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PubMed1980

Calcium and pancreatic beta-cell function. IX. Demonstration of lanthanide-induced inhibition of insulin secretion independent of modifications in transmembrane Ca2+ fluxes.

beta-Cell-rich pancreatic islets were microdissected from noninbred ob/ob-mice and used to examine the mode of action of trivalent lanthanide ions on insulin secretion. La3+, Sm3+, and Tm3+ were equally effective inhibitors of basal and glucose-stimulated insulin release. As indicated by perifusion experiments with Tm3+, the inhibitory action was prompt, sustained, and readily reversible. Despite the similarities among the lanthanides in inhibiting insulin secretion, these cations differed considerably in their ability to impair transmembrane 45Ca fluxes. Using 10 different members of the lanthanide series, it was possible to demonstrate that their effectiveness to inhibit 45Ca uptake increased with ionic radius. La3+ markedly inhibited intracellular uptake and superficial binding of 45Ca at both 3 and 20 mM glucose. However, Tm3+ failed to affect intracellular 45Ca uptake and only reduced superficial binding of 45Ca at 3 mM glucose. In efflux experiments, Tm3+ did not affect basal or glucose-stimulated 45Ca washout from islets perifused with a medium containing 1.28 mM Ca2+. In a Ca2+-deficient medium, Tm3+ caused a slight transient increase, followed by reduction of 45Ca washout. However, when glucose was omitted, there was a prompt increase in the washout of radioactivity in the presence of Tm3+. Accordingly, the potent inhibitory action of Tm3+ on insulin secretion is not matched by changes in transmembrane Ca2+ fluxes. Since the lanthanides do not penetrate intracellularly, we propose the existence of cationic binding sites in the beta-cell plasma membrane with direct inhibitory effects on insulin secretion.

Animals↗
PubMed1988

Non-enzymatic glycation of antithrombin III in vitro.

Non-enzymatic glycation of antithrombin III (AT-III) has been proposed as a significant contributor to the increased incidence of thrombo-occlusive events in diabetics. AT-III, isolated from normal human plasma by means of heparin affinity and ion-exchange chromatography, was incubated with 0-0.5 M glucose in neutral phosphate buffer at 37 degrees C. The extent of non-enzymatic glycation could be monitored by uptake of radioactivity as well as by binding to a phenylboronate affinity resin, which effectively retards AT-III containing ketoamine-linked glucose. Non-enzymatically glycated AT-III (approx. 1 mol glucose/mol protein) bound heparin nearly as efficiently as non-glycated AT-III. The two AT-III preparations were equally active in inhibiting thrombin cleavage of chromogenic substrate. Following incubation with [14C]glucose, structural analyses of cyanogen-bromide-cleaved peptides of enzymatically glycated AT-III showed that the [14C]glucose adducts were distributed over many sites on the molecule. This lack of specificity contrasts with the restricted sites of modification on hemoglobin, albumin and ribonuclease A, and explains why non-enzymatic glycation of AT-III has little if any effect on its function.

Amino Acid Sequence↗
PubMed1973

Method for Simultaneous Measurement of Radioactive and Inactive CO(2) Evolved from Soil Samples During Incubation with Labeled Substrates.

An apparatus is described which allows the simultaneous, continuous, and highly sensitive analysis of inactive and radioactive CO(2) evolved from C-supplemented soils or other materials. The apparatus consists of a control unit, a commercially available conductometric CO(2) analyzer, and fraction collector. A number of model experiments were conducted to demonstrate the potentials of the apparatus. These included analysis of the time course of priming action, when C-glucose was added to soil, separation of CO(2) respiration peaks caused by simultaneous degradation of radioactive and inactive soil supplements, and study of the effects of a fungicide, Benomyl, on degradation of C-labeled glucose. In the last experiment, partial degradation of the fungicide could also be followed.

Journal Article↗
PubMed1993

Vasopressin and oxytocin but not glucose stimulate hydrolysis of phosphatidylinositol and phosphatidylcholine in a hamster insulinoma.

HIT-T15 cells prelabeled with [3H]-arachidonate were incubated for 15 minutes at 37 degrees C in Krebs Ringer buffer (pH 7.1) in the presence and absence of various agonists. Radioactivity remaining in major phospholipids was measured at the end of incubation period. Oxytocin (1 microM), vasopressin (1 microM), and A23187 (5 microM) stimulated loss of radioactivity from phosphatidylinositol and phosphatidylcholine. No loss of radioactivity from either of the phospholipids, however, was detected in the presence of 10 mM D-glucose, an insulin secretagogue in HIT-T15 cells. The lack of phosphatidylinositol response to glucose was also evident when the cells were prelabeled with myo-[3H] inositol. The formation of inositol phosphates at 15 minutes was readily observed upon the treatment of myo-[3H] inositol-labeled cells with oxytocin or vasopressin but not glucose or A23187. Inability of glucose to stimulate phosphatidylinositol and phosphatidylcholine hydrolysis in beta cell-derived HIT-T15 cells contrasts sharply with results from studies with pancreatic islets, where hydrolysis of these two phospholipids is readily observed and thought to contribute to the signaling mechanism responsible for stimulation of insulin secretion.

Animals↗
PubMed1979

Effects of glucose, chloromercuribenzene-p-sulphonic acid and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid on phosphate efflux from pancreatic islets.

Collagenase-isolated pancreatic islets of non-inbred ob/ob mice, containing more than 90% beta-cells, were labelled with radioactive orthophosphate (32P or 33P) and then subjected to non-recirculating perifusion. The basal D-glucose concentration in the perifusion medium was 2.8 mM. When the concentration was suddenly raised to 5.6, 8.3 or 16.7 mM, D-glucose promptly elicited a transient and dose-dependent release of radiophosphate. In the presence of 2.8 mM D-glucose, 0.1 mM of the poorly permeating sulphydryl blocker, chloromercuribenzene-p-sulphonic acid, also evoked a phosphate flush resembling the one induced by D-glucose. The basal radiophosphate release was partially inhibited by 1 mM 4-acetamido-4-'-isothiocyanostilbene-2,2'-disulphonic acid. However, the phosphate flush induced by 16.7 mM D-glucose was not noticeably inhibited by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid. It is concluded that the phosphate flush emanates from beta-cells and that membrane sulphydryl groups may participate in its regulation. Although at least the basal phosphate release may in part represent transmembrane transport through 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid-sensitive anion channels, other mechanisms are also likely to participate in the glucose-induced phosphate flush.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed2003

Rapid screening of 2-[18F]-fluoro-2-deoxy-D-glucose infusions for volatile organic compound contaminants by solid phase microextraction with gas chromatography-selective ion monitoring mass spectrometry (SPME-GC-SIMMS).

A method compatible with radioactive samples, capable of detecting trace volatile components in a sample volume of ca. 1cm(3) of 2-[18F]-fluoro-2-deoxy-D-glucose solution is described. The approach, based on solid phase micro-extraction gas chromatography-mass spectrometry with a carboxen/polydimethylsiloxane based fibre, was optimised with respect to extraction time (10 min), extraction temperature (60 degrees C) and phase volume ratio (1). The analysis time, including extraction, was less than 20 min with linear responses for acetonitrile and ethanol over the ranges: 0.09-80 microg cm(-3) (22 degrees C, acetonitrile) and 0.78-79 microg cm(-3) (22 degrees C, ethanol). The detection limits were estimated to be ca. 0.78 microg cm(-3) for ethanol and 0.09 microg cm(-3) for acetonitrile. Stability studies indicated analyte losses of up to 75% over 24h and analysis of aged 2-[18F]FDG samples showed that levels of ethanol and acetonitrile were not less than 100 microg cm(-3), indicative of levels substantially greater than this in the original infusions given to human subjects.

Calibration↗
PubMed1984

Effect of glucose on the major testosterone-maintained protein in the cultured rat ventral prostate.

The effect of glucose on the androgen-maintained protein synthesis was studied in the cultured rat ventral prostate. The explants were cultivated for 5 days in the glucose-free medium containing 10% fetal calf serum with or without 10 mM glucose and 10(-7) M testosterone. In some experiments tunicamycin, a specific inhibitor of protein glycosylation was added to the glucose-containing medium. The morphological integrity of the tissue was maintained in all the mediums used. At the end of the culture, the explants were incubated with [35S]methionine. Soluble radioactive proteins were separated by the SDS-polyacrylamide gel electrophoresis and analyzed further by the fluorography. Glucose was necessary for the testosterone-maintained accumulation of three components (Mr less than 14,000) of the major prostatic secretory protein. The electrophoretic migration, glycosylation pattern and immunological data (not shown) indicated that it was the well-known prostatic binding protein. On the other hand, two prominent polypeptides (Mr 70,000 and 100,000) appeared in the absence of glucose. Glucose starvation and the inhibition of glycosylation with tunicamycin caused similar effects on the labelling of the newly-synthesized soluble proteins. The mechanisms of glucose maintenance of the major prostatic protein and suppression of two high molecular weight proteins seemed to be different, although glycosylation was probably involved in both glucose effects.

Animals↗
PubMed1977

Origin and metabolic fate of plasma glycerol in the rat and rabbit fetus.

On day 21.5 a pregnant rat received a single injection of [1-14C]glycerol. The purpose was to study the transfer of glycerol through the placenta from the maternal to fetal plasma. From 3-20 min after injection the specific activity of glycerol in maternal and fetal plasma was measured. The results indicate that the mother can provide this molecule to the fetus. Similar results were obtained with the rabbit on day 28 of pregnancy. The possibility of the conversion of plasma glycerol to glucose has been investigated in the rat and rabbit fetus. This molecule was chosen chiefly to see whether the gluconeogenic pathway was functioning in the fetus above the triose phosphate step. At two stages of fetal development the capacity of the fetus to incorporate [1-14C]glycerol into glucose plus glycogen has been shown in the two species. In the rat fetus the conversion of [1-14C]glycerol to [14C]glucose increases from 19.5 to 21.5 days of gestation. For the rabbit this parameter increases from 25 to 28 days of gestation. On day 25 in the rabbit and day 19.5 in the rat the liver glycogen was labeled, but it did not accumulate the [14C]glucose from [1-14C]glycerol during the time that we have studied. In contrast, on day 28 in the rabbit and day 21.5 in the rat the incorporation of radioactivity increased as function of the time. However, the relative importance of glycerol as precursor of the glucose plus glycogen in the fetus remains to be elucidated.

Animals↗
PubMed1980

Effect of stimulation on the incorporation of 14C from glial and neuronal specific substrates into brain proteins in vivo and in vitro.

The incorporation of amino acids into brain proteins following brachial plexus stimulation (BPS) was studied in anaesthetised Sprague-Dawley rats following injection of radioactive precursors of both neuronal and glial compartments. Following intraperitoneal injection of [14C]glucose, which is the major neuronal pool precursor, BPS resulted in a significant increase of 37% (P < 0.001) in the incorporation of carbon from [14C]glucose into TCA-insoluble proteins in the contralateral sensorimotor cortex as compared with the ipsilateral area of the same animal. This increase was abolished totally when tetrodotoxin (10 micrograms ml-1) was applied topically to the surface of the stimulated area. Following intraperitoneal injection of [14C]acetate, which is considered to by mainly a glial cell precursor, the same afferent electrical stimuli caused a significant decrease of 21% in the incorporation of amino acids into proteins in the stimulated versus unstimulated sensorimotor cortex. With [4-(3)H]phenylalanine or [1-(14)C]leucine as precursors a significant decrease (12%) or no change was recorded, respectively. A similar decrease in protein synthesis in the stimulated sensorimotor cortex was achieved using different routes of injection. No significant changes were observed in the ratio of the specific radioactivities of the total amino acids of the two hemispheres using either precursor. In vitro, synaptosomes showed a large increase in incorporation into proteins after treatment with electrical pulses, both with [14C]glucose and with [U-14C]acetate as precursors.

Acetates↗
PubMed1975

[Comparative study of the effect of free and combined glucose and fructose on the absorption and retention of calcium].

Many carbohydrates play an important role in the process of absorption of several inorganic compounds, particularly of calcium, as shown by FOURNIER. VAUGHAN and FILER have proved that all carbohydrates are equally active under particular conditions: the difference of their action on calcium absorption is related to their absorption speed rate. Fructose appears in an intermediate situation between rapidly and slowly absorbed oses according to Cori's classification based on rates of absorption. In order to examine more closely the extent of differences of carbohydrates on calcium absorption, we chose to compare the action of free and combined fructose with that of carbohydrates found in normal feeding. Six months old rats in a state of calcium equilibrium received orally 2 ml aquous mixture containing 0,01 mM/ml calcium supplemented by 0,12 mu Ci 45 Ca and 0,075 mM/ml carbohydrate. The rats were divided into groups according to the carbohydrate they received, which was one of the following: D-glucose, D-mannose, D-fructose, L-sorbose, maltose, lactose, sucrose, starch, glycogen or inulin. Both absorption and retention of calcium were observed. A coefficient of absorption of 45Ca and the specific radioactivity of bone were established by the measurements of radioactivity of blood samples, which were taken 1 1/2 hour, 3 hours and 24 hours after ingestion, of intestinal content and feces, and of a femur after nitroperchloric mineralization. The results showed that values of absorption coefficient and of specific radioactivity are high for slowly absorbed sugars (mannose, sorbose, lactose) but are very low for free glucose or for glucose easily released by intestine enzymatic activity. Free fructose possesses in intermediate activity equivalent to that of inulin. However, sucrose administered at the same concentration has little effect on calcium absorption.

Absorption↗
PubMedDate not supplied

Nonradioisotope assay of glucose uptake activity in rat skeletal muscle using enzymatic measurement of 2-deoxyglucose 6-phosphate in vitro and in vivo.

We investigated a nonradioisotope method for the evaluation of glucose uptake activity using enzymatic measurement of 2-deoxyglucose 6-phosphate (2DG6P) content in isolated rat soleus muscle in vitro and in vivo. The 2DG6P content in isolated rat soleus muscle after incubation with 2-deoxyglucose (2DG) was increased in a dose-dependent manner by insulin (ED(50) = 0.6 mU/ml), the maximum response being about 5 times that of the basal content in vitro. This increment was completely abolished by wortmannin (100 nM), with no effect on basal 2DG6P content. An insulin-mimetic compound, vanadium, also increased 2DG6P content in a dose-dependent manner. In isolated soleus muscle of Zucker fa/fa rats, well known as an insulin-resistant model, insulin did not increase 2DG6P content. The 2DG6P content in rat soleus muscle increased after 2DG (3 mmol/kg) injection in vivo, and conversely, the 2DG concentration in plasma was decreased in a dose-dependent manner by insulin (ED(50) = 0.11 U/kg). The maximum response of the accumulation of 2DG6P in soleus muscle was about 4 times that of the basal content. This method could be useful for evaluating glucose uptake (transport plus phosphorylation) activity in soleus muscle in vitro and in vivo without using radioactive materials.

Androstadienes↗
PubMed1973

Control of synthesis and release of radioactive acetylcholine in brain slices from the rat. Effects of neurotropic drugs.

1. Studies of the synthesis and release of radioactive acetylcholine in rat brain-cortex slices incubated in Locke-bicarbonate-[U-(14)C]glucose media, containing paraoxon as cholinesterase inhibitor, revealed the following phenomena: (a) dependence of K(+)-or protoveratrine-stimulated acetylcholine synthesis and release on the presence of Na(+) and Ca(2+) in the incubation medium, (b) enhanced release of radioactive acetylcholine by substances that promote depolarization at the nerve cell membrane (e.g. high K(+), ouabain, protoveratrine, sodium l-glutamate, high concentration of acetylcholine), (c) failure of acetylcholine synthesis to keep pace with acetylcholine release under certain conditions (e.g. the presence of ouabain or lack of Na(+)). 2. Stimulation by K(+) of radioactive acetylcholine synthesis was directly proportional to the external concentration of Na(+), but some synthesis and release of radioactive acetylcholine occurred in the absence of Na(+) as well as in the absence of Ca(2+). 3. The Na(+) dependence of K(+)-stimulated acetylcholine synthesis was partly due to suppression of choline transport, as addition of small concentrations of choline partly neutralized the effect of Na(+) lack, and partly due to the suppression of the activity of the Na(+) pump. 4. Protoveratrine caused a greatly increased release of radioactive acetylcholine without stimulating total radioactive acetylcholine synthesis. Protoveratrine was ineffective in the absence of Ca(2+) from the incubation medium. It completely blocked K(+) stimulation of acetylcholine synthesis and release. 5. Tetrodotoxin abolished the effects of protoveratrine on acetylcholine release. It had blocking effects (partial or complete) on the action of high K(+), sodium l-glutamate and lack of Ca(2+) on acetylcholine synthesis and release. 6. Unlabelled exogenous acetylcholine did not diminish the content of labelled tissue acetylcholine, derived from labelled glucose, suggesting that no exchange with vesicular acetylcholine took place. In the presence of 4mm-KCl it caused some increase in the release of labelled acetylcholine. 7. The barbiturates (Amytal, pentothal), whilst having no significant effects on labelled acetylcholine synthesis in unstimulated brain except at high concentration (1mm), diminished or abolished (at 0.25 or 0.5mm) the enhanced release of acetylcholine, due to high K(+) or lack of Ca(2+). The fall in tissue content of acetylcholine, due to lack of Ca(2+), was diminished or abolished by pentothal (0.25 or 0.5mm) or Amytal (0.25mm).

Acetylcholine↗
PubMed1978

Preparation of internally labelled rat pituitary somatotropin (growth hormone).

Rat somatotropin (growth hormone) was labelled biosynthetically by incubating anterior pituitary lobes with radioactive amino acids for 24 h in a simple buffered salts medium containing glucose. The labelled hormone was isolated by preparative polyacrylamide-gel electrophoresis or by chromatography on Sephadex G-100 and then DEAE-cellulose. The labelled material was pure by several criteria and cross-reacted immunologically with unlabelled rat somatotropin. When a mixture of 14C-labelled amino acids was used for labelling the protein, label could be introduced into these same amino acids of somatotropin, though relative specific radioactivities varied considerably. Somatotropin labelled by the procedures described in the present paper was suitable for structural studies and could be used for a variety of other biochemical experiments.

Amino Acids↗
PubMed1992

Time course of skeletal muscle glucose uptake during euglycemic hyperinsulinemia in the anesthetized rabbit: a fluorine-18-2-deoxy-2-fluoro-D-glucose study.

Since skeletal muscle has been implicated as the major site of insulin resistance, the purpose of this study was to examine in detail the time course of muscle glucose uptake during the onset and maintenance of euglycemic hyperinsulinemia. Uptake of 18F-2-deoxy-2-fluoro-D-glucose (FDG) by the thigh muscle of an anesthetized rabbit was monitored by a single pair of coincidence photon detectors. Graphical analysis of tissue and plasma radioactivity concentrations was performed to derive fractional rates of FDG phosphorylation continuously. FDG phosphorylation rates were determined during rest (glucose 7 mM, insulin 5-10 microU/ml) and subsequent 5-min intervals under conditions of euglycemic hyperinsulinemia (glucose 6-8 mM; insulin 350-400 microU/ml plasma). FDG phosphorylation did not increase above resting control levels until 5.5 +/- 1.5 min after intravenous insulin administration. After 20-30 min of hyperinsulinemia, FDG phosphorylation and calculated glucose metabolic rates were increased by 50%. At 35-40 min of the clamp in place, there was a second increase in tracer phosphorylation which plateaued at 200% of control (p less than 0.01) and remained at this level for the remainder of the experiment. In conclusion, we have described a method for making rapid, serial estimates of insulin-mediated skeletal muscle glucose uptake. We suggest that appraisal of the time course of glucose uptake with FDG will aid in the understanding of normal and pathophysiologic states of insulin action in vivo.

Animals↗
PubMed2004

[Positron emission tomography: a clinical and biological research tool].

Medical and biological imaging has undergone a revolution in the past decade. Positron emission tomography (PET) has been developed to visualize biochemical and physiological phenomena in living humans and animals. For instance, blood flow, blood volume, glucose metabolism, amino acid metabolism, can be quantitatively estimated by means of PET with various radioactive tracers. This functional and molecular imaging technique has progressed rapidly from being a research technique in laboratories to a routine clinical imaging modality. The most widely used radiotracer in routine is 18F-fluorodeoxyglucose (18FDG), which is an analogue of glucose. Since glucose metabolism is increased many fold in malignant tumors, PET has a major role in the field of clinical oncology and recently in cardiology and neurology. PET is also a valuable tool to study cerebral or cardiac binding sites and to image the expression of reporter genes in small animals. In this review, we summarize the most recent developments in PET imaging with particular reference to the radiotracers available and their application.

Animals↗
PubMed1981

Levels and syntheses of cerebrosides and sulfatides in subcellular fractions of jimpy mutants.

During the period of brain development, the levels of nonhydroxy- and hydroxycerebrosides in the cytosol from brains of jimpy mutants were determined by high-performance liquid chromatography and compared to those in the rest of the particulates from the same brains as well as those in the littermate controls. The concentrations of cerebrosides in jimpy brain preparations were much lower than in controls at all ages. In another experiment, [U-14C]glucose was injected intraperitoneally into jimpy mutants and their littermate controls. The amounts of radioactivity incorporated into cerebrosides and sulfatides in brain cytosol, the microsome-rich fraction, and the rest of the heavier particles were determined. Although the total radioactivity incorporated into these lipids was much lower in jimpy, the specific activities were 2-3 times the control value in all subcellular fractions.

Animals↗
PubMed2002

Endometrial carcinoma diagnosed by positron emission tomography: a case report.

BACKGROUND: Positron emission tomography (PET) scanning utilizes the recognized tumor metabolic property of increased glycolysis with the radioactive decay of 2-[(18)F]fluoro-2-deoxy-D-glucose (FDG) and generation of gamma radiation to provide quantitative tumor imaging. PET scanning has proven useful in the evaluation and staging of malignancies including malignant melanoma. CASE REPORT: As part of the preoperative staging evaluation a PET scan was performed in a 76-year-old white female with a biopsy-proven malignant melanoma on the posterior thorax. Physiologic uptake was delineated, and in addition, an area of increased uptake of FDG in the pelvis anatomically consistent with the uterus was observed. Subsequent endometrial sampling revealed a moderately differentiated endometrioid adenocarcinoma. Surgical staging revealed a stage IB, grade 2 lesion. CONCLUSION: This is the first case report of an endometrial carcinoma diagnosed incidentally by positron emission tomography in an asymptomatic patient.

Aged↗