Metabolic and secretory response of tumoral-insulin producing cells to D-fructose and D-galactose

At variance with normal islet cells, tumoral insulin-producing cells of the RINm5F line were found to display a positive secretory response not solely to D-glucose and D-mannose, but also to D-fructose and D-galactose. All hexoses increased the ATP/ADP ratio, exerted a sparing action upon the oxidation of endogenous nutrients in cells prelabelled with either L-[U-¹⁴C]glutamine or [U-¹⁴C]palmitate, increased the output of lactic acid and, as judged from data collected in the presence of D-[U-¹⁴C]hexoses, underwent oxidation in the RINm5F cells. The secretory response to these four hexoses appeared commensurate with the extent of their metabolic effects.     

Inhibition by a nonmetabolized analogue of L-leucine of protein biosynthesis in tumoral pancreatic islet cells

The effect of L-leucine, its deaminated metabolite 2-ketoisocaproate and its nonmetabolized analogue b(+/-)2-aminobicyclo[2,2,1]-heptane-2-carboxylic acid (BCH) upon protein labelling was examined in tumoral islet cells (RINm5F line) exposed to L-[4-3H]phenylalanine or L-[3-3H]serine. The interpretation of the results, in terms of changes in biosynthetic activity, was obscured by a possible interference of the tested nutrients with the uptake and further metabolism of the tracer tritiated amino acids. Nevertheless, when the cells were preincubated with the nutrient secretagogues and then incubated in the sole presence of L-[3-3H]serine, BCH, but not L-leucine or 2-ketoisocaproate, still inhibited protein labelling, this coinciding with a decrease in the ratio between TCA-precipitable and total radioactivity in the RINm5F cells. The inhibitory action of BCH was antagonized, to a limited extent, by D-glucose. It is proposed that BCH could be used as a tool to interfere with the function and growth of insulinoma cells.     

The stimulus-secretion coupling of amino acid-induced insulin release. Insulinotropic action of L-alanine

Available information on the fate and insulinotropic action of L-alanine in isolated pancreatic islets is restricted to data collected in obese hyperglycemic mice. Recent data, however, collected mostly in tumoral islet cells of either the RINm5F line or BRIN-BD11 line, have drawn attention to the possible role of Na(+) co-transport in the insulinotropic action of L-alanine. In the present study conducted in islets prepared from normal adult rats, L-alanine was found (i) to inhibit pyruvate kinase in islet homogenates, (ii) not to affect the oxidation of endogenous fatty acids in islets prelabelled with [U-14C]palmitate, (iii) to stimulate 45Ca uptake in islets deprived of any other exogenous nutrient, and (iv) to augment insulin release evoked by either 2-ketoisocaproate or L-leucine, whilst failing to significantly affect glucose-induced insulin secretion. The oxidation of L-[U-14C]alanine was unaffected by D-glucose, but inhibited by L-leucine. Inversely, L-alanine decreased the oxidation of D-[U-14C]glucose, but failed to affect L-[U-14C]leucine oxidation. It is concluded that the occurrence of a positive insulinotropic action of L-alanine is restricted to selected experimental conditions, the secretory data being compatible with the view that stimulation of insulin secretion by the tested nutrient(s) reflects, as a rule, their capacity to augment ATP generation in the islet B cells. However, the possible role of Na(+) co-transport in the secretory response to L-alanine cannot be ignored.     

The stimulus–secretion coupling of amino acid-induced insulin release. Insulinotropic action of l-alanine

Available information on the fate and insulinotropic action of l-alanine in isolated pancreatic islets is restricted to data collected in obese hyperglycemic mice. Recent data, however, collected mostly in tumoral islet cells of either the RINm5F line or BRIN-BD11 line, have drawn attention to the possible role of Na⁺ co-transport in the insulinotropic action of l-alanine. In the present study conducted in islets prepared from normal adult rats, l-alanine was found (i) to inhibit pyruvate kinase in islet homogenates, (ii) not to affect the oxidation of endogenous fatty acids in islets prelabelled with [U-¹⁴C]palmitate, (iii) to stimulate ⁴⁵Ca uptake in islets deprived of any other exogenous nutrient, and (iv) to augment insulin release evoked by either 2-ketoisocaproate or l-leucine, whilst failing to significantly affect glucose-induced insulin secretion. The oxidation of l-[U-¹⁴C]alanine was unaffected by d-glucose, but inhibited by l-leucine. Inversely, l-alanine decreased the oxidation of d-[U-¹⁴C]glucose, but failed to affect l-[U-¹⁴C]leucine oxidation. It is concluded that the occurrence of a positive insulinotropic action of l-alanine is restricted to selected experimental conditions, the secretory data being compatible with the view that stimulation of insulin secretion by the tested nutrient(s) reflects, as a rule, their capacity to augment ATP generation in the islet B cells. However, the possible role of Na⁺ co-transport in the secretory response to l-alanine cannot be ignored.     

Crabtree effect in tumoral pancreatic islet cells

The relationship between glycolysis and respiration was examined in a model of pancreatic B-cell dysfunction, namely in tumoral insulin-producing cells of the RINm5F line. A rise in D-glucose concentration from 2.8 to 16.7 mM increased the utilization of D-[5-3H]glucose and production of [14C]lactate from D-[U-14C]glucose, whereas decreasing the oxidation of either D-[U-14C]glucose or D-[6-14C]glucose. Whereas 2.8 mM D-glucose augmented O2 uptake above basal value, a further rise in D-glucose concentration to 16.7 mM decreased respiration, which remained higher, however, than basal value. Whether at low or high concentration, D-glucose exerted a pronounced sparing action upon the oxidation of endogenous nutrients in cells prelabeled with either L-[U-14C]glutamine or [14C]palmitate and, nevertheless, augmented above basal value the rate of lipogenesis, ATP/ADP content, adenylate charge, and cytosolic NADH/NAD+ and NADPH/NADP+ ratios. The generation of ATP resulting from the catabolism of either exogenous D-glucose or endogenous nutrients was not affected by the rise in hexose concentration from 2.8 to 16.7 mM. Thus, in sharp contrast with the situation found in normal islet cells, a rise in D-glucose concentration, instead of stimulating mitochondrial oxidative events, caused, through a Crabtree effect, inhibition of hexose oxidation and O2 consumption in tumoral islet cells.     

Resistance to alloxan of tumoral insulin-producing cells

Rat pancreatic islets and insulin-producing cells of the RINm5F line were incubated for 5 min at 7 or 23 degrees C in media containing 3H2O and either L-[1-14C]glucose or [2-14C]alloxan. In the islets the intracellular distribution space of [2-14C]alloxan represented, at 7 and 23 degrees C respectively, 11.4 +/- 1.0 and 25.5 +/- 2.3% of the intracellular 3H2O space. In the RINm5F cells, the distribution space of [2-14C]alloxan failed to be affected by the ambient temperature and represented, after correction for extracellular contamination, no more than 5.2 +/- 0.5% of the intracellular 3H2O space. Preincubation for 30 min at 7 degrees C in the presence of alloxan (10 mM) failed to affect subsequent D-[U-14C]glucose oxidation in the tumoral cells, whilst causing a 70% inhibition of glucose oxidation in the islets. It is proposed that RINm5F cells are resistant to the cytotoxic action of alloxan, this being attributable, in part at least, to poor uptake of the diabetogenic agent.     

Effect of cytochalasin B on glucose uptake, utilization, oxidation and insulinotropic action in tumoral insulin-producing cells

Cytochalasin B (17-3 microM) virtually abolished 3-O-methyl-D-[U-14C]glucose uptake and D-[5-3H]glucose utilization in tumoral insulin-producing cells of the RINm5F line. This coincided with a marked decrease in D-[U-14C]glucose oxidation and suppression of the stimulant action of D-glucose upon insulin release. Cytochalasin B, however, augmented basal insulin release by the tumoral cells. The RINm5F cells appeared much more sensitive than normal islet cells to cytochalasin B, as judged by the relative magnitude of inhibition in either hexose uptake or utilization. In both cell types, the inhibitory action of cytochalasin B upon glucose metabolism seemed to be competitive, being more marked at low than high glucose concentration. These results are interpreted in support of the view that a decreased efficiency of hexose transport across the plasma membrane represents an essential deficiency of the RINm5F cells.     

d-Glucose Metabolism in BRIN-BD11 Islet Cells

A novel insulin-secreting cell line, BRIN-BD11, was recently established following electrofusion of RINm5F cells with NEDH rat pancreatic islet cells. In the present study,d-glucose metabolism was compared in BRIN-BD11 and RINm5F cells. The concentration dependency ofd-[5-³H]glucose utilization displayed a comparable pattern in the two cell lines, but the absolute values were lower in BRIN-BD11 than RINm5F cells. Except in the case ofd-[1-¹⁴C]glucose, the ratio between¹⁴C labeledd-glucose oxidation andd-[5-³H]glucose utilization was higher, however, in BRIN-BD11 than RINm5F cells. Moreover, BRIN-BD11 cells were less affected than RINm5F cells by a rise ind-glucose concentration, in terms of the inhibitory action of the hexose upon oxidative variables, such as oxidative glycolysis, pyruvate decarboxylation, and oxidation of glucose-derived acetyl residues in the Krebs cycle. The total energy yield fromd-glucose catabolism appeared similar, however, in BRIN-BD11 and RINm5F cells. These findings extend the knowledge that BRIN-BD11 cells display an improved metabolic and secretory behavior, when considering the difference otherwise found between normal and tumoral islet cells.     

32P, 86Rb+ and 45Ca2+ handling by tumoral insulin-secreting cells (RINm5F line)

In perifused tumoral islet cells (RINm5F line), which were prelabelled with either [32P]orthophosphate, 86Rb+ or 45Ca2+, the administration of D-glucose (1.4, 2.8 or 16.7 mM) increased the efflux of 32P, decreased the outflow of 86Rb, increased slightly the efflux of 45Ca from cells perifused in the presence of Ca2+, and decreased modestly the outflow of 45Ca from cells perifused in the absence of Ca2+. D-glucose also stimulated the net uptake of 45Ca2+. When Ba2+ (2 mM) was used, in the absence of Ca2+, instead of D-glucose as an insulin secretagogue, the efflux of 32P was little affected, but the outflow of 45Ca was dramatically increased. These changes are qualitatively similar to those occurring in normal islet cells. Nevertheless, the ionic response to D-glucose appeared, as a rule, less marked in tumoral than normal islet cells. Moreover, the concentration-response relationship was shifted to a lower range of hexose concentrations in the RINm5F cells.     

Channeling of alpha-D-glucose 6-phosphate in tumoral islet cells exposed to D-galactose

In human erythrocytes, in which the fractional turnover rate of glucose 6-phosphate is rather low, menadione increases to almost the same relative extent the oxidation of D-[U-14C]glucose and D-[U-14C]galactose. However, in pancreatic tumoral islet cells (RINm5F line), in which the fractional turnover rate of glucose 6-phosphate is considerably higher, menadione increases the oxidation of D-[1-14C]glucose but not that of D-[1-14C]galactose. These results suggest that alpha-D-glucose 6-phosphate generated from exogenous D-galactose is channeled preferentially into the glycolytic rather than pentose phosphate pathway. Such was no more the case, however, when the RINm5F cells were exposed simultaneously to both D-glucose and D-galactose.     

Participation of endogenous fatty acids in the secretory activity of the pancreatic B-cell.

The pancreatic B-cell may represent a fuel-sensor organ, the release of insulin evoked by nutrient secretagogues being attributable to an increased oxidation of exogenous and/or endogenous substrates. The participation of endogenous fatty acids in the secretory response of isolated rat pancreatic islets was investigated. Methyl palmoxirate (McN-3716, 0.1 mM), an inhibitor of long-chain-fatty-acid oxidation, suppressed the oxidation of exogenous [U-14C]palmitate and inhibited 14CO2 output from islets prelabelled with [U-14C]palmitate. Methyl palmoxirate failed to affect the oxidation of exogenous D-[U-14C]glucose or L-[U-14C]glutamine, the production of NH4+ and the output of 14CO2 from islets prelabelled with L-[U-14C]glutamine. In the absence of exogenous nutrient and after a lag period of about 60 min, methyl palmoxirate decreased O2 uptake to 69% of the control value. Methyl palmoxirate inhibited insulin release evoked by D-glucose, D-glyceraldehyde, 2-oxoisohexanoate, L-leucine, 2-aminobicyclo[2.2.1]heptane-2-carboxylate or 3-phenylpyruvate. However, methyl palmoxirate failed to affect insulin release when the oxidation of endogenous fatty acids was already suppressed, e.g. in the presence of pyruvate or L-glutamine. These findings support the view that insulin release evoked by nutrient secretagogues tightly depends on the overall rate of nutrient oxidation, including that of endogenous fatty acids.     

Mechanism of 3-phenylpyruvate-induced insulin release. Metabolic aspects.

1. The metabolism and metabolic effects of 3-phenylpyruvate were examined in rat pancreatic islets. 2. Islet homogenates catalysed transamination reactions between 3-phenylpyruvate and L-glutamate, L-leucine, L-norleucine or L-valine. 3-Phenylpyruvate failed to activate glutamate dehydrogenase. 3. 3-Phenylpyruvate rapidly entered into islet cells, was extensively converted into phenylalanine but slowly oxidized. 4. The conversion of phenylpyruvate into phenylalanine coincided with a fall in the content of several amino acids (especially glutamate and aspartate) in the islets and incubation medium, the accumulation of 2-oxoglutarate and a modest fall in the NH4+ production rate. 5. 3-Phenylpyruvate failed to affect 14CO2 output from islets prelabelled with [U-14C]palmitate, but augmented 14CO2 output from islets prelabelled or incubated with L-[U-14C]glutamine. 6. In the presence of L-glutamine, 3-phenylpyruvate augmented the ATP/ADP ratio and NAD(P)H islet content, and caused a rapid and sustained decrease in the outflow of radioactivity from islets prelabelled with [2-3H]adenosine. 7. These data support the view that the insulin-releasing capacity of 3-phenylpyruvate coincides with an increase in the catabolism of endogenous amino acids acting as 'partners' in transamination reactions leading to the conversion of 3-phenylpyruvate into phenylalanine.     

Fructose metabolism via the pentose cycle in tumoral islet cells

In tumoral islet cells (RINm5F line) the phosphorylation of D-fructose is catalyzed by hexokinase rather than fructokinase. Fructose 6-phosphate appears to be preferentially channelled into the pentose cycle, as suggested by a ratio of D-[1-14C]fructose/D-[U-14C]fructose oxidation close to 2.7, the failure to generate 14C-labelled lactate from D-[1-14C]fructose and a poor metabolic response to menadione. When the islet cells are exposed to both D-fructose and D-glucose, however, the metabolism of the former hexose is dramatically modified, fructose 6-phosphate being now formed at a lower rate and preferentially channelled into the glycolytic pathway. These findings illustrate the existence of regulatory steps in fructose catabolism located distally to its site of phosphorylation.     

Underestimation of D-glucose phosphorylation as measured by 3H2O production from D-[2-3H]glucose

In rat pancreatic islets, tumoral islet cells (RINm5F line), parotid gland, and in human erythrocytes, but not in rat hepatocytes, the production of 3H2O from D-[2-3H]glucose is 20-30% lower than from D-[5-3H]glucose. This coincides with the production of tritiated lactic acid from D-[2-3H]glucose and may be attributable to an intramolecular hydrogen transfer in the phosphoglucoisomerase reaction. It is concluded that the production of 3H2O from D-[2-3H]glucose is not a reliable tool to assess the total rate of hexose phosphorylation.     

Pathways of reducing equivalents in hepatocytes from rats. Estimation of cytosolic fluxes by means of 3H-labelled substrates for either A- or B-specific dehydrogenases.

The rat insulinoma-derived RINm5F cell line retains many differentiated functions of islet beta-cells. However, it fails to recognize glucose as an insulin secretagogue in the physiological concentration range. With this cell line, glucose-transport kinetics were investigated, by using a double-label technique with the non-metabolizable glucose analogue 3-O-methylglucose (OMG). RINm5F cells possess a passive glucose-transport system with high capacity and low affinity. Equilibration across the plasma membrane of extracellular OMG concentrations up to at least 20 mM is achieved within 2 min at 37 degrees C. The half-saturation of OMG uptake occurs at 32 mM. At lower temperatures OMG uptake is markedly retarded, with a temperature coefficient (Q10) of 2.9. As indicated by efflux measurements, transport is symmetrical. Cytochalasin B at micromolar concentrations and phlorrhizin in millimolar concentrations are potent inhibitors of OMG uptake. Neutralization of the secreted insulin with antibodies does not alter OMG uptake kinetics. The glucose metabolism of RINm5F cells is much exaggerated compared with that of islet beta-cells. Nonetheless, when measured in parallel to uptake, transport exceeds by far the rate of metabolism at glucose concentrations above 3 mM. Measurements of intracellular D-glucose reveal a lower intracellular glucose concentration relative to the extracellular in RINm5F cells. This seems to be due to abnormalities in the subsequent steps of glucose metabolism, rather than to abnormalities in hexose uptake. The loss of glucose-induced insulin release in RINm5F cells cannot be explained by alterations in hexose transport.     

L-glutamine and palmitate catabolism in pancreatic islets from rats depleted in long-chain polyunsaturated omega 3 fatty acids

The catabolism of D-glucose was recently found to be impaired in pancreatic islets from rats depleted in long-chain polyunsaturated omega3 fatty acids. The specificity of this alteration was now investigated by characterizing the oxidative fate of endogenous nutrients in islets preincubated with either L-[U-14C]glutamine or [U-14C]palmitate and then incubated variously in the absence of D-glucose, presence of the hexose or presence of metabolic poisons. Relative to their radioactive content after preincubation, the production of 14CO2 by islets prelabelled with [U-14C]glutamine was higher in omega3-depleted rats than control animals. The enhancing action of D-glucose upon such production was impaired, however, in the omega3-depleted rats. The net uptake of 14C-palmitate and absolute value for 14CO2 output were both increased in omega3-depleted rats, whilst the ratio between 14CO2 output and islet radioactive content was decreased in the same animals. The inhibition of 14CO2 production by metabolic poisons was comparable in all cases. These results are consistent with recent findings on such items as the availability of endogenous amino acids and uptake of unesterified fatty acids in extrapancreatic sites of the omega3-depleted rats. They also support the view that the alteration of D-glucose metabolism in the islets of the latter animals may be attributable, in part at least, to alteration of glucokinase kinetics by high intracellular acyl-CoA levels.     

Metabolic and secretory responses of parotid cells to cationic amino acids. Oxidation of the amino acids and interference with the oxidation of D-glucose or endogenous nutrients.

Cationic amino acids were recently found to stimulate amylase release from rat parotid cells. The possible relevance of their oxidative catabolism to such a secretory stimulation was investigated. D-Glucose, which was efficiently metabolized in parotid cells and which augmented O2 uptake above basal value, failed to affect basal or stimulated amylase release. L-Arginine, L-lysine and L-histidine failed to stimulate the oxidation of either exogenous D-[6-14C]glucose or endogenous nutrients in cells pre-labelled with [U-14C]palmitate or L-[U-14C]glutamine. The oxidation of L-[U-14C]arginine, L-[U-14C]ornithine, L-[U-14C]lysine and L-[U-14C]histidine, all tested at a 10 mM concentration, was much lower than that of D-[U-14C]glucose (5.6 mM). These findings argue against the view that the stimulation of amylase release by cationic amino acids would be related to their role as a source of energy in the parotid cells.     

Effect of exogenous ATP upon inositol phosphate production, cationic fluxes and insulin release in pancreatic islet cells

Endogenous ATP is thought to play a key regulatory role in nutrient-stimulated insulin release. The present study deals with the effect of exogenous ATP and its stable analog alpha, beta-methylene ATP upon pancreatic islet function. Both alpha, beta-methylene ATP (5.0 microM to 0.2 mM) and ATP (0.3-3.0 mM) caused a rapid and concentration-related increase in insulin output by rat islets incubated or perfused at an intermediate concentration of D-glucose (8.3 mM). The effect of the ATP analog faded out at both lower and higher D-glucose concentrations. In the presence of 8.3 mM D-glucose, ATP also increased both 86Rb and 45Ca outflow from prelabelled islets. The cationic response to ATP persisted in the absence of extracellular Ca2+ and, hence, was reminiscent of that evoked by cholinergic agents. Like carbamylcholine, ATP caused a dose-related increase in the production of [3H]inositol phosphates from prelabelled islets or tumoral islet cells (RINm5F line). The latter effect was duplicated by alpha, beta-methylene ATP and unaffected by atropine. It is speculated that ATP, liberated together with insulin at the exocytotic site, might participate in a positive feedback control of insulin release.     

Hexose metabolism in pancreatic islet cells: the coupling between hexose phosphorylation and mitochondrial respiration

The possible relevance of D-glucose phosphorylation by mitochondria-bound hexokinase to the control of respiration was examined in mitochondria prepared from either tumoral pancreatic islet cells (RINm5F line) or normal rat liver. In both systems, ATP generated by mitochondria exposed to ADP and succinate could serve as a substrate for the phosphorylation of D-glucose. However, after exposure to exogenous ADP in the presence of succinate, only mitochondria isolated from RINm5F cells displayed a sizeable increase in O2 consumption in response to a subsequent administration of D-glucose. In this respect, the discrepancy between mitochondria from islet cells and liver, respectively, was found to be attributable to the much lower hexokinase activity, relative to respiratory rate, in liver than in RINm5F cell mitochondria. It is speculated that the coupling between hexose phosphorylation and respiration in islet cells may prime the mitochondria to generate ATP during the early metabolic and secretory response to a rise in extracellular D-glucose concentration.     

Na+-Independent Transporters, LAT-2 and b0,+, Exchange L-DOPA with Neutral and Basic Amino Acids in Two Clonal Renal Cell Lines

The present study examined the functional characteristics of L-DOPA transporters in two functionally different clonal subpopulations of opossum kidney (OKLC and OKHC) cells. The uptake of L-DOPA was largely Na+-independent, though in OKHC cells a minor component (approximately 15%) required extracellular Na+. At least two Na+-independent transporters appear to be involved in L-DOPA uptake. One of these transporters has a broad specificity for small and large neutral amino acids, is stimulated by acid pH and inhibited by 2-aminobicyclo(2,2,l)-heptane-2-carboxylic acid (BCH; OKLC, Ki = 291 mM; OKHC, Ki = 380 mM). The other Na+-independent transporter binds neutral and basic amino acids and also recognizes the di-amino acid cystine. [14C]-L-DOPA efflux from OKLC and OKHC cells over 12 min corresponded to a small amount of intracellular [14C]-L-DOPA. L-Leucine, nonlabelled L-DOPA, BCH and L-arginine, stimulated the efflux of [14C]-L-DOPA in a Na+-independent manner. It is suggested that L-DOPA uses at least two major transporters, systems LAT-2 and b0,+. The transport of L-DOPA by LAT-2 corresponds to a Na+-independent transporter with a broad specificity for small and large neutral amino acids, stimulated by acid pH and inhibited by BCH. The transport of L-DOPA by system b0,+ is a Na+-independent transporter for neutral and basic amino acids that also recognizes cystine. LAT-2 was found equally important at the apical and basolateral membranes, whereas system b0,+ had a predominant distribution in apical membranes.