Maintenance of pancreatic endocrine cells of the neonatal rat: IV. The effect of 3-amino-3-deoxyglucose

Monolayer islet cells prepared from neonatal rat pancreases were cultured in media with 5.5 mM glucose alone or further supplemented with 5 mM 3-amino-3-deoxyglucose (3A3dG) for a total of 7 days. After culture for 7 days, 3A3dG-supplementation maintained the recovery of insulin released into the medium during the last 2 days of a 7-day culture at a level 2.9 fold higher that at day 0. Similarly, the insulin content of the cells was significantly higher than the initial level at day 0 (2.8-fold) and that of the cells grown in medium with glucose alone (4.5-fold). The maximum secretory responses to glucose (2.8-16.7 mM), leucine (2.5-10 mM) and 2-ketoisocaproate (2.5-10 mM) were several times as high as the initial. Further, 3A3dG caused a selective deletion of fibroblasts mostly consisting of endocrine cells. In these monolayer cells, either the cAMP response to glucose or the cellular cAMP content were significant. In conclusion, it is suggested that the beneficial effect of 3A3dG may be associated with an increase in either the oxidative catabolism of amino acids or the activity of adenylate cyclase in the B cell.     

Maintenance of pancreatic endocrine B cells of neonatal rat: Part-XIV--Effect of maternal hyperglycemia on the secretion of insulin and glucagon

Effects of gestational hyperglycemia on A and B cells were examined in pancreatic monolayer islet cell cultures of neonatal rats from mothers of normoglycemia (C) and made slightly (SH), moderately (MH) and highly hyperglycemic (HH) by streptozotocin injection. Monolayer cultures were maintained for 7 days in the medium with 5.5 mM glucose plus 1 mM 2-deoxyglucose. On day 0, B cells of the SH group were more responsive to glucose and 2-ketoisocaproate than those of other groups. On day 7, the response of B cells in the C and SH groups was remarkably enhanced, thus displaying a dose-dependent increasing pattern of insulin secretion in response to glucose, 2-ketoisocaproate and arginine, and a convex-type secretion to leucine. However, there was no response by B cells in the MH and HH groups. Further, a dose-dependent inhibition of glucagon secretion due to glucose was seen in A cells of the C and SH groups on day 0 and day 7. The responses of these A cells to other nutrients were slightly decreased or were of a low convex-type. In the MH group, however, the glucagon secretion was remarkably enhanced due to leucine and 2-ketoisocaproate on day 0 and day 7, and due to arginine on day 7, although it remained suppressed by glucose. A cells of the HH group were unresponsive through the whole culture period. These results suggest that the development of A- and B-cell responses in vitro of neonates was differently affected by the degree of maternal hyperglycemia.     

Maintenance of neonatal endocrine cells of the rat. I. Effects of 2-deoxyglucose and 2-deoxy-2-fluoroglucose

Pancreatic endocrine cells of the neonatal rat cultured in medium with 5.5 mM glucose for 7 days showed no response to glucose. By contrast, the supplementation of the medium with 1.0 mM 2-deoxyglucose or with 0.1-5.0 mM 2-deoxy-2-fluoroglucose maintained the capacity of glucose-induced insulin release and biosynthesis, and the recovery of insulin in cells at day 7 at levels significantly higher than in basal medium; the highest responses were recorded for 1.0 mM deoxysugars. Moreover, the addition of 1.0 mM deoxysugars caused a selective deletion of fibroblasts and yielded monolayers mostly consisted of endocrine cells at the end of the culture study period. In these monolayer cells, the stimulating level of c-AMP release was significantly higher than the basal. On the other hand, the in vitro function of A cells in culture was also better preserved in media with 1.0 mM deoxysugars.     

Regulated secretion of mature cathepsin B from rat exocrine pancreatic cells.

By indirect immunofluorescence and immunogold electron microscopy with an antibody that recognizes specifically the two forms of native mature rat cathepsin B (31 kDa and 5:25 kDa) but not the proenzyme, we detected cathepsin B not only in lysosomes of adult rat exocrine pancreatic cells but also in the trans Golgi condensing vacuoles, the zymogen granules and the pancreatic juice in the intralobular ducts. In contrast, immunocytochemistry with an antibody specific for rat cathepsin D showed the latter to be present in the same cells only in lysosomal compartments as expected. The same pattern of labeling with these two antibodies was found in the first zymogen granules to form in 17-day-old fetal rat pancreas. Counts of the extent of immunogold labeling of cathepsin B in the adult exocrine cells showed that the concentration of the enzyme was only two-fold higher in the lysosomal compartments than in the zymogen granules. To confirm these observations, rat pancreatic postnuclear supernatant (PNS), a fraction enriched in zymogen granules and rat pancreatic juice obtained by catheterization of the pancreatic duct, were subjected to 2D gel electrophoresis followed by immunoblotting with the cathepsin B antibody. All three samples contained a 31 kDa protein recognized by the antibody with a pI of about 4.5, the single chain mature form of cathepsin B. We then radiolabeled pancreatic PNS and zymogen granule fractions with benzyloxycarbonyl-Tyr[125I]-Ala-CHN2, an affinity label that covalently binds to the active sites of mature forms of both cathepsin B and cathepsin L. In both PNS and zymogen granule fractions this reagent labeled cathepsin B. Immunoprecipitation experiments showed that the antibody to cathepsin B recognized specifically both the single chain and the double chain mature forms of cathepsin B in the native state. Finally, Northern blots with a cDNA of rat cathepsin B showed that the concentration of cathepsin B mRNA in total pancreatic RNA increased following in vivo stimulation of the exocrine pancreatic cells with optimal doses of cerulein, a cholecystokinin analogue. We conclude that significant amounts of mature cathepsin B are secreted from exocrine pancreatic cells via the apical regulated exocytotic pathway, and we discuss this in terms of models for sorting of proteins to the cores of dense cored secretory granules.     

The effect of 2-deoxy-D-glucose on insulin release by neonatal rat B cells in culture

The culture for 7 days in medium with 5.5 mM glucose and 1 mM 2-deoxy-D-glucose enhanced the glucose sensitivity of neonatal rat B cells, and even stimulated their growth in vitro. Also, 2-deoxy-D-glucose supplementation maintained insulin release evoked by leucine and 2-ketoisocaproate from B cells at day 7 at levels several times higher than at day 1. The effect of leucine was greatly augmented by glutamine, whereas that of the 2-keto acid remained almost unchanged irrespective of the presence of glutamine. These results suggest an increase in oxidative catabolism of medium nutrients in B cells grown in medium with 2-deoxy-D-glucose for 7 days, and such metabolic changes may promote the growth of B cells in vitro.     

Effect of iodoacetic acid on maintenance of pancreatic endocrine cells of the neonatal rat

This report describes the specific cytotoxicity of iodoacetic acid (IAA) in selectively destroying the fibroblastoid cells and stimulating the in vitro function of neonatal B cells prepared from rat pancreases. Under culture conditions with a basal medium containing 5.5 mM D-glucose alone, the responses to insulin secretagogues tested were abolished by day 7 of culture. In contrast, the addition of 10 microM IAA enhanced either insulin release evoked by D-glucose (16.7 mM), L-leucine (10 mM) and 2-ketoisocaproate (10 mM) or the cellular insulin content to approximately twice the initial levels (day 0). L-Glutamine (10 mM) augmented the stimulatory effect of L-leucine or 2-ketoisocaproate. Moreover, the continuous application of IAA significantly increased the rates of glutamine oxidation in endocrine cells after 7 days of culture. On the other hand, the IAA-supplemented medium did not preserve the function of A cells. The phase-contrast microphotograph examination revealed the selective removal of fibroblasts from the monolayer cultures. This corresponded very closely with a remarkable reduction in culture DNA content.     

The effect of insulin on amino acid incorporation into exocrine pancreatic cells of the rat.

The rate of incorporation of radioactive leucine per cell in the acinar pancreatic cells of the rat increases by 50 per cent within one hour after subcutaneous administration of insulin, an effect that lasts for at least one more hour. The rate of incorporation has been measured by quantitative radioautography and by determination of the radioactivity per mug DNA in TCA-precipitable material from tissue homogenates. The capacity for amino acid (leucine and lysine) incorporation as measured by incubating pancreatic fragments in vitro is not enhanced by insulin treatment of the rat in vivo during one or more hours. Insulin was found to lower the serum concentration of most amino acids significantly, leucine by 50 per cent. The apparent effect of insulin on the incorporation of radioactive leucine in vivo can be explained by the difference in the specific radioactivity of the circulating amino acid in the treated rats as compared to the untreated ones. A change in amino acid concentration in the serum may likewise be the explanation of the decrease in amino acid incorporation rate in alloxan diabetic rats. The absence of a short term effect of insulin on the rate of protein synthesis does not exclude a long term effect as suggested by the higher rate of incorporation in the cells of peri-insular acini.     

Effects of glucose,exogenous insulin,and carbachol on C-peptide and insulin secretion from isolated perifused rat islets

Isolated perifused rat islets were stimulated with glucose, exogenous insulin, or carbachol. C-peptide and, where possible, insulin secretory rates were measured. Glucose (8-10 mm) induced dose-dependent and kinetically similar patterns of C-peptide and insulin secretion. The addition of 100 nm bovine insulin had no effect on C-peptide release in response to 8-10 mm glucose stimulation. The addition of 100 nm bovine insulin or 500 nm human insulin together with 3 mm glucose had no stimulatory effect on C-peptide secretion rates from perifused rat islets. Stimulation with carbachol plus 7 mm glucose enhanced both C-peptide and insulin secretion, and the further addition of 100 nm bovine insulin had no inhibitory effect on C-peptide secretory rates under this condition. Perifusion studies using pharmacologic inhibitors (genistein and wortmannin) of the kinases thought to be involved in insulin signaling potentiated 10 mm glucose-induced secretion. The results support the following conclusions. 1) C-peptide release rates accurately reflect insulin secretion rates from collagenase-isolated, perifused rat islets. 2) Exogenously added bovine insulin exerts no inhibitory effect on release to several agonists including glucose. 3) In the presence of 3 mm glucose, exogenously added bovine or human insulin do not stimulate endogenous insulin secretion.     

Sensitized effect of prestimulation on insulin release from the perifused rat islets with a slow-rise glucose stimulation.

The sensitized effect of prestimulation with 16.7 mM glucose on insulin release with a slow-rise glucose stimulation from the perifused rat islets of Langerhans was studied, together with the kinetic analysis of insulin release, and the interrelationship between the prestimulation time and the maximal rate of insulin release. All dose-response relationships which were derived from the dynamics of insulin release from islets prestimulated over various time periods within 60 min, showed sigmoidal profiles. Kinetic analyses were performed with Lineweaver-Burk's and Hill's equations. The 30-min prestimulation significantly reduced Hill's constant (n) from 6.2 +/- 0.7 of the control to 3.7 +/- 0.6 (p less than 0.05) and also enhanced the logarithmic equilibrium constant (log K) from -5.4 +/- 0.6 mM-n to -3.7 +/- 0.6 mM-n (p less than 0.05). However, the Km value was almost the same as that of the control (7.3 +/- 0.5 mM). On the other hand, the 60-min prestimulation remarkably diminished the Km value and the maximal rate of insulin release to 5.3 +/- 0.4 mM (p less than 0.05) and 0.6 +/- 0.08 muU/ml/islet/min (p less than 0.005), respectively. The maximal rate of insulin release linearly increased in proportion to the prestimulation time within 30 min. In conclusion, these results suggested that there would be some regularity depending on the prestimulation time in the process of transmission of the insulin-releasing signal in the pancreatic B cell and the accumulation of insulin into the provisional pool such as the labile insulin.     

The effect of pancreatic mesenchyme on the differentiation of endocrine cells from gastric endoderm

To determine whether mesenchyme plays a part in the differentiation of gut endocrine cells, proventricular endoderm from 4- to 5-day chick or quail embryos was associated with mesenchyme from the dorsal pancreatic bud of chick embryos of the same age. The combinations were grown on the chorioallantoic membranes of host chick embryos until they reached a total incubation age of 21 days. Proventricular or pancreatic endoderm of the appropriate age and species reassociated with its own mesenchyme provided the controls. Morphogenesis in the experimental grafts corresponded closely to that in proventricular controls, i.e. the pancreatic mesenchyme supported the development of proventricular glands from proventricular endoderm. Insulin, glucagon and somatostatin cells and cells with pancreatic polypeptide-like immunoreactivity differentiated in the pancreatic controls. The latter three endocrine cell types, together with neurotensin and bombesin/gastrin-releasing polypeptide (GRP) cells, developed in proventricular controls and experimental grafts. The proportions of the major types common to proventriculus and pancreas (somatostatin and glucagon cells) were in general similar when experimental grafts were compared with proventricular controls but different when experimental and pancreatic control grafts were compared. Hence pancreatic mesenchyme did not materially affect the proportions of these three cell types in experimental grafts, induced no specific pancreatic (insulin) cell type and allowed the differentiation of the characteristic proventricular endocrine cell types, neurotensin and bombesin/GRP cells. However, an important finding was a significant reduction in the proportion of bombesin/GRP cells, attributable in part to a decrease in their number and in part to an increase in the numbers of endocrine cells of the other types. This indicates that mesenchyme may well play a part in determining the regional specificity of populations of gut endocrine cells.     

Wortmannin, a PI3-kinase inhibitor: promoting effect on insulin secretion from pancreatic beta cells through a cAMP-dependent pathway

To determine the role of phosphatidylinositol 3-kinase (PI3-kinase) in the regulation of insulin secretion, we examined the effect of wortmannin, a PI3-kinase inhibitor, on insulin secretion using the isolated perfused rat pancreas and freshly isolated islets. In the perfused pancreas, 10(-8) M wortmannin significantly enhanced the insulin secretion induced by the combination of 8.3 mM glucose and 10(-5) M forskolin. In isolated islets, cyclic AMP (cAMP) content was significantly increased by wortmannin in the presence of 3.3 mM, 8.3 mM, and 16.7 mM glucose with or without forskolin. In the presence of 16.7 mM glucose with or without forskolin, wortmannin promoted insulin secretion significantly. On the other hand, in the presence of 8.3 mM glucose with forskolin, wortmannin augmented insulin secretion significantly; although wortmannin tended to promote insulin secretion in the presence of glucose alone, it was not significant. To determine if wortmannin increases cAMP content by promoting cAMP production or by inhibiting cAMP reduction, we examined the effects of wortmannin on 10(-4) M 3-isobutyl-1-methylxantine (IBMX)-induced insulin secretion and cAMP content. In contrast to the effect on forskolin-induced secretion, wortmannin had no effect on IBMX-induced insulin secretion or cAMP content. Moreover, wortmannin had no effect on nonhydrolyzable cAMP analog-induced insulin secretion in the perfusion study. These data indicate that wortmannin induces insulin secretion by inhibiting phosphodiesterase to increase cAMP content, and suggest that PI3-kinase inhibits insulin secretion by activating phosphodiesterase to reduce cAMP content.