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PubMed1982

Properties of N-maleoylmethionine sulphone, a novel impermeant maleimide, and its use in the selective labelling of the erythrocyte glucose-transport system.

1. The synthesis of N-maleoylmethionine sulphone (MMS), a membrane-impermeant protein-labelling reagent, is described. Radioactively labelled MMS can be readily prepared at high specific radioactivity from [35S]methionine. 2. The permeability of the erythrocyte membrane to the reagent was assessed by determining the extent of inactivation of glyceraldehyde 3-phosphate dehydrogenase after treatment of erythrocytes with MMS. Some inactivation of this enzyme was found when high concentrations (20mM) of the compound were used, but this could be prevented by pretreatment of the erythrocytes with 4,4'-di-isothiocyanatostilbene-2,2'-disulphonic acid, suggesting that MMS slowly enters the cells via the anion-transport system. 3. Treatment of erythrocytes with [35S]MMS resulted in the labelling of six major components. Labelling of erythrocyte membranes resulted in the intense labelling of many additional components. 4. MMS inhibited erythrocyte glucose transport. Cytochalasin b protected glucose transport against inactivation by MMS. Labelling experiments in erythrocytes in the presence and in the absence of cytochalasin b showed that the cytochalasin b-protected material was a broad band in the band-4.5 region.

Biological Transport↗

PubMed1977

Effect of insulin and glucose on the activity of insulin-degrading enzymes in rat liver.

The degradation of insulin by insulin protease and glutathion-insulin transhydrogenase (glutathioneproteindisulphide oxidoreductase--EC 1.8.4.2, GIT) was measured in rat liver either after replacing food and water by 15% glucose solution, or after daily insulin administration 8 U daily for 3 days or after fasting. The breakdown of radioiodinated insulin was followed by measuring the increase of TCA soluble radioactivity during incubation of cell fractions with 125I insulin at 37 degrees C. The highest GIT activity was observed in liver microsomes of rats after glucose feeding and after insulin administration, whereas enzyme activity of fasted animals did not essentially differ from corresponding values of normally fed controls. The insulin protease in cytosol of liver cells remained unchanged after these procedures. The important role of GIT in insulin degradation seems to be conclusively demonstrated.

Animals↗

PubMed1995

Correction of spillover radioactivities for estimation of the blood time-activity curve from the imaged LV chamber in cardiac dynamic FDG PET studies.

In dynamic cardiac PET FDG studies for measurement of myocardial metabolic rate of glucose (MMRGlc), the plasma FDG time-activity curve (input function) is commonly obtained from the left ventricular (LV) region on the PET images. The input function is contaminated by spillover of radioactivity from the surrounding myocardium and this could cause significant error in the estimated MMRGlc. In this study, we determined the effect of myocardial to blood pool spillover on MMRGlc and developed a method to correct for this spillover of activity. The method is based on a reformulation of the FDG model equation in terms of the spillover contaminated input function that includes both the myocardium to blood pool and blood pool to myocardium spillover fractions as variable parameters (Fmb and Fbm). The reformulated model equation can be used to fit the global myocardial tissue activity curve to estimate Fmb and thus yields a spillover corrected input function. The MMRGlc estimate with the corrected input function was within 95% of the true value (compared to 85% using the uncorrected input function) in a set of computer simulation studies. Dynamic PET FDG data were obtained in eight human studies and blood samples were obtained during the study. As compared to the results with the uncorrected input function, the estimates of k4 by the new method were reduced by 69% into a range consistent with in vitro results. The method is effective in correcting Fmb spillover and leads to more accurate estimates of MMRGlc. The method also allows larger regions of interest (up to 150 mm2) to be drawn over the LV in dynamic PET images, thereby reducing the noise level in the input function.

Computer Simulation↗

PubMed1995

Noradrenaline-induced stimulation of glutamine metabolism in primary cultures of astrocytes.

Effects of noradrenaline and of adrenergic subtype specific agonists on the uptake and metabolism of [14C]glutamine and [14C]glutamate in primary cultures of mouse astrocytes have been investigated. The total uptake of radioactivity from extracellular [14C]glutamine into the cells was enhanced during exposure to 100 microM noradrenaline, isoproterenol, or clonidine. This is partly due to an increased radioactivity in the glutamine pool and partly due to an increased formation of labeled glutamate from glutamine, which had become very marked (66%) after 240 min of incubation. The CO2 formation from labeled glutamine during 4 hr of incubation was enhanced about twofold in the presence of noradrenaline. Ten millimolar amino oxyacetic acid (AOAA), a transamination inhibitor, had no effect on CO2 formation from glutamine, indicating that the formation of alpha-ketoglutarate from glutamate occurs as an oxidative deamination. The stimulation of 14CO2 production from labeled glutamine was at least as large when glucose was deleted from medium, suggesting that the increased 14CO2 formation represents a stimulation of glutamine metabolism as such and is not only a reflection of an increase in oxidative metabolism of glucose and a bidirectional exchange between alpha-ketoglutarate and glutamate. The opposite process, incorporation of radioactivity from labeled glutamate into glutamine, was not enhanced in the presence of noradrenaline. The findings suggest that noradrenaline stimulates the rates of glutamine uptake, glutamate synthesis, and CO2 production from glutamine and thus increases energy supply to astrocytes but has no effect on the opposite reaction, i.e., glutamine formation from glutamate, a reaction of importance for neuronal-astrocyte interations.

Adrenergic alpha-Agonists↗

PubMed1982

Role of an acidic compartment in synthesis of disaturated phosphatidylcholine by rat granular pneumocytes.

A possible role for an acidic subcellular compartment in biosynthesis of lung surfactant phospholipids was evaluated with granular pneumocytes in primary culture. Incubation with chloroquine (100mum) was used to perturb this compartment. With control cells, incorporation of [9,10-(3)H]palmitic acid into total lipids and into total phosphatidylcholines increased linearly with time up to 4h. Total incorporation into phosphatidylcholine during a 1h incubation was 999+85pmol of [9,10-(3)H]palmitic acid, 458+/-18pmol of [1-(14)C]oleic acid and 252+/-15pmol of [U-(14)C]glucose per mug of phosphatidylcholine phosphorus. The cellular content of either disaturated phosphatidylcholine or total phosphatidylcholines did not change during a 2h incubation with chloroquine. In the presence of chloroquine, the specific radioactivity of [(3)H]palmitic acid in disaturated phosphatidylcholine increased by 40%, and that of disaturated-phosphatidylcholine fatty acids from [U-(14)C]glucose increased by 125%. Incorporation of [1-(14)C]oleic acid into phosphatidylcholine was decreased by chloroquine by 79% and 33% in the presence or absence of palmitic acid respectively. Chloroquine stimulated phospholipase activity in intact cells, and in sonicated cells at pH4.0, but not at pH8.5. The observations indicate that chloroquine stimulates synthesis of disaturated phosphatidylcholine in granular pneumocytes from fatty acids, both exogenous and synthesized de novo, which can be due to stimulation of acidic phospholipase. This stimulation of acidic phospholipase A activity by chloroquine appears to be coupled to the synthesis of disaturated phosphatidylcholine, thereby enhancing remodelling of phosphatidylcholine synthesized de novo. Our findings, therefore, implicate the involvement of an acidic subcellular compartment in the remodelling pathway of disaturated phosphatidylcholine synthesis by granular pneumocytes.

Animals↗

PubMed1988

Serum from diabetic patients enhances synthesis of arterial basement membrane-like material in cultured smooth muscle cells.

The effect of serum from both type I and type II diabetic subjects on the metabolism of arterial basement membrane (BM)-like material was studied in cultures of rabbit aortic smooth muscle cells. The basement membrane-like material was isolated from the cell-layer by a combined sonication and centrifugation technique. Serum from type I diabetic persons added to the incubation medium increased statistically significantly the incorporation of L-[4,5]-3H-leucine into the basement membrane-like material as compared to serum from non-diabetic subjects (2P less than 0.05). The same effect was seen with serum from type II diabetic patients as compared to serum from nondiabetic subjects (2P less than 0.05). No effect of serum from type I diabetic persons was seen in degradation experiments. Incubation medium supplemented with normal serum and extra glucose neither changed the production of basement membrane-like material nor the disappearance rate of radioactive leucine from the basement membrane-like material in degradation experiments. The present study indicates that serum from diabetic subjects enhances the production of arterial basement membrane-like material from arterial smooth muscle cells in culture. The obtained data may be relevant for the understanding of the development of macroangiopathy among diabetic patients.

Adult↗

PubMed1973

Abolition of crypticity of Arthrobacter pyridinolis toward glucose and alpha-glucosides by tricarboxylic acid cycle intermediates.

Arthrobacter pyridinolis cannot grow on glucose as sole carbon source, although the cells possess catabolic enzymes of the Embden-Meyerhof and pentose phosphate pathways as well as a complete tricarboxylic acid cycle. Crypticity toward glucose is abolished by a period of growth in a medium containing malate, succinate, citrate, or fumarate in addition to glucose. Other carbon sources, which support as rapid growth as does malate (e.g. asparagine), do not enable the cells to use glucose. Malate, succinate, citrate, and fumarate abolish crypticity toward glucose only in the second phase of diauxic growth after the tricarboxylic acid cycle intermediate has been depleted. This sequence of events, first observed in growth curves, has been verified by experiments in which the incorporation of radioactive substrates into trichloroacetic acid-insoluble cellular material was followed. The tricarboxylic acid cycle intermediates which confer the ability to utilize glucose also enhance the utilization of the alphaglucosides sucrose and maltose. The mechanism whereby growth on certain tricarboxylic acid cycle intermediates confers the subsequent ability to grow on glucose is related to a transport system for glucose and alpha-glucosides. This transport system has been assayed by measuring the uptake of [1-(14)C]-2-deoxyglucose. Cells grown for varying periods of time in asparagine, asparagine plus glucose, or malate do not transport 2-deoxyglucose. Cells from malate-glucose cultures that are in the exponential phase of growth on glucose can transport 2-deoxyglucose. Transport of 2-deoxyglucose shows Michaelis-Menten kinetics with a K(m) of 2.9 x 10(-4) M. It is competitively inhibited by glucose, alpha-methylglucopyranoside, and maltose. The transport of 2-deoxyglucose is inhibited by cyanide, dinitrophenol, azide, and N-ethylmaleimide, but not by malonate or fluoride. No phosphoenolpyruvate: d-glucose phosphotransferase activity has been detected, and the 2-deoxyglucose transported into the cell is not phosphorylated.

Alcohol Oxidoreductases↗

PubMed1999

[Utilization of glucose, valine, glycerin and palmitic acid by energy and anabolic pathways of metabolism in rats].

Possibility of evaluation of nutrients use by energy and anabolic ways by measurement in blood of radioactivity of water formed from labelled by tritium precursors was studied. The experiment was made on rats, which consecutively with interval 1.5-2 weeks were given intravenous indicative amount of 3H2(2,3)-valine, 1-14C-glucose, 6-3H-glucose, 3H2(1,3)-glycerin and 3H2(9,10)-palmitic acid. Half-time of glucose in blood was 46 min, glycerin--16 min, valine and palmitic acid--24 min. Plastic and energy characteristics of these substances were studied. The proposed approach to the evaluation of plastic and energy functions of nutrients can be useful for development of specialized products for enteral and parenteral feeding.

Animals↗

PubMed1999

Dietary glycotoxins: inhibition of reactive products by aminoguanidine facilitates renal clearance and reduces tissue sequestration.

Evidence indicates that the metabolic turnover of food-derived reactive orally absorbed advanced glycation end products (AGEs) or glycotoxins (GTs) is delayed, possibly contributing to the tissue damage induced by endogenous AGEs, especially in patients with diabetes and kidney disease. The aim of this study was to explore whether pharmacologic inhibition of dietary AGE bioreactivity by aminoguanidine (AG) can improve turnover and renal excretion of these substances. Normal Sprague-Dawley rats were fed single-labeled [14C]AGE-ovalbumin, double-labeled [14C-125I]AGE-ovalbumin, or control 125I-labeled ovalbumin diet plus free [14C]glucose, with or without AG (0.2% in water). [14C]AGE- and 125I-labeled peptide-associated radioactivity (RA) were compared with AGE immunoreactivity (by enzyme-linked immunosorbent assay) in tissues, serum, and 72-h urine samples. The effect of AG on dietary AGE bioreactivity was assessed by monitoring the inhibition of covalent complex formation between fibronectin (FN) peptide fragments and serum components, after a meal of labeled dietary AGE with or without AG. The radiolabeled AGE diet produced serum absorption and urinary excretion peaks kinetically distinct from those of free [14C]glucose or [125I]ovalbumin. Some 26% of the orally absorbed AGE-ovalbumin was excreted in the urine, whereas after AG treatment, urinary excre-tion of dietary AGEs increased markedly (to >50% of absorbed). More than 60% of tissue-bound RA was found covalently deposited in kidneys and liver, whereas after treatment with AG, tissue AGE deposits were reduced to <15% of the amount found in untreated AGE-fed controls. Sera enriched for dietary GTs formed covalently linked complexes with FN, a process completely inhibitable by AG cotreatment. Amelioration of dietary GT bioreactivity by AG improves renal elimination and prevents tissue deposition of food GTs. This may afford a novel and potentially protective use of AG against excessive tissue AGE toxicity in diabetic patients with renal disease.

Animals↗

PubMed1976

The effect of glucose, insulin and adrenaline on glycerol metabolism in vitro in rat adipose tissue.

The uptake and utilization of [1-14C]glycerol was determined in pieces of rat epididymal fat-pads incubated in Krebs--Ringer bicarbonate buffer containing albumin. Insulin (200 muunits/ml), adrenaline (epinephrine; 0.5 mug/ml) and glucose (0, 5, 15 and 20 mM) were added to the medium. Changes in the specific radioactivity of the tracer during the incubation were taken into account in calculating the rate of glycerol utilization. Adrenaline decreased glycerol uptake, whereas insulin plus adrenaline increased it. The rate of incorporation of glycerol into glycerides was decreased by adrenaline and insulin, singly or together. Insulin increased the rate of formation of CO2 and fatty acids from glycerol. The formation of CO2 and fatty acids was further enhanced by insulin plus adrenaline. The decrease in glycerol uptake induced by adrenaline, the decrease in incorporation of glycerol into glycerides induced by insulin and insulin plus adrenaline and the synthesis of fatty acids were dependent on the presence of glucose in the medium. Thus insulin and adrenaline act on glycerol utilization in adipose tissue and some of their effects are mediated by action on glucose metabolism, but others are independent of this.

Adipose Tissue↗

PubMed1980

Noninvasive determination of local cerebral metabolic rate of glucose in man.

A method for the determination of local cerebral metabolic rates of glucose (LCMRGlc) in normal man is described. The method employs [18F]2-fluoro-2-deoxy-D-glucose (FDG) and emission-computed tomography (ECT). FDG was injected intravenously as a bolus. Radioactivities in separate brain regions were measured with ECT. Plasma FDG concentration following injection was measured from blood samples. A mathematical model that describes the kinetics of FDG transports was employed to determine the transport rate constants of FDG and to convert the radioactivity measurements to metabolic rates. The model has taken into account the possible dephosphorylation reaction from FDG-6-PO4 (FDG-6-P) to free FDG in brain tissues. Experiments were performed in 13 normal volunteers. The rate constants of FDG in man were found to be comparable to those of deoxyglucose in rat and in rhesus monkey. The average LCMRGlc in gray and in white matter were found to be 7.30 +/- 1.18 (SD) and 3.41 +/- 0.64 mg/min per 100 g brain tissue, respectively. The subject-to-subject variation of LCMRGlc as measured by the present method was comparable to those of other methods that measure whole-brain CMRGlc.

Biological Transport↗

PubMed1965

Pathways of fructose conversion into glucose in foetal rat liver.

1. Glucose, formed from [1-(14)C]fructose or [6-(14)C]fructose in rat-liver slices, has been isolated as gluconate and degraded to give the radioactivity in C-1, C-2-5 and C-6. 2. By using this method it has been shown that, in liver from foetal rats younger than 20 days, glucose is formed from fructose without splitting of the molecule by the aldolase reaction. The rate of glucose formation from fructose in liver from these foetuses is approximately half of the rate in adult liver. 3. The direct conversion of fructose into glucose in foetal rat liver is not via sorbitol as in seminal vesicles, as this pathway cannot be detected. 4. When liver slices are incubated with [U-(14)C]fructose of high specific activity, the labelled intermediates are similar whether from liver from 18-day foetal, newborn or adult rats. 5. These findings are discussed with reference to the changing pathways of fructose metabolism during perinatal development of the liver in the rat.

Journal Article↗

PubMed1977

The effect of glycerol and other intermediates on the incorporation of 14C-fructose and 14C-glucose to hepatic triglyceride.

Mature male rats were given, orally, 14C-fructose either alone or in conjunction with dihydroxyacetone, glyceraldehyde, or glycerol. Glycerol was also administered with 14C-glucose in a dose equimolar with fructose. After 2 hr the rats were killed and the liver triglyceride fraction was separated. Estimations of the degree of incorporation of 14C from the radioactive labeled carbohydrate into the glycerol and fatty acid moieties were carried out. Dihydroxyacetone and glyceraldehyde reduced the incorporation of 14C-fructose into hepatic triglyceride to approximately equal extents, suggesting that both trioses contribute equally to alpha-glycerophosphate formation. Glycerol, however, brought about a profound increase in the amount of fructose converted to triglyceride. This effect of glycerol was not apparent to any measurable extent on glucose incorporation into triglyceride and none of the intermediates affected the proportional distribution of radioactive counts between the fatty acid and glycerol moieties of the triglyceride after 14C-fructose administration.

Animals↗

PubMed2005

[Effect of scalp acupuncture on glucose metabolism in brain of patients with depression].

OBJECTIVE: To observe the effect of scalp acupuncture (SA) on the glucose metabolism in different regions of brain in patients with depression by positron emission computed tomography (PET). METHODS: Twelve depressive patients were treated by scalp acupuncture on middle line of vertex (MS5), middle line of forehead (MS1) and bilateral lateral line 1 of forehead (MS2), once a day for six days per week, and received PET detection on different region of brain before and after 6 weeks acupuncture treatment. Semiquantitative analysis was used to compare the average values of radioactive count gotten from various brain regions before and after treatment, which could reflect the condition of glucose metabolism at the brain region detected. RESULTS: SA could increase the glucose metabolism at bilateral frontal lobes, bilateral parietal lobes, right occipital lobe, right caudate nucleus, right cingulated gyrus and left cerebellum and decrease that at right temporal lobe and bilateral thalamus. CONCLUSION: SA on MS5, MS1 and MS2 in depressive patients could influence the glucose metabolism in various brain regions. It primarily illustrated that the mechanism of SA in treating depression is related with its regulation on cortex-limbic circuitry dysfunction and increase the glucose metabolism in various brain regions.

Adult↗

PubMed1999

Modulation of gastrin-releasing peptide (GRP) receptors in insulin secreting cells.

Gastrin-releasing peptide (GRP) receptors are present in pancreatic islets, though their regulation is unknown except for homologous desensitization. The modulation of binding of GRP to mouse pancreatic islets and INS-1 cells was studied. At 60 min (steady-state), total binding of [(125)I-Tyr(15)] GRP was 1.62 per cent of total radioactivity per 50 islets; non-specific binding (presence of 1 mM unlabelled GRP(1-27)) was 0.05 to 0.61 per cent of total radioactivity. A preincubation with 1000 nM cholecystokinin (CCK(8)) or with 1000 nM glucose-dependent insulinotropic peptide (GIP) augmented the number of GRP binding sites but not their affinity. [(125)I-Tyr(15)]GRP binding to INS-1 cells was saturable (90 min) and specific with respect to compounds that are not chemically related to GRP (e.g. calcitonin gene-regulated peptide-CGRP and atrial natriuretic peptide-ANP). Displacement studies showed one binding site with a K(d) of 0.39 nM and a B(max) of 13.2 fmoles mg(-1) protein. When the cells were pretreated for 24 h with 10 nM GIP or CCK(8), only GIP but not CCK(8) increased the B(max) of the GRP binding site. The affinity (K(d)) was not changed by either compound. This effect of GIP pretreatment was not affected by downregulating PKC by TPA (phorbol ester; long-term pretreatment). These data indicate that: (1) specific binding sites for GRP are present in mouse pancreatic islets and INS-1 cells; (2) the GRP binding is upregulated by GIP in both islets and INS-1 cells and additionally by CCK(8 ), albeit only in islets; and (3) PKC does not seem to be involved in the up-regulation process. Thus a positive interplay between both the incretins GIP and CCK(8) and the neurotransmitter GRP is obvious.

Animals↗

PubMed1981

Biotransformation of aloenin, a bitter glucoside constituent of Aloe arborescens, by rats.

Aloenin has been established to be 4-methoxy-6-(2-beta-D-glucopyranosyloxy-4-hydroxy-6-methylphenyl)-2-pyrone; it shows an inhibitory activity for gastric juice secretion. Rats metabolized it to 4-methoxy-6-(2,4-dihydroxy-6-methylphenyl)-2-pyrone, 2,5-dimethyl-7-hydroxychromone and glucose, which were excreted in the feces and the urine. The distribution of the radioactivity originating from 14C-labeled aloenin was studied. The tracer found in the kidney and the liver reached 60% of the amount administered 24 h after feeding and decreased rapidly in the next 24 h.

Animals↗

PubMed1981

Glucose-6-phosphate dehydrogenase: partial characterization of the rat liver and uterine enzymes.

Some properties of rat liver and uterine glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49) have been determined. A procedure has been used for the purification of rat liver glucose-6-phosphate dehydrogenase to homogeneity (spec. act. 210-225 units/mg protein) from large amounts of liver (0.5-2 kg) with yields of up to 30%. Uterine glucose-6-phosphate dehydrogenase was obtained by immunoprecipitation methods and the properties of radioactively-labeled forms of this enzyme were then determined. The amino acid composition of the liver enzyme was found to be similar to that for the enzyme from other mammalian tissues. The liver and uterine enzymes have a subunit molecular weight of 57000 and a pI of 6.5. The NH2-terminal amino acid of both enzymes was found to be pyroglutamate.

Amino Acid Sequence↗

PubMed1991

Selective stimulation of brush border glutamine transport in the tumor-bearing rat.

Intestinal extraction of circulating glutamine across the basolateral membrane is diminished in the tumor-bearing rat (TBR). This study was designed to investigate the effects of progressive malignant growth on brush border glutamine transport in order to gain further insight into the adaptive/regulatory changes in intestinal glutamine metabolism that occur in the tumor-bearing rat. Fischer 344 rats (225 +/- 5 g) were implanted with fibrosarcoma cells and were studied at various time points after implantation when the tumors comprised 7%, 20%, and 29% of total body weight. Control and tumor-bearing rats were pair-fed throughout the study. Jejunal brush border membrane vesicles (BBMVs) were prepared by magnesium aggregation/differential centrifugation and transport of radioactively labeled L-glutamine, L-leucine, L-alanine, and D-glucose by BBMVs was measured using a Millipore filtration technique. BBMVs were enriched 15-fold in alkaline phosphatase, indicating brush border vesicle purity. Uptake of all substrates occurred into an osmotically active space, exhibited overshoots, and had similar 1-hr equilibrium values. The rate of glutamine uptake by BBMVs from all tumor-bearing rats was significantly greater than controls, regardless of tumor size. The increase in transport activity was not due to a change in carrier affinity but rather to an increase in maximal transport velocity. In rats with small tumors (7% of body weight), the Vmax was 431 +/- 40 pmole/mg protein/10 sec compared to 259 +/- 30 in control animals (P less than 0.01). In marked contrast, the mean transport of alanine was diminished in BBMVs from TBR (31 +/- 3 pmole/mg protein/10 sec in TBR vs 23 +/- 2 in controls, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗