derived cells.     

Verapamil reduces glucose and fatty acid metabolism of beating and nonbeating rat heart cells

The effects of various dosages of verapamil on glucose or fatty acid utilization by beating or nonbeating rat heart myocytes in tissue culture were determined. Myocytes were incubated with verapamil and either D-6-14C glucose or 1-14C palmitic acid as substrate. After incubation the subsequently generated 14CO2 was captured with hyamine hydroxide and the equivalent oxygen values were calculated. Low doses of verapamil (50 ng/ml) treatment produced a 52% reduction in myocyte glucose utilization and a 16% reduction in fatty acid utilization that appeared to be independent of its effect on myocyte contractile rate since these effects were evident in both beating and nonbeating myocytes. In addition, verapamil treatment caused differences in the myocyte handling of substrate. Verapamil (50 ng/ml) lowered cellular accumulation of glucose by 21% compared to controls. Contrary to glucose, myocyte concentrations of palmitic acid were significantly increased by 117% relative to controls in verapamil treated cultures. These results suggest that verapamil may have a direct effect on basal heart cell metabolism in a way that is unrelated to myocyte contractile activity. In addition, verapamil may interfere with glucose membrane transport.