Effects of glucose on the ultrastructure and insulin biosynthesis of isolated mouse pancreatic islets maintained in tissue culture

Abstract

Summary. Isolated mouse pancreatic islets were mainrained in tissue culture for up to 12 days in glucose concentrations varying between 3.3 and 28 raM. A satisfactory ultrastruetural preservation of the islet cells was found irrespective of the glucose concentration of the culture medium. While the B-cells of islets cultured in the lower glucose concentration showed slight degranulation, there was extensive degranulation and increased amounts of rough-surfaced endoplasmic retieulum after culture in the higher glucose concentration. The immunoreactive insulin content of islets cultured at 28 mM glucose was markedly decreased and the insulin secretion during the culture period was much higher than that of islets cultured at 3.3 mM glucose. The insulin biosynthesis, as reflected in the incorporation of 3I-I-leucine into gel chromategraphed extracts of acid-ethanol soluble islet proteins, was studied either during the culture period or at the end of a 6-day culture in short-term incubations lasting for 90 or 180 rain. The results consistently showed tlhat the biosynthesis of insulin proceeded at a high rate and remained regulated by glucose throughout the culture period. The eontinfious addition of newly synthesized and labelled insulin to the small intracellular insulin pool of the high-glucose cultured B-cells produced a very high specific radioactivity of the insulin.Key words : Tissue culture, pancrea tic islets, degranulation, endoplasraie reticulum, insulin content, insulin release, insulin degradation, insulin biosynthesis.Although the responses of the B-cell to an acute glucose challenge have been studied in great detail there is still little known about the long-term reactions of these cells to an increased extraeellular glucose coneentration. Thus, it is unclear whether the deficient insulin response to glucose and the diminished insulin reserves in diabetes mellitus are caused by a primary dysfunction of the cells or secondary functional exhaustion due to the hyperglycemic state. The experimental approach to this problem has been difficult mainly due to the lack of suitable techniques for long-term studies of iso]ated B-cells in a system separate from all the complex influences which occur in rive. In the present study a recently developed technique for in vitro culture of intact isolated islets [1] has been utilized in an attempt to overcome these difficulties. The insulin content and the biosynthesis of insulin have been analysed in mouse pancreatic islets maintained in culture at widely different glucose concentrations. In addition, the insulin release to the culture medium and the glucose-dependent variations of the ultrastructure of the cultured ]3-cells have been evaluated. Materials and Methods A. Tissue Culture TechniqueMale NMI~I mice (Anticimex AB, Stockholm, Sweden) weighing 30--35 g were starved over-night before the experiments. Isolated islets were obtained by digestion of the pancreas with collagenase (Worthington Biochemical * Presented in part at the Congress of the European Association f...

Function of microdissected pancreatic islets cultured in a chemically defined medium. I. Insulin content and release

“…The attached islets can be removed by scraping or trypsination. Although trypsin treatment was reported not to affect the insulin-releasing capacity of cultured islets isolated by collagenase [6], freshly isolated islets displayed decreased secretory and metabolic responses to glucose after treatment with trypsin or collagenase [6,8,15]. These observations point to the advantage of isolating islets without using enzymes.…”

“…Disintegration of the islets can be prevented by the addition of serum to the culture medium [6]. However, when present in the culture medium, serum induces an outgrowth of cells from the islet periphery, leading to a firm attachment of the islets to adjacent surfaces [6]. The attached islets can be removed by scraping or trypsination.…”

“…used a very mild collagenase treatment, leaving the islets surrounded by acinar tissue [14]. Disintegration of the islets can be prevented by the addition of serum to the culture medium [6]. However, when present in the culture medium, serum induces an outgrowth of cells from the islet periphery, leading to a firm attachment of the islets to adjacent surfaces [6].…”

“…A characteristic feature of these islets, when cultured in media containing calf serum, is that they attach firmly to adjacent surfaces. In order tO remove these islets from their support, further enzymatic digestion or mechanical means have to be used [6].…”

Superior beta cell proliferation, function and gene expression in a subpopulation of rat islets identified by high blood perfusion

“…Islet isolation Islets were isolated by collagenase digestion [16] and hand picked. They were then dichotomously sorted under a fluorescence microscope by the presence or absence of microspheres.…”