Benigna Blondel

Monolayer cell culture of neonatal rat pancreas: An ultrastructural and biochemical study of functioning endocrine cells

Cells obtained by enzymatic dissociation of newborn rat pancreases grown in monolayer culture for periods of 14 hours to 20 days were utilized for ultrastructural and physiological studies. Small clusters of epithelioid cells, first recognized at 14 hours, increased in size with time, frequently merging to form large sheets of cells surrounded by fibroblastoid cells. Endocrine B and A cells were the most numerous elements in the clusters. The integrity of their differentiated ultrastructure was documented by the accumulation of stored immunoreactive insulin and immunoreactive glucagon within the cultures and the ability to regulate their release into the medium by means of normal physiological stimuli. The morphophysiological integrity of the B cells was also demonstrated in a pulse-chase experiment by closely correlating the movement of 3 H-leucine into proinsulin and insulin with sequential labeling by autoradiography of the rough endoplasmic reticulum, Golgi, and secretory granules. Furthermore, the presence of mitotic division among granulated cells provided evidence of B-cell replication. In contrast, surviving pancreatic exocrine cells underwent progressive morphologic simplification, giving rise to a population of dedifferentiated epithelioid cells whose ultimate fate requires further study.

Calcium-induced insulin release in monolayer culture of the endocrine pancreas. Studies with ionophore A23187.

SummaryThe possible role of Ca2+ in glucagon release has been investigated by the use of ionophore A23187. This ionophore permits Ca2+ entry down a suitable concentration gradient by complexing and releasing Ca2+, thereby acting as a carrier in plasma membranes. Cultured cells obtained by enzymatic digestion of pancreases from newborn rats were studied on the third day of culture. As expected the effects of the ionophore were dependent upon the presence of Ca2+ in the medium. However, either stimulation or inhibition of glucagon release resulted when different concentrations of ionophore and Ca2+ were used. With 1.0 mM Ca2+ in the medium, glucagon release was stimulated in the presence of 0.01 and 0.1 μg/ml ionophore, but inhibited in the presence of 3.0 and 10.0 μg/ml. With 0.1 μg/ml ionophore, glucagon release was stimulated by 0.3 and 1.0 mM Ca2+ but not by 2.5 mM Ca2+. With 10 μg/ml ionophore glucagon release was stimulated by 0.03, 0.1 and 0.3 mM Ca2+, whereas at 1.0 mM, glucagon release was depressed. These findings suggest that by increasing Ca2+, glucagon is released from the A-cells, whereas too large an increase in Ca2+ is inhibitory. The effect to stimulate release was not completely specific for Ca2+ in that while the ionophore did not stimulate release in the presence of either Mg2+ or Sr2+ in the absence of Ca2+, it did stimulate release when Ba2+ was tested. Furthermore Ba2+ at 0.3 mM was stimulatory even in the absence of ionophore. Glucagon release in the absence of ionophore was also enhanced by addition of 30 mM Ca2+ or by omission of Ca2+ from the medium. It is concluded that Ca2+, which plays an essential role in the stimulus-secretion coupling in several different cell types, may be involved in the stimulation of glucagon release from the A-cells of the pancreas.

Long term exposure of isolated pancreatic islets to mannoheptulose : evidence for insulin degradation in the β-cell

Abstract The possibility that the β cell may degrade insulin to regulate pancreatic insulin stores was studied. Isolated rat islets were maintained in tissue culture for 6 days with 8.3 mM glucose and mannoheptulose (0.5, 5 and 20 mM), an inhibitor of glucose phosphorylation. Mannoheptulose inhibited glucose utilization, insulin accumulation in the culture medium, and insulin biosynthesis, and decreased islet insulin content. The total recoverable insulin from the culture system (islets plus culture medium, corrected for extracellular insulin degradation) was 251, 127, 56 and 24 per cent of the content of freshly isolated islets for 0, 0.5, 5 and 20 mM mannoheptulose, respectively. Thus both 5 and 20 mM mannoheptulose caused a net loss of insulin accounted for by intracellular degradation. After 6 days in culture with 20 mM mannoheptulose, apart from β cell degranulation, there were no ultrastructural changes indicative of the mechanism of intracellular insulin destruction. When the inhibition of insulin biosynthesis was taken into account, intracellular insulin degradation could even be demonstrated in the presence of 0.5 mM mannoheptulose. Since such degradation occurred under conditions allowing for a net increase in recoverable insulin, this may be a mechanism normally operative in islets to control insulin stores.

Insulin and Glucagon Release from Monolayer Cell Cultures of Pancreas from Newborn Rats

Abstract. Monolayer culture of pancreatic cells from newborn rats has been shown to yield ultrastructurally normal endocrine cells, virtual absence of differentiated exocrine cells, and the maintenance of the capacity to synthesize immuno‐reactive insulin (IRI) and glucagon (ERG). Such a preparation has potential advantages for the study of mechanisms of hormone synthesis and release. Therefore, a survey of factors influencing hormone content and release from cultured cells was undertaken. The following general features were demonstrated: (1) highly reproducible responses within a given preparation of cells despite (2) some variations of absolute hormone content and release between preparations, (3) suitability for preparing sufficient quantities of cells to permit the simultaneous comparison of several factors within a given preparation, and (4) persistence of physiological responses to known modulators of IRI and IRG release. Thus, IRI release was stimulated in concentration‐related fashion by glucose. Amino acids, tolbutamide and glucagon augmented release, and 2‐deoxy‐D‐glucose, mannoheptulose and diazoxide inhibited glucose‐induced release. Epinephrine inhibited glucose‐induced IRI release through stimulation of an α‐adrenergic receptor mechanism, and the presence of probable β‐receptor stimulation of release was also demonstrated. Agents affecting the microtubular‐microfilamentous system exerted effecte similar to those demonstrated in other preparations. Ouabain, as well as the absence or augmented potassium in the medium increased IRI release in the presence of non‐stimulatory 2.75 mM glucose, whereas absence of calcium inhibited the response to 11 mM glucose without affecting baseline, non‐stimulated release. IRG release was shown to be inversely related to the glucose concentration in the medium. It was stimulated by epinephrine, arginine, alanine, lactate and pyruvate, and inhibited by β‐hydroxybutyrate. Diazoxide alone had no effect on IRG release. The monolayer culture employed in these studies provides a convenient, reproducible system for the further study of adult‐type IRI and IRG secretory behaviour. In addition to acute or short‐term regulation it may be especially suited for the study of long‐term modulating effects during the culture period.

The beta cell and its responses: summarizing remarks and some contributions from Geneva.

In summarizing the proceedings concerned with the /? cell, it will be my aim to condense into short statements my necessarily prejudiced reactions and to underline newer developments or shifts in emphasis. Further, I shall try to indicate where work originating from our Geneva group either has led to slightly different conclusions or perhaps has added or emphasized specific points. Dr. Paul Lacys film provided a superb and coherent summary of the ultrastructural substrate of insulin biosynthesis, storage and release in the pancreatic ft cell. Surely the central aspect of his views of the secretory process remains that of emiocytosis of /? granules, although he would be the first to admit, I believe, that classical electron microscopy has experienced difficulties in quantifying this process or in even approaching an estimation of whether emiocytosis is the only quantitatively important mechanism of insulin release. Accordingly, I should like to present a sample of the work of Dr. Lelio Orci, from the Department of Histology and Embryology in Geneva. Dr. Orcis novel approach in its

Inhibition of insulin release by somatostatin: no evidence for interaction with calcium

Abstract Somatostatin (SRIF) inhibits hormone release in response to different stimuli.1 Because of the well-known importance of Ca++ for hormone release, it has been postulated that the action of SRIF on insulin release is exerted by an inhibitory effect on Ca++ uptake by the pancreatic β cell.2 The present study was undertaken to examine the effect of a wide range of Ca++ concentrations on SRIF inhibition of glucose- or arginine-stimulated insulin release in pancreatic monolayer cultures. In addition, we studied the effect of SRIF on insulin release stimulated by the ionophore A23187, which acts as a Ca++ carrier in the plasma membrane. Finally, SRIF action on Ba++-stimulated insulin release was investigated. In contrast to the other three stimuli, Ba++ is able to elicit release in the absence of Ca++.3

Stimulatory and inhibitory effects of cyclic AMP on pancreatic glucagon release from monolayer cultures and the controlling role of calcium

SummaryWhen glucagon release from monolayer cultures of newborn rat pancreas was measured over four hours in media containing 2.5 mM Ca++, a significant cyclic AMP-related inhibition of release was observed. This was noted whether intracellular cyclic AMP levels were raised by the addition of exogenous cyclic AMP or dibutyryl cyclic AMP, by phosphodiesterase inhibition with theophylline, or by the stimulation of adenylate cyclase with cholera toxin. The inhibition was concentration dependent for cyclic AMP and could not be reproduced by the addition of AMP, ADP or ATP. Adenosine also inhibited glucagon release while ATP was stimulatory. From time course studies it appeared that the inhibitory effects of cyclic AMP and cholera toxin were progressive after two hours of incubation. With cholera toxin an early stimulation of glucagon release was observed. The effects of cyclic AMP and cholera toxin on argininestimulated glucagon release were to stimulate further the glucagon release during the first hour of the incubation. Thus, the effects of raising intracellular cyclic AMP levels were biphasic in that both an early stimulation and a late inhibition of glucagon release were observed. In examining the nature of these responses a remarkable controlling role for Ca++ was uncovered: at Ca++ concentrations of 0.3 mM and lower no effect of cyclic AMP on glucagon release was found. With 1 mM Ca++ in the medium cyclic AMP stimulated glucagon release early (30 min) and thereafter had no further effect. In the presence of 2.5 mM Ca++ cyclic AMP did not stimulate early but did cause the delayed inhibition of release. It is concluded that the effect of cyclic AMP on glucagon release can be either stimulatory or inhibitory depending upon the Ca++ concentration of the medium and the duration of exposure to raised cyclic AMP levels.

Streptozotocin-induced Functioning Islet Cell Tumor in the Rat: High Frequency of Induction and Biological Properties of the Tumor Cells

After a long latency, hormone-producing islet cell tumors are induced with high frequency by a single administration of streptozotocin and nicotinamide in the rats. Thus, pancreatic islets must be considered a target tissue for the tumorigenic, as well as diabetogenic, action of streptozotocin. These tumors are well-differentiated and resemble normal islet tissue, both morphologically and functionally, as they are rich in typical β cells and release insulin in response to glucose, both in vivo and in culture. However, some noteworthy differences have been observed in comparison to normal tissue: (a) the release of large amounts of proinsulin after glucose stimulation, (b) the presence of granulated cells of uncertain classification, similar but not identical, to δ cells, and (c) the altered proportion in hormone content, which suggests a different sensitivity of the different islet cell types to the chemically induced transformation.

Studies in vivo and in vitro on chemically-induced primary islet cell tumours and non-tumour endocrine pancreatic tissue

SummaryRat islet cell tumours induced by injection of streptozotocin and nicotinamide have been studied in vivo and after the establishment of monolayer cultures of tumour cells. During an intravenous glucose tolerance test, tumour-bearing rats had increased release of immunoreactive insulin, with a high proportion of proinsulin, as well as accelerated glucose disposal relative to control rats. The tumours were rich in immunoreactive insulin and somatostatin, poor in glucagon. Non-tumour pancreatic tissue or isolated islets contained 10% or less of the corresponding normal amounts of insulin whereas the islet content of somatostatin was unchanged and that of glucagon increased. This is best interpreted as a selective suppression of non-tumour B cells, further supported by the observation that the initially reduced insulin release and content of non-tumour islets were partially restored after 2 days in tissue culture. In monolayer culture, tumour cells maintained insulin production and acute responsiveness to glucose for prolonged periods. There was no sign of cell proliferation. It is concluded that primary, chemically-induced insulin-producing pancreatic islet cell tumours retain several features characteristic of normal B cells and continue to influence glucose homeostasis in vivo.

Inverse relationship between glucose metabolism and glucose-induced insulin secretion in rat insulinoma cells.

Slowly growing X-ray-induced rat insulinomas and derived cell lines have been used as a model system for glucose-induced insulin release. During perfusions of tumors transplanted under the kidney capsule, the carbohydrates glucose and D-glyceraldehyde increased insulin secretion. These stimuli and the amino acids leucine and alanine also provoked insulin release in freshly isolated tumor cells. Under these conditions, glucose utilization had a Km of 4.6 mM and maximal velocity of 0.9 nmol/min/10(6) cells. A continuous cell line was established from such a preparation. In culture, glucose-induced insulin secretion was no longer detectable while responses to D-glyceraldehyde and amino acids were retained. Glucose metabolism in the cell line showed a decrease in Km to 0.7 mM glucose and an increased maximal velocity of 1.4 nmol/min/10(6) cells. Attempts to revert these alterations were undertaken using glucose-deficient culture medium to diminish glycolytic flux. Basal insulin release was lowered, while the growth pattern of the cells remained unchanged. Another approach involved the use of sodium butyrate which has been demonstrated to promote differentiation in other cell systems. Whereas sodium butyrate markedly increased cellular insulin content, the secretory responses were not improved. These results provide evidence that the loss of glucose-induced insulin secretion is paralleled by alterations in glucose metabolism.