Biomedical subjects

A Sener

Publications and source records attributed to A Sener.

At least 145 records · Page 8Linked to original sources

PubMed1992

A sensitive radioisotopic assay of myo-inositol: its application to rat pancreatic islets.

A radioisotopic procedure for the assay of myo-inositol is presented. It is based on the generation of NADH from NAD+ in the reaction catalyzed by myo-inositol dehydrogenase and the subsequent NADH-dependent conversion of 2-[U-14C]ketoglutarate to 14C-labeled L-glutamate in the reaction catalyzed by glutamate dehydrogenase. This method was applied to the measurement of myo-inositol in rat pancreatic islets. The myo-inositol islet content was decreased when the animals were fed a diet deprived of myo-inositol. When incubated in the absence of exogenous D-glucose, pancreatic islets, like parotid cells, released myo-inositol in the incubation medium. Over 90 min of incubation, a rise in extracellular D-glucose concentration increased the myo-inositol islet content, which was decreased, however, after incubation in the presence of carbamylcholine. These findings indicate that the myo-inositol content of islets is affected by nutritional and other environmental factors.

Animals↗

PubMed1992

Occurrence of the purine nucleotide cycle in rat pancreatic islets.

The presence of the purine nucleotide cycle is investigated in rat pancreatic islets. Adenylosuccinase, adenylate deaminase, and adenylosuccinate synthetase activities are characterized in islet homogenates. In the assay of the latter enzyme, evidence is obtained for operation of the full cycle in islet extracts. The activities of the three enzymes are not vastly different in islet and brain. These findings are discussed in the light of the role currently ascribed to the purine nucleotide cycle in producing ammonia from amino acids, in adjusting the concentration of Krebs cycle intermediates, in regulating the relative concentrations of ATP, ADP, and AMP, and in controlling the activity of phosphofructokinase.

AMP Deaminase↗

PubMed1992

Enzymic and metabolic anomalies in islets of diabetic rats: relationship to B cell mass.

A preferential impairment of the pancreatic B cell secretory response to D-glucose occurs in adult rats injected with streptozotocin during the neonatal period. Three possible explanations for such a preferential defect were investigated in the present study. First, the time course for 3-O-methyl-D-glucose uptake by islets suggested that the anomaly in hexose transport was mainly attributable to a decrease in the space accessible to the D-glucose analog commensurate with the decrease in B cell mass, rather than to a delayed equilibration of hexose concentration across the B cell plasma membrane. Second, the activity of glucose-6-phosphatase was found to be equally low in islets from diabetic and control rats, ruling out the futile cycling between D-glucose and D-glucose 6-phosphate as a cause for the preferential alteration of the secretory response to the hexose. Third, the activity of flavine adenine dinucleotide-linked glycerophosphate dehydrogenase was found to be decreased to a greater relative extent than the B cell mass. This coincided with an impaired generation of 3HOH from L-[2-3H] glycerol in intact islets. It is proposed, therefore, that an altered circulation in the glycerol phosphate shuttle may play a major role in the impaired process of glucose-stimulated insulin release in this model of noninsulin-dependent diabetes.

3-O-Methylglucose↗

PubMed1991

Hexose metabolism in pancreatic islets. Activation of the Krebs cycle by nutrient secretagogues.

In rat pancreatic islets, a rise in extracellular D-glucose concentration is known to cause a greater increase in the oxidation of D-[6-14C]glucose than utilization of D-[5-3H]glucose. In the present study, such a preferential stimulation of acetyl residue oxidation relative to glycolytic flux was mimicked by nutrient secretagogues such as 2-aminobicyclo[2,2,1]heptane-2-carboxylate, 3-phenylpyruvate, L-leucine, 2-ketoisocaproate, D-fructose and ketone bodies. The preferential stimulation of D-[6-14C]glucose oxidation by these nutrients was observed at all hexose concentrations (0.5, 6.0 and 16.7 mM), coincided with an unaltered rate of D-[3,4-14C]glucose oxidation, was impaired in the absence of extracellular Ca2+, and failed to be affected by NH4+. Although the ratio between D-[6-14C]glucose oxidation and D-[5-3H]glucose utilization in islets exposed to other nutrient secretagogues could be affected by factors such as isotopic dilution and mitochondrial redox state, the present data afford strong support to the view that the preferential stimulation of oxidative events in the Krebs cycle of nutrient-stimulated islets is linked to the activation of key mitochondrial dehydrogenases, e.g. 2-ketoglutarate dehydrogenase. The latter activation might result from the mitochondrial accumulation of Ca2+, as attributable not solely to stimulation of Ca2+ inflow into the islet cells but also to an increase in ATP availability.

Amino Acids↗

PubMed1991

Arginine metabolism in rat enterocytes.

Rat enterocytes exposed to L-arginine in the absence of any other exogenous substrate were found to actively metabolize this cationic amino acid. L-Arginine was converted to L-citrulline either directly in a NADPH-sensitive manner thought to be coupled with the generation of NO, or indirectly through the sequence of reactions catalyzed by arginase and ornithine transcarbamylase. A large fraction of L-citrulline and L-ornithine generated from exogenous L-arginine was released in the incubation medium. The production of CO2 and (poly)amines from L-arginine occurred at rates 2 to 3 orders of magnitude lower than that characterizing the net uptake of the cationic amino acid, and this despite the fact that enterocytes were equipped to allow the interconversion of L-ornithine and L-glutamate. It is concluded that the oxidative catabolism of L-arginine in enterocytes is quantitatively negligible relative to its conversion to L-citrulline and L-ornithine.

Animals↗

PubMed1991

Hexose metabolism in pancreatic islets: the glucose-6-phosphatase riddle.

Glucose-6-phosphatase activity was measured in rat liver or pancreatic islet crude homogenates and microsomes. The data recorded in the liver were comparable to those reported in prior studies. However, in the islets, the hydrolysis of D-glucose 6-phosphate by disrupted microsomes represented, when expressed relative to the protein content, less than 2% of the value recorded in liver microsomes. Moreover, no phosphotransferase activity was detected in the islets. These findings impose reservation on both the presence of glucose-6-phosphatase in rat islets and its participation to stimulus-secretion coupling.

Animals↗

PubMed1991

Metabolic and secretory response of parotid cells to cationic amino acids. Uptake and catabolism of L-arginine and L-ornithine.

L-Arginine and L-ornithine, which stimulate amylase release, are taken up by rat parotid cells. L-Arginine is converted, in an NADPH-dependent manner and to a limited extent to L-citrulline in parotid cell homogenates, despite the absence of ornithine transcarbamylase activity. L-Arginine is largely converted to urea and L-ornithine. The generation of putrescine and polyamines from L-ornithine occurs at a very low rate, relative to the cell content in performed amines. The major fate of exogenous or arginine-derived ornithine consists in its conversion to L-glutamate, which is then further metabolized. These findings raise several hypotheses for the secretory response of the parotid cells to cationic amino acids, including their accumulation as positively charged molecules inside the cell and the generation of either NO, amines, substrates for a transglutaminase-catalyzed reaction, or ATP through oxidative catabolism. However, each of these hypotheses meets with objections, the modality for the stimulation of amylase release by cationic amino acids being eventually considered as an unsettled matter.

Amylases↗

PubMed1991

Stimulus-secretion coupling of arginine-induced insulin release: significance of changes in extracellular and intracellular pH.

The possible relevance of changes in extracellular and/or intracellular pH to the insulinotropic action of L-arginine and L-homoarginine was investigated in rat pancreatic islets. A rise in extracellular pH from 7.0 to 7.4 and 7.8 augmented the secretory response to these cationic amino acids whilst failing to affect the uptake of L-arginine by islet cells and whilst decreasing the release of insulin evoked by D-glucose. Under these conditions, a qualified dissociation was also observed between secretory data and 45Ca net uptake. Moreover, at high extracellular pH, the homoarginine-induced increase in 86Rb outflow from prelabelled islets rapidly faded out, despite sustained stimulation of insulin release. The cationic amino acids failed to affect the intracellular pH of islet cells, whether in the absence or presence of D-glucose and whether at normal or abnormal extracellular pH. These findings argue against the view that the secretory response to L-arginine would be related to either a change in cytosolic pH or the accumulation of this positively charged amino acid in the beta-cell. Nevertheless, they suggest that the yet unidentified target for L-arginine and its non-metabolized analogue in islet cells displays pH-dependency with optimal responsiveness at alkaline pH.

Animals↗

PubMed1991

Hexose metabolism in pancreatic islets. Regulation of aerobic glycolysis and pyruvate decarboxylation.

1. D-Glucose (0.5-16.7 mM) preferentially stimulates aerobic glycolysis and D-[3,4-14C]glucose oxidation, relative to D-[5-3H]glucose utilization in rat pancreatic islets, the concentration dependency of such a preferential effect displaying a sigmoidal pattern. 2. Inorganic and organic calcium antagonists, as well as Ca2+ deprivation, only cause a minor decrease in the ratio between D-[3,4-14C]glucose oxidation and D-[5-3H]glucose utilization in islets exposed to a high concentration of the hexose (16.7 mM). 3. Non-glucidic nutrient secretagogues such as 2-aminobicyclo[2,2,1]heptane-2-carboxylate (BCH), 2-ketoisocaproate and 3-phenylpyruvate fail to stimulate aerobic glycolysis and D-[3,4-14C]glucose oxidation in islets exposed to 6.0 mM D-glucose. Nevertheless, BCH augments [1-14C]pyruvate and [2-14C]pyruvate oxidation. 4. The glucose-induced increment in the paired ratio between D-[3,4-14C]glucose oxidation and D-[5-3H]glucose utilization is impaired in the presence of either cycloheximide or ouabain. 5. These findings suggest that the preferential effect of D-glucose upon aerobic glycolysis and pyruvate decarboxylation is not attributable solely to a Ca(2+)-induced activation of FAD-linked glycerophosphate dehydrogenase and/or pyruvate dehydrogenase, but may also involve an ATP-modulated regulatory process.

Aerobiosis↗

PubMed1991

Neonatal streptozotocin injection: a model of glucotoxicity?

Adult rats injected with streptozotocin during the neonatal period displayed in the fed state moderate hyperglycemia. However, the percentages of glycated hemoglobin in erythrocytes and glycated lactate dehydrogenase in liver and pancreatic islets, as well as the sorbitol and glycogen content of the islets, were not significantly increased. Likewise, in intact islets, the ouabain-sensitive inflow of 86Rb+, and the ratio between 3H2O production from D-[2-3H]glucose and D-[5-3H]glucose were not different in control and streptozotocin-injected rats. These findings suggest that the alteration in both the mitochondrial catabolism of D-glucose and secretory response to the hexose previously documented in the islets of the latter animals are not attributable to factors such as the excessive nonenzymatic glycation of cytosolic proteins, sorbitol or glycogen accumulation, or impaired Na+, K(+)-adenosine triphosphatase (ATPase) activity. Although a contributive role of glucotoxicity in the impaired function of beta cell in this model of non-insulin-dependent diabetes should not be ruled out, it is speculated that streptozotocin might also cause a long-term damage of key mitochondrial dehydrogenases in the pancreatic beta cells and, possibly, their precursor cells.

Animals↗

PubMed1991

Hexose metabolism in pancreatic islets. Effect of (-)-hydroxycitrate upon fatty acid synthesis and insulin release in glucose-stimulated islets.

Anaplerotic reactions leading to the de novo synthesis of fatty acids, were recently proposed to participate in the coupling of metabolic to secretory events in the process of glucose-stimulated insulin release. In an attempt to validate such a proposal, the effect of (-)-hydroxycitrate upon fatty acid synthesis and insulin release was investigated in glucose-stimulated rat pancreatic islets. The inhibitor of ATP citrate-lyase, when tested in the 1.0-2.0 mM range, failed to affect glucose-stimulated insulin release, but also failed to inhibit the incorporation of 14C-labelled acetyl residues derived from L-[U-14C]leucine into islet lipids. A partial inhibition of fatty acid labelling by 3H2O was only observed in islets incubated for 120 min in the presence of 5.0 mM (-)-hydroxycitrate and absence of CaCl2. These findings suggest that (-)-hydroxycitrate is not, under the present experimental conditions, a useful tool to abolish fatty acid synthesis in intact pancreatic islets.

Animals↗

PubMed1991

Hexose metabolism in pancreatic islets. Regulation of D-[6-14C]glucose oxidation by non-nutrient secretagogues.

In rat pancreatic islets, a rise in D-glucose concentrations increases the oxidation of hexose-derived acetyl residues relative to glycolytic flux, an effect possibly attributable, in part at least, to the activation of key mitochondrial dehydrogenases by Ca2+ accumulated in the mitochondria of glucose-stimulated islet cells. The effects of non-nutrient insulinotropic agents upon D-[6-14C]glucose oxidation and D-[5-3H]glucose utilization were investigated. At an intermediate concentration of D-glucose (6 mM), the oxidation of D-[6-14C]glucose was unaffected by hypoglycemic sulfonylureas, an organic Ca2+ agonist, a cholinergic agent, forskolin, theophylline and cytochalasin B. At a higher concentration of the hexose (17 mM), however, the 14CO2/3H2O production rate was decreased by organic and inorganic Ca(2+)-antagonists and by ouabain, whilst being increased by NH+4 (10 mM) and aminooxyacetate. These findings suggest that the preferential stimulation of oxidative events in the Krebs cycle is largely independent of the rate of insulin release, and not merely consequential to the stimulation of Ca2+ inflow into the B-cell. It might be regulated, in a feedback process, by the rate of ATP utilization and, both directly and indirectly, by the mitochondrial redox state. The glucose-induced mitochondrial accumulation of Ca2+ and subsequent activation of the Krebs cycle appear to require an increase in both cytosolic Ca2+ activity and ATP availability.

Ammonia↗

PubMed1991

Assay of 3-hydroxybutyrate in the picomolar range.

A radioisotopic procedure for the assay of 3-hydroxybutyrate is presented. It is based on the measurement of NADH, generated in the 3-hydroxybutyrate dehydrogenase reaction, through the conversion of 2-[U-14C]ketoglutarate to 14C-labeled L-glutamate in the presence of beef liver glutamate dehydrogenase. The assay is linear in the range of 2.5 to 20.0 pmole/sample and about 100-times more sensitive than previous methods. The procedure proved useful for the measurement of 3-hydroxybutyrate in liver samples not exceeding 25 micrograms wet weight.

3-Hydroxybutyric Acid↗

PubMed1991

Generation of 3HOH from D-[6-3H]glucose by erythrocytes: role of pyruvate alanine interconversion.

Human and rat erythrocytes were found to generate 3HOH from D-[6(N)-3H]glucose. The rate of 3HOH production represented 7-10% of the glycolytic flux. The generation of 3HOH appeared attributable, in part at least, to the detritiation of [3-3H]pyruvate during the interconversion of the 2-keto acid and L-alanine in the reaction catalyzed by glutamate-pyruvate transaminase. Indeed, purified pig heart glutamate-pyruvate transaminase, as well as homogenates prepared from rat erythrocytes or pancreatic islets, catalyzed the generation of 3HOH from L-[3-3H]alanine. When the production of tritiated pyruvate from L-[3-3H]alanine was coupled to the conversion of the 2-keto acid to L-lactate, the production of 3HOH accounted for one-third of the reaction velocity, the latter failing to display isotopic discrimination. In these experiments, the production of 3HOH was abolished by amino-oxyacetate. Likewise, in intact rat erythrocytes, aminooxyacetate inhibited the generation of 3HOH and tritiated L-alanine from D-[6-3H]glucose (or D-[1-3H]glucose), as well as the generation of 3HOH from L-[3-3H]alanine. In pancreatic islets, however, aminooxyacetate failed to affect significantly the generation of 3HOH from D-[6-3H]glucose. These findings indicate that the generation of 3HOH from D-[6-3H]glucose is mainly attributable to an intermolecular tritium transfer in transaminase reaction, at least in cells devoid of mitochondria.

Alanine↗

PubMed1991

Impairment of glycerol phosphate shuttle in islets from rats with diabetes induced by neonatal streptozocin.

In islets from adult rats injected with streptozocin during the neonatal period, the oxidative and secretory responses to D-glucose are more severely affected than those evoked by L-leucine. A possible explanation for such a preferential defect was sought by comparing the rate of aerobic glycolysis, taken as the sum of D-[3,4-14C]glucose conversion to labeled CO2, pyruvate, and amino acid, with the total glycolytic flux, as judged from the conversion of D-[5-3H]glucose to 3H2O. A preferential impairment of aerobic relative to total glycolysis was found in islets from diabetic rats incubated at either low or high D-glucose concentration. This coincided in islet mitochondria of diabetic rats with a severe decrease in both the basal (no-Ca2+) generation of 3H2O from L-[2-3H]glycerol-3-phosphate and the Ca2(+)-induced increment in [3H]glycerophosphate detritiation. The mitochondria of diabetic rats were also less efficient than those of control animals in generating 14CO2 from [1-14C]-2-ketoglutarate. The diabetes-induced alteration of 2-ketoglutarate dehydrogenase in islet mitochondria was less marked, however, than that of the FAD-linked glycerophosphate dehydrogenase and was not associated with any change in responsiveness to Ca2+. Sonicated islet mitochondria of diabetic rats displayed normal to slightly elevated glutamate dehydrogenase activity. We propose, therefore, that the preferential impairment of the oxidative and secretory responses of islet cells to D-glucose in this experimental model of diabetes may be at least partly attributable to an altered transfer of reducing equivalents into the mitochondria as mediated by the glycerol phosphate shuttle.

Animals↗

PubMed1991

Hexose metabolism in pancreatic islets. Insignificance of D-glucose futile cycling in rat islets.

When rat pancreatic islets were incubated in the presence of unlabelled D-glucose (16.7 mM) and 3HOH, the production of 3H-labelled material susceptible to be phosphorylated by yeast hexokinase and then detritiated by yeast phosphoglucoisomerase did not exceed 2.66 +/- 0.21 pmol/islet per 180 min, i.e. about 1% of the rate of exogenous D-[5-3H]glucose utilization. Such a material accounted for 43 +/- 4% of the total radioactivity, associated with tritiated hexose(s). It is proposed, therefore, that the futile cycling of D-glucose in the reactions catalyzed in the islet cells by the hexokinase isoenzymes and glucose-6-phosphatase represents a negligible fraction of the total rate of D-glucose phosphorylation.

Animals↗

PubMed1991

Stimulation of 45Ca uptake and amylase release by cationic amino acids in parotid cells.

L-arginine, L-ornithine, L-lysine and L-histidine (each 10 mM) stimulated amylase release from rat parotid cells. The secretory response to the cationic amino acids was suppressed in the absence of extracellular Ca2+ and, at physiological Ca2+ concentration, coincided with stimulation of 45Ca net uptake by the parotid cells. All cationic amino acids also accumulated inside the parotid cells. Nevertheless, the concept that the stimulation of amylase release is merely attributable to depolarization of the plasma membrane, secondary to the accumulation of these positively charged amino acids in the parotid cells, is questioned in view of both the inverse correlation found between their secretory effects and degree of ionization and the knowledge that parotid cells are electrically inexcitable.

Amino Acids↗

PubMed1991

Variable expression of isotopic discrimination in metabolic flows.

Isotopic discrimination in reaction velocity may affect to a variable extent the estimation of metabolic flow when a metabolic intermediate is catabolized by two pathways with different degrees of discrimination. This was explored in erythrocytes exposed to 14C- or 3H-labelled D-glucose in the absence or presence of menadione. In the absence of menadione, when the pentose phosphate pathway accounted for only 5% of the D-glucose 6-phosphate turnover, the oxidation of C1-protonated or C1-deuterated D-[U-14C]glucose and D-[1-14C]glucose, mixed with the homologous non-radioactive D-[1-1H]glucose or D-[1-2H]glucose, indicated that, relative to the phosphorylation of the hexose, C1-deuterated D-glucose was less efficiently converted to 14CO2 than C1-protonated D-glucose. Moreover, in the absence of menadione, non-deuterated D-[U-14C]glucose and D-[1-14C]glucose were more efficiently oxidized in cells exposed to D-[1-2H]glucose rather than D-[1-1H]glucose. In the presence of menadione, which increased more than ten-fold the flow rate through the pentose phosphate pathway, the phenomenon of isotopic discrimination was either revealed or masked. These data indicate that the phenomenon of isotopic discrimination may indeed affect to a variable extent the estimation of a given metabolic flow.

Animals↗