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PubMed1995

Molecular interactions of levonorgestrel and its 5 alpha-reduced derivative with androgen receptors in hamster flanking organs.

The 5 alpha-reduction of levonorgestrel (LNG) as well as its binding capacity to the androgen receptors of the hamster flank organ were investigated. Furthermore, the effects of LNG and its 5 alpha-reduced metabolite in the flank organ test and on [U-14C]glucose incorporation into lipids by this tissue were determined. Homogenates from female hamster flank organs were incubated in the presence of [3H]LNG at pH 7.4. The radioactive 5 alpha-LNG metabolite was isolated and its purity was assessed. Competition experiments for androgen binding receptors were carried out with 1.38 nM [3H-7 alpha-17 alpha]dimethyl-19- nortestosterone (DMNT), Kd, plus a range of increasing concentrations of the different unlabeled steroid hormones. The flank organ test was performed in vivo, and [U-14C]glucose incorporation into lipids was determined under organ culture conditions. The 5 alpha-LNG had the same binding capacity to androgen receptors (AR) as LNG in male flank organs. The flank organ test demonstrated that 5 alpha-LNG activity was similar to that observed for levonorgestrel and testosterone (T) on gonadectomized male hamster flank organs. Topical applications of LNG or 5 alpha-LNG increased [U-14C]glucose incorporation into lipids in a way similar to that of T. The overall data indicate that LNG and 5 alpha-LNG produced androgenic activity in the lipid pathway of male flank organs, and that 5 alpha-reduction is not essential for the LNG effect on this tissue.

Animals↗
PubMed1970

Uptake of long-chain fatty acid methyl esters by mammalian cells.

Albumin-bound long-chain fatty acid methyl esters (ME) were taken up and utilized by Ehrlich ascites tumor cells and slices of rat heart, liver, and kidney. Much more ME than albumin was taken up by the tumor cells, indicating that ME dissociated from the carrier protein during their uptake. 70-80% of the radioactivity associated with the cells after 1 min of incubation at 37 degrees C remained as ME. The results of studies with metabolic inhibitors and glucose suggest that uptake of ME is an energy-independent process. Changes in incubation medium pH between 7.8 and 6.5 did not markedly alter uptake of ME. Cells incubated with FFA and methanol did not synthesize ME. These findings indicate that ME are taken up intact, and they suggest that the presence of an anionic carboxyl group is not essential for the binding of a long-chain aliphatic hydrocarbon to a mammalian cell. When incubation with labeled ME was continued for 1 hr, increasing amounts of radioactivity were recovered in FFA, phospholipids, neutral lipid esters, and CO(2). ME radioactivity associated with the cells after a brief initial incubation was released in the form of ME and FFA when the cells were incubated subsequently in a medium containing albumin. If the second incubation medium contained no albumin, most of the ME radioactivity initially associated with the cells was incorporated into phospholipids, neutral lipid esters, and CO(2). These results suggest that much of the ME which is taken up, is hydrolyzed to FFA, and that the fatty acids derived from ME are available for further metabolism.

Albumins↗
PubMed1973

Brush border disaccharidases in dog kidney and their spatial relationship to glucose transport receptors.

The localization of disaccharidases in kidney has been studied by means of the multiple indicator dilution technique. A pulse injection of a solution containing Evans blue dye (plasma marker), creatinine (extracellular marker), and a (14)C-labeled disaccharide (lactose, sucrose, maltose, and alphaalpha-trehalose), is made into the renal artery of an anesthetized dog, and the outflow curves are monitored simultaneously from renal venous and urine effluents. Lactose and sucrose have an extracellular distribution. Trehalose and maltose remain extracellular from the postglomerular circulation. About 75% of filtered tracer maltose or trehalose is extracted by the luminal surface of the nephron. Thin-layer chromatography of urine samples shows that all of the excreted (14)C radiolabel is in the form of the injected disaccharide. Following the administration of phlorizin, all of the filtered radioactivity is recoverable in the urine, but chromatography of the urine samples now reveals that there is a significant excretion of [(14)C]glucose, approximating the amount previously extracted under control conditions (in the absence of phlorizin). It has been verified that no hydrolysis of maltose or trehalose to their constituent glucose subunits occurred during the transit of tracer between the point of injection (renal artery), and the point of filtration (glomerular basement membrane). Similarly, after addition of [(14)C]disaccharides to fresh urine there is no chromatographically recoverable [(14)C]glucose. It is concluded that there exist alpha-glucosidases with maltase and trehalase activity along the brush border of the proximal tubule and that these disaccharidases are located spatially superficial to the glucose transport receptors.

Animals↗
PubMed1980

Prolonged effect of insulin on glucose uptake by rat skeletal muscle.

The fate of insulin, as it relates to its action on skeletal-muscle glucose uptake, was studied in non-cyclically perfused rat hindlimbs. Insulin (1m-i.u./ml) with and without (125)I-labelled insulin was infused intra-arterially for 5 or 6min. Net glucose uptake and the release of (125)I-labelled insulin into the venous effluent were evaluated by arteriovenous-difference measurements for an additional 24-32min. The infusion of insulin for 5min promoted glucose uptake, an effect that persisted throughout a subsequent 25min of perfusion in the absence of insulin. The addition of insulin antibody to the perfusate in the presence of insulin blocked the action of insulin on glucose uptake, but it failed to alter insulin action if the muscle tissue had been exposed to insulin before addition of antibody. When (125)I-labelled albumin was infused for 6min, venous effluent radioactivity decayed rapidly and remained HClO(4)-insoluble and there was no significant tissue retension of radioactivity. Comparable experiments in which (125)I-labelled insulin was infused for 6min revealed that the venous effluent radioactivity decayed more slowly, a significant amount of the (125)I-labelled insulin appeared as fragments (HClO(4)-soluble) and there was a significant retention of radioactivity in the tissue. Radioactivity in muscle tissue biopsies obtained 28min after infusion of (125)I-labelled insulin was associated largely with intact insulin and a peptide of mol.wt. 2400. The total radioactivity retained in the muscle at this time was 7% of the amount infused. An insulin bolus (1i.u.) failed to increase the discharge of this tissue-associated radioactivity. These results suggest that insulin and a product of insulin metabolism persists in muscle tissue long after the arterial presence of insulin ends. This tissue residence and processing of insulin may be important components of insulin's prolonged action on glucose uptake by skeletal muscle.

Animals↗
PubMed1976

Red blood cell metabolism in AKR mice in the prelymphomatous phase.

The purpose of this study was to evaluate erythrokinetics and in vitro red blood cell (RBC) glucose utilization, 2,3-diphosphoglycerate production, and adenosine triphosphate levels following incubation in AKR mice from early in life until the onset of AKR lymphoma. Normal BALB/c mice served as controls. While hemoglobin concentration and RBC survival remained constant and similar in both groups of mice, the half disappearance time of injected radioactive 59Fe was longer and the 48-hr reappearance of 59Fe was less in AKR mice, compared with those of BALB/c mice. In vitro RBC metabolic studies indicated increased glucose utilization and 2,3-diphosphoglycerate production and decreased adenosine triphosphate levels following incubation in AKR RBC, in contrast to those in BALB/c RBC. RBC metabolic studies were also done in a small group of low leukemic C3H mice, and were similar to BALB/c mice. These differences became most marked in RBC from mice aged 15 to 30 weeks. Overt lymphoma began to occur after age 40 weeks. Hence, these erythropoietic changes occurred prior to the onset of lymphoma. The data imply a direct effect of virus infection on RBC or their precursors. The results are similar to changes in RBC metabolism noted in Rauscher-infected BALB/c mice. The broader implication of these findings in reference to viral host interactions and human leukemogenesis is discussed.

Adenosine Triphosphate↗
PubMed1976

Cholestero metabolism in rats sensitive to high cholesterol diet.

There was great individual variation in the elevations of serum cholesterol concentrations in a Wistar strain of rats by the ingestion of a large amount of cholesterol, although their cholesterol concentrations were almost identical while under the feeding of a regular stock diet. Their serum total phospholipid and dextran precipitable beta-lipoproteins showed the same tendencies, but serum triglycerids concentration was not affected by the dietary supplement in both groups. So, the rats with the different elevation rates of serum cholesterol concentrations were divided into good, normo and poor responding groups to cholesterol ingestion by the degrees of the elvations and bred for several generations. Serum lipid levels in the descendants from hyperresponding rats were not different from those from hyporesponding rats during the observed period, when they were given a stock diet. However, the response of serum cholesterol level to oral cholesterol ingestion in the former was larger than that in the latter. Therefore, it is suggested that the susceptibility of serum cholesterol to cholesterol ingestion may be heritable from parents to their offsprings. By the results of the tracer experiments, it was demonstrated that neither hepatic cholesterol synthesis nor absorption was affected in hyperresponding rats. On the other hand, a half life of labeled cholesterol was prolonged in this selected group. The relative fractional turnover rate was 17.8 per cent in hyporesponding rats and 15.8 per cent in hyperresponding rats. Excretion of the radioactivity from labeled cholesterol into the bile in good responding rats with bile fistula was slower than that in poor responding rats. When 1 g per 100 g body weight of glucose was given to the rats after 40 hours fasting, hepatic cholesterol synthesis increased at the same rate in both groups of rats, but the induction of hepatic cholesterol 7alpha-hydroxylation in hyperresponding groups was slower than that in hyporesponding groups. The distribution rates of the radioactivities into livers seemed to be delayed in good responders after the labeled cholesterol was ingested orally.It means that some disturbances in cholesterol transport may exist which induce the metabolic abnormality in such animals. It was shown that cholesterol metabolism was impaired in aged animals as compared with young ones. Serum cholesterol concentrations were elevated in good responders more than in poor responders by aging. The inborn errors of cholesterol metabolism in such animals might be emphasized from the fact of the impairment of cholesterol metabolism by the aging process.

Aging↗
PubMed1984

Enzymatic glucosylation of dolichol monophosphate and transfer of glucose from isolated dolichyl-D-glucosyl phosphate to ceramides by BHK-21 cell microsomes.

Enzymatic glucosylation of dolichol monophosphate (dolichol-P) from UDP-D-[3H]glucose was studied using the microsomal fraction of BHK-21 cells. The reaction product was separated by preparative thin-layer chromatography, further purified by DEAE-cellulose acetate column chromatography, and characterized as dolichyl-beta-D-glucosyl phosphate (Dol-P-Glc). The microsomal fraction of BHK cells catalyzed the incorporation of glucose from UDP-[3H]glucose into ceramides (endogenous and exogenous) and Dol-P; both reactions required Mn2+. Maximal glucosylation of Dol-P was achieved at pH 5.6-5.8 in the presence of a non-ionic detergent, Zonyl A. Glucosylation of exogenous Dol-P, from UDP-Glc, was non-competitively inhibited by exogenous ceramides. Incubation of Dol-P-[3H]Glc or Dol-P-[14C]Glc with liposomes (containing ceramides) and the microsomal fraction of BHK-21 cells resulted in the formation of a radioactive glucolipid which comigrated with the same RF value as glucosylceramide (Glc-Cer) on silica gel thin-layer chromatography. Transfer of [14C]glucose from Dol-P-[14C]Glc to exogenous ceramides was confirmed by double-labeling techniques. The pH dependence for transfer of radio-labeled glucose from Dol-P-[3H]Glc to ceramides was multi-phasic (optima at pH 4.0 and 7.0); glycosylation occurred within 5 min and Zonyl A was absolutely essential for the transfer reaction. These results indicate that Dol-P-Glc may also participate in the synthesis of ceramide hexosides.

Animals↗
PubMed1988

Evidence of a defect in insulin-receptor recycling in adipocytes from older rats.

Although insulin-stimulated glucose uptake is known to be decreased in adipocytes isolated from old obese rats, the cause of this defect is not totally understood. In the present study, we examined the possibility that insulin resistance is associated with defects in the intracellular processing of the insulin-receptor complex. Adipocytes were isolated from control (2-mo-old rats) and obese, insulin-resistant rats (12-mo-old rats), and the following measurements were made: 1) insulin-stimulated glucose uptake; 2) insulin binding; 3) insulin-receptor internalization and recycling; 4) accumulation of insulin within the cell; and 5) rate of loss of insulin from the cell. The results indicated that maximal insulin-stimulated glucose uptake was significantly reduced in adipocytes from obese, insulin-resistant rats (increase over basal value was 500 +/- 53% in obese rats and 1,200 +/- 96 in control rats, P less than 0.01). 125I-insulin (A14) binding (cell-associated radioactivity) and the internalization of the hormone-receptor complex were not different in the two groups of animals studied. In contrast, insulin-receptor recycling was significantly decreased in adipocytes from obese rats (72.0 +/- 6.1 vs. 93.6 +/- 2.6%, P less than 0.01). In addition, loss of intracellular radioactivity was significantly prolonged in insulin-resistant rats (t1/2 = 12.05 +/- 0.9 vs. 9.4 +/- 0.3 min, P less than 0.05). Thus adipocytes isolated from the older rats were resistant to the insulin effect on glucose uptake, and this defect was not associated with a reduction in insulin binding. However, there was a decrease in insulin receptor recycling, and this phenomenon may be related to the insulin resistance present in these cells.

Adipose Tissue↗
PubMed1986

Biosynthesis of lipid-linked oligosaccharides in embryonic liver. Glucosylation of mannose containing dolichyl diphosphate oligosaccharide.

A maximum rate of dolichyl phosphate [14C]glucose synthesis from 55-day embryos was achieved at 16 nM concentration of exogenous dolichyl phosphate and exceeded about 3 times that without addition of dolichyl phosphate. The highest values of [14C]glucose incorporation from UDP-[14C]glucose into dolichyl phosphate [14C]glucose, dolichyl diphosphate [14C]Glc-oligosaccharides and protein were reached at 5 min time point of incubation of liver microsomes both from embryos and sows. The radioactive incorporation into proteins was about 7-fold higher in liver microsomes from sows compared to that from embryos, probably due to the greater content of acceptor proteins in microsomes from sows. The enzymatic transfer of Glc3-oligosaccharide from a lipid carrier to endogenous protein acceptor in microsomes from pig embryonic and adult livers was considerably faster than the removal of glucose residues during the initial stages of processing of protein-bound oligosaccharides. One labelled compound was discovered in the CHCl3-CH3OH-H2O (1:1:0.3, by vol) extract after incubation of liver microsomes from embryos and sows with UDP-[14C]glucose. On the basis of its mobility on the chromatogram it appears to be GlcNAc2Man9Glc3.

Animals↗
PubMed1995

Age-dependent differences in insulin secretion and intracellular handling of insulin in isolated pancreatic islets of the rat.

Insulin secretory response to glucose changes with age. To elucidate age-dependent differences in pancreatic islet responsiveness to glucose, isolated islets from rats one week, three months and 14 months old were investigated in vitro. At three months of age, islet insulin secretion was increased five-fold by an acute glucose challenge. There was a significantly lower insulin response in both younger and older age groups. Islet insulin biosynthesis, as determined by the rate of incorporation of radioactive leucine into immunoprecipitable insulin, was lower at three months of age than at one week or 14 months. Insulin content was lowest in islets from the youngest rats and increased with age. The capacity for islet intracellular degradation of insulin was estimated according to the disappearance of tritiated leucine-labelled insulin during a 24-hour chase incubation. At a high glucose concentration, virtually no insulin was degraded intracellularly in islets from three-month-old rats, whereas islets from both younger and older animals showed a significant degradative capacity. High activities of a number of lysosomal enzymes in islets from one-week-old rats could account for the high degradative capacity and relatively low insulin content of these islets. Thus, low insulin response to glucose during early development may depend primarily on low insulin stores. However, during ageing, when islets are characterised by high insulin content, low response may depend primarily on impairment of beta-cell stimulus-secretion coupling, with high intracellular degradation of insulin resulting secondarily from an accumulation of insulin in the islets.

Aging↗
PubMed1997

Dissociation of neutrophil emigration and metabolic activity in lobar pneumonia and bronchiectasis.

In animal models of pulmonary inflammation, neutrophils exhibit a dramatic influx of glucose in periods of high metabolic activity. This information was utilized to develop a technique, involving positron emission tomography (PET) of 2-[18F]-fluoro-2-deoxy-D-glucose (18FDG), which measures neutrophil activity in situ. This technique was applied in a comparative study of neutrophil function in patients with acute lobar pneumonia or bronchiectasis. Neutrophil emigration was measured by gamma-scintigraphy of intravenously injected 111In-labelled granulocytes and neutrophil activity determined by PET of 18FDG. Neutrophil emigration was evident in 4 out of 5 bronchiectasis patients, whilst no emigration was apparent in the two pneumonia patients studied, consistent with animal studies showing maximum emigration soon after challenge. In contrast, 18FDG uptake was markedly increased in 4 out of 5 pneumonia patients but not in the patients with bronchiectasis. Localization of radioactivity to neutrophils was confirmed by microautoradiography of lavage fluid in a patient with pneumonia. These results suggest that the elevated uptake of glucose by neutrophils during the inflammatory response is a postmigratory event, most likely reflecting the respiratory burst, and that high levels of neutrophil emigration are not necessarily associated with significantly increased metabolic activity of these cells.

Adult↗
PubMed1994

Acute effects of endotoxin (lipopolysaccharide) on tissue lipid metabolism in the lactating rat. The role of delivery of intestinal glucose.

The aim of this study was to compare the effects of endotoxin on lipid metabolism and, in particular, lipogenesis in virgin and lactating rats. Intraperitoneal administration of bacterial endotoxin (lipopolysaccharide, LPS; 3 mg/kg body wt.) to fed virgin rats caused a 4-fold increase in lipogenic rate in liver in vivo. The stimulatory effect was not seen when glucose (6 mmol) was administered either orally or intraperitoneally to increase the basal rate. In contrast, the rate of lipogenesis in interscapular brown adipose tissue was inhibited, after LPS, and this was relieved by intraperitoneal glucose. In the lactating rat there were no significant changes in hepatic lipogenesis after the administration of endotoxin. However, LPS decreased the lipogenic rate in mammary gland of lactating rats and intraperitoneal glucose administration, but not oral, was able to restore the rate. In both virgin and lactating rats, LPS decreased glucose removal from the intestinal tract. In lactating rats, LPS induced a rise in blood concentrations of lactate, and plasma triacylglycerols and non-esterified fatty acids, similar to those in endotoxin-treated virgin rats. The administration of LPS did not decrease the accumulation of radioactivity in lipid in either liver or in mammary gland after injection of 3H-oleate. In contrast, LPS decreased the accumulation of radioactivity in mammary gland after injection of 3H-chylomicrons and increased it in liver and plasma. These changes were accompanied by a decrease in mammary gland activity of lipoprotein lipase. Intraperitoneal glucose partially reversed these changes in chylomicron disposition.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Use of short-lived 18F and long-lived 14C in double tracer autoradiography for simultaneous measurement of LCBF and LCGU.

We have developed a quantitative autoradiographic technique for the simultaneous measurement of local cerebral blood flow (LCBF) and local cerebral glucose utilization (LCGU) using a combination of 14C-long-lived and 18F-short-lived radionuclides as labels. To obtain the LCGU image, a 50-fold greater radioactivity of 18F than of 14C was administered and the first exposure was done for 2 hours. Three days later, when most of the 18F had decayed, a second exposure was done for 5 to 6 days to obtain the LCBF image. 18F standards were prepared in each experiment. The technique provides, for the first time, the local glucose flow ratio (LGFR). LGFR, obtained by dividing the LCGU by the LCBF image, was expressed as percent mumol/ml (multiplied by 100). Measurement of these three values in the same animal is expected to prove useful in the investigation of the pathophysiology of the brain. The advantages of this method are that cross contamination is less than 4%, chemical or physical manipulation of the slices is unnecessary, and final results can be obtained within a week.

Animals↗
PubMedDate not supplied

[Features of the effects of disaccharide structure of saccharose on kinetic parameters of hepatic lipid synthesis from glucose (mechanism of disaccharide effect)].

Effects of saccharose or invert sugar on kinetics of lipid synthesis in the rat liver were investigated using 1-14C-glucose as a precursor. The diet containing 30% of saccharose (of the total energy value) for 40 days was found to be sufficient to increase the radioactive carbon flux through total liver lipids more than twice, and through rapidly metabolized neutral lipids and phospholipids more than 4 and 3 times, consequently. On the contrary, when the invert sugar diet was used the radioactive carbon flux through total lipids in the liver was reduced up to 54% of the control level, the lowest radioactive carbon flux was observed through the rapidly metabolized fraction of phospholipids--6% of the control level, only. These changes were caused by the corresponding alterations in lipid synthesis constants.

Animals↗
PubMed1975

Absorption and biotransformation of topically applied 8-(methylthio) cyclic AMP. Studies in dogs and humans.

8-(Methylthiocyclic AMP-35S was applied topically to the intact or abraded skin of dogs at a dose of about 2-5 mg/kg. In this species, an average of 7% of the dose was absorbed through the intact and 62% through abraded skin. Half-lives for radioactivity in plasma averaged 3-6 h. After the topical application of the drug to the intact skin of dogs, the average concentration in plasma of glucose, but not of insulin, was increased by as much as 21%; after application to abraded skin, glucose and insulin were increased, on average, by as much as 84 and 221%, respectively. 8-(methylthio)adenosine and two unidentified compounds were present as metabolites in the urine of dogs; some unchanged 8-(methylthio)cyclic AMP-35S was also excreted. 8-(methylthio)cyclic AMP-35S was also applied topically (25 mg; 0-32 mg/kg) to the intact and stripped skin of normal human subjects under conditions similar to those used for dogs. Three subjects with intact skin did not absorb any of the drug, whereas three subjects with stripped skin absorbed 0-5, 8-3, and 23-3% of the dose. The half-life of radioactivity in the plasma of the subject with the greatest absorption was 0-5 h during the first 2 h, and 11 h for the next 10 h. During the first 2 h, this same subject excreted unchanged drug and 8-(methylthio)adenosine in his urine. No changes in glucose or insulin concentrations in plasma were observed in any of the subjects nor was there any apparent irritation of the skin.

Adenosine↗
PubMed1998

Distribution of 14C-labelled carbon from glucose and glutamate during anaerobic growth of Saccharomyces cerevisiae.

The distribution of carbon from glucose and glutamate was studied using anaerobically grown Saccharomyces cerevisiae. The yeast was grown on glucose (20 g l-1) as the carbon/energy source and glutamic acid (3.5 g l-1) as additional carbon and sole nitrogen source. The products formed were identified using labelled [U-14C]glucose or [U-14C]glutamic acid. A seldom-reported metabolite in S. cerevisiae, 2-hydroxyglutarate, was found in significant amounts. It is suggested that 2-hydroxyglutarate is formed from the reduction of 2-oxoglutarate in a reaction catalysed by a dehydrogenase. Succinate, 2-oxoglutarate and 2-hydroxyglutarate were found to be derived exclusively from glutamate. Based on radioactivity measurements, 55%, 17% and 14% of the labelled glutamate was converted to 2-oxoglutarate, succinate and 2-hydroxyglutarate, respectively, and 55%, 9% and 3% of the labelled glucose was converted to ethanol, glycerol and pyruvate, respectively. No labelled glucose was converted to 2-oxoglutarate, succinate or 2-hydroxyglutarate. Furthermore, very little of the evolved CO2 was derived from glutamate. Separation of the amino acids from biomass by paper chromatography revealed that the glutamate family of amino acids (glutamic acid, glutamine, proline, arginine and lysine) originated almost exclusively from the carbon skeleton of glutamic acid. It can be concluded that the carbon flow follows two separate paths, and that the only major reactions utilized in the tricarboxylic acid (TCA) cycle are those reactions involved in the conversion of 2-oxoglutarate to succinate.

Amino Acids↗
PubMed1993

Micro-autoradiographic method to study [18F]FDG uptake in mouse tissue.

A new micro-autoradiographic method using 18F (half-life: 109.8 min) was developed to detect 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) in mouse tissues. A linear relationship between the number of silver grains and corresponding 18F-radioactivity was observed. The half-distance of spatial resolution with 18F-micro-autoradiography was about 2.1 microns. The grain number increased with exposure time until 6 h, but 7 h or longer exposure induced a characteristic latent image fading. After i.v. injection of [18F]FDG into normal mice, the highest grain level among the hippocampal regions of the brain was found in the stratum lacunosum-moleculare followed by the dentate molecular and the stratum oriens. All granular and pyramidal cell layers showed lower grain levels than neuropil fields. In the pyramidal cell fields, the CA3a contained the highest grain level and the CA4 the lowest. This method requires only 4 h exposure and can be applied to all 18F-tracers to assess the tracer-tissue association.

Animals↗