Early Differentiation of Glucagon-Producing Cells in Embryonic Pancreas: A Possible Developmental Role for Glucagon

Abstract

Glucagon and insulin are first detectable at the onset of rat pancreas organogenesis. Initially, the specific activity of glucagon is approximately 100-fold higher than that of insulin. At this early stage, endocrine storage granules, similar to a granules, are identifiable in electron micrographs. The granule characteristics, as well as the relative hormone levels, suggest that the early population of differentiated endocrine cells is in fact composed of glucagon-producing (A) cells. This high level of glucagon is present in the embryo much earlier than the metabolic processes thought to be controlled by this hormone. Moreover, glucagon-producing cells may be the first endocrine cells to differentiate. Other known endocrine products accumulate later, during the terminal stages of organogenesis. These observations suggest that glucagon may have a regulatory function in early embryogenesis.The islets of the differentiated rat pancreas contain A and B cells, which are responsible for the production of glucagon and insulin, respectively, and D cells, whose function has yet to be defined. Until recently, the endocrine cells have been assumed to differentiate well after the appearance of the pancreatic diverticulum (3-5). However, Clark and Rutter (6), using a sensitive immunoassay, detected low levels of insulin at the time of the initial formation of the pancreatic diverticulum. Moreover, Wessells and Evans (7) (8) were decapitated, and the embryos of an appropriate gestational age were transferred to dissecting dishes containing Earle's balanced salt solution and 1.0% bovine-serum albumin. Pancreatic rudiments, excluding most of the associated mesenchyme, were excised and placed in polyethylene tubes (microfuge tubes, Spinco) on dry ice. The samples were stored at -70°until the time of assay, when they were thawed and subsequently sonicated in an aliquot of glass-distilled water. The crude homogenates were immediately monitored for hormone activity and protein content. Routinely, three dilutions were made of each sample, and each dilution was assayed in duplicate.Insulin Assay. Insulin was assayed according to a micromodification of the double-antibody technique of Morgan and Lazarow (9). Previously (6) beef insulin was used as a standard, while our more recent studies use the homologous rat insulin and a different combination of reagents to enhance the precision and sensitivity of the assay. In the current procedure, dilutions of reagents, standards, and samples are made with 0.5% crystalline bovine-serum albumin (Miles Laboratories) in 0.05 M phosphate (pH 7.0). The assay is carried out in three successive stages on each sample:

Efficient restricted gene expression in beta cells by lentivirus-mediated gene transfer into pancreatic stem/progenitor cells

“…Prior to the grafting of these pancreases, we characterised the presence of endocrine and exocrine tissue in the explant by immunofluorescence analysis. In accordance with previously reported results [34], at E16.5 the vast majority of the endocrine cells in the pancreas are glucagon-expressing cells, in addition to rare insulin-expressing cells (Fig. 2a).…”

Proteins synthesized and secreted during rat pancreatic development.

“…During the next 3-4 d, acinar structures form and the low levels of exocrine secretory proteins and insulin are maintained ("protodifferentiated state") (22, 23) . A second differentiative transition is detectable beginning at 14-15 d gestation when there is a rapid increase in rough endoplasmic reticulum, a 103-to 10'-fold increase in the accumulation of the specific exocrine enzymes and insulin, coupled with the appearance of zymogen granules in acinar cells and,8 granules in B cells (4,21,23) accumulate in a parallel fashion but slightly preceding the secretory proteins (10,11,20) .…”

“…Normal development requires interaction between epithelial and mesenchymal tissues (8,25). Early investigations suggested that pancreatic differentiation is a multiphasic process (4,21,23) . In the rat, the formation of the pancreatic rudiment from the gut at -11 d gestation is coupled to the appearance of very low levels of exocrine proteins and insulin (the primary transition) .…”

Ontogeny of Somatostatin Cells in the Rat Fetal Pancreas.

“…Somatostatin inhibits the release of insulin and glucagon from B and A cells, respectively, in the pancreatic islet both in vivo and in vitro [1,7,8,10,11]. Early development of somatostatin cells has not been much studied in detail, although that of A and B cells has been widely investigated in the rat fetus [5,6,[16][17][18]. The present study was performed to determine when somatostatin cells would appear and in what fashion they would grow in the rat fetus, by means of the immunocytochemical technique.…”

“…It is proposed that B cells of the fetal pancreas differentiate by 3 steps [9,13]. In the first step up to day 15, B cells reveal no signs of cytological differentiation with constantly low insulin content and with undetectable insulin granules by ultrastructural analysis [16,17]. In the second step around day 15, B cells reveal a cytodifferentiation characterized by the appearance of secretory granules and an exponential increase in the cell volume and the insulin content [12].…”