Beta-cell Secretory Granules Research Articles

Immunocytochemical localization of heat-shock protein 60-related protein in beta-cell secretory granules and its altered distribution in non-obese diabetic mice.

Immuno-electron microscopy technique was employed to investigate the cellular distribution of 60 kDa heat-shock protein (HSP60) in pancreatic Beta cells of control and non-obese diabetic mice. In thin sections prepared from control mice, antibody to mammalian HSP60 cross-reacted with protein(s) located to mitochondria and secretory granules. In particular, prominent binding of the antibody was seen to the insulin core of the mature insulin-secreting granules. In comparison, very little immunoreactivity was observed with immature secretory granules or with the Golgi apparatus. No binding to secretory granules or mitochondria was observed with normal mouse serum or with unrelated sera. On Western blots, HSP60 antibody specifically interacted with a single 62 kDa islet cell protein. These results suggest the existence of an HSP60-related protein with a novel location in mature secretory granules of Beta cells. The preferential association of the HSP60-related protein with the insulin core was gradually lost in Beta cells of pre-diabetic non-obese diabetic mice, and correlated with the progression of insulitis. The decrease in the granular binding of the HSP60 antibody was accompanied by an increase in cytoplasm staining, and was concomitant with a significant expansion of the insulin core diameter. The altered distribution of the HSP60-related protein in prediabetic mice, together with our observation that immature secretory granules accumulate in these animals indicate that the presence of HSP60-related protein in secretory granules might be associated with the secretory function of Beta cells.

Immunoelectron microscopic localization of antigens which react with islet cell cytoplasmic antibodies within human pancreatic beta cells.

The presence of circulating autoantibody to islet cell cytoplasm is considered to be an important marker of Type 1 (insulin-dependent) diabetes mellitus. In the present study using islet cell cytoplasmic antibody positive patient sera as the first antibody, we studied the intracellular distribution of its antigen at the electron microscopic level using the pre-embedding immunoperoxidase method. Specific immunoreactivity was found in the membranes of beta cell-secretory granules and cytoplasmic membranes. This result is compatible with the interpretation that the antigen(s) on the membranes of beta cell secretory granules is (are) the target of islet cell cytoplasmic antibody.

Expression of reg protein in rat regenerating islets and its co-localization with insulin in the Beta cell secretory granules

Regenerating islets can be induced by the administration of poly(ADP-ribose) synthetase inhibitors to 90% depancreatized rats. In screening a regenerating islet-derived cDNA library, we previously isolated a novel gene. reg (regenerating gene), which encodes a 165-amino acid protein with a 21-amino acid signal sequence. In the present study, we have examined the expression and localization of reg protein in the regenerating islets by immunocytochemical techniques using a monoclonal antibody against a recombinant rat reg protein of 144 amino acids without the signal sequence. Light microscopy examination showed strong immunoreactivity for reg protein in the regenerating islets of the rats at two weeks and two months after 90% pancreatectomy, whereas reg protein was almost undetectable in normal rat islets or in the islets of the rats one year after the pancreatectomy. Almost all the reg protein-positive cells were stained for insulin. By applying the immunogold technique at the ultrastructural level, it was demonstrated that both reg protein and insulin occur in the central granular core of the regenerating Beta cell secretory granules. These results suggest that reg protein is synthesized in and secreted from the regenerating Beta cells and that its expression is closely associated with Beta-cell regeneration.

External ATP mimics carbachol in initiating calcium mobilization from pancreatic β‐cells conditioned by previous exposure to glucose

1 Exposure to ATP (2-200 microM) resulted in a prominent peak of 45Ca efflux, when beta-cell-rich pancreatic islets from ob/ob-mice were perifused with a Ca2+-deficient medium. ADP and the stable alpha/beta-methylene analogues of ATP and ADP also had stimulatory effects. 2 The nucleotide initiation of 45Ca efflux mimicked that obtained with carbachol both in requiring previous exposure to glucose and in being more pronounced after replacing extracellular Na+ by K+. 3 It was possible to induce repeated peaks of stimulated 45Ca efflux, when the exposure to ATP was interrupted with intervals of perifusion with glucose-containing media. 4 The observations are consistent with the existence of P2-purinoceptors in islets, suggesting that these receptors mediate a similar mobilization of calcium as noted when activating polyphosphoinositide breakdown with carbachol. In view of the high contents of ATP and ADP in the beta-cell secretory granules, activation of P2-purinoceptors should be considered as a possible mechanism for amplification of the initial insulin secretory response.

Insulinoma. An immunocytochemical and morphologic analysis of 95 cases.

One hundred twenty-seven insulinomas from 95 cases (1 malignant and 94 benign) were studied pathologically. Thirty-six tumors (35 cases) were examined by electron microscopy. Typical beta-cell secretory granules of crystalloid-form cores and/or atypical secretory granules were discerned in all tumors examined. A new type of secretory granule, with high electron-dense crystalloid-form cores and moderate electron-dense granular substance filling the space between the core and the limiting membrane, were observed in two cases. Among 68 insulinomas (67 cases) subjected to immunocytochemical investigations with ten peptide hormones (insulin, glucagon, somatostatin, pancreatic polypeptide (PP), gastrin, motilin, secretin, vasoactive intestinal polypeptide (VIP), gastric inhibitory polypeptide (GIP), and neurotensin), 42 were found to be multihormonal, varying from two to four peptides secreted. The hormones contained were insulin, glucagon, PP, somatostatin, and gastrin in different combinations. One patient had hyperinsulinemia and hypergastrinemia concurrently, and two islet tumors were excised at an interval of 10 months. Both electron microscopy and immunocytochemistry confirmed the presence of beta- and alpha-cells in the first tumor, whereas the second tumor revealed only G-cells by electron microscopy, and G- and beta-cells on immunocytochemical staining. The morphologic and immunocytochemical characteristics of the insulinomas in this series are discussed.

Glucose-induced proton uptake in secretory granules of beta-cells in monolayer culture.

The weak base acridine orange (AO) has been shown to be accumulated by the insulin-containing secretory granules of cultured beta-cells in response to high glucose. Various lines of evidence indicate that this accumulation is due to a pH gradient. Thus ionophores such as monensin and nigericin abolish the glucose-induced accumulation, and a high concentration of the weak base, benzylamine, results in swelling of the granules. In the absence of glucose, ATP addition to digitonin-permeabilized cells also results in dye uptake. These data also suggest that a primary or secondary active accumulation mechanism for hydrogen ions exists across the granule membrane.

Quick-freeze fixation and freeze-drying of isolated rat pancreatic islets: application to the ultrastructural localization of inorganic phosphate in the pancreatic beta cell.

A bounce-free mechanical quick-freeze assembly and a Coulter-Terracio freeze-dry apparatus were successfully coupled to obtain high quality ultrastructural preservation of pancreatic beta cells in a simple and dependable manner. Except for obvious shrinkage spaces, morphological relationships at the tissue, cellular, and subcellular levels were all intact. Beta cell secretory granules demonstrated a dense core surrounded by an electron lucent halo as typically described in specimens after aqueous fixation. Cell membranes and intracellular membranes demonstrated a trilaminar appearance. Golgi apparatus were well preserved. Two clearly defined populations of mitochondria were found. One group of very dark mitochondria had extremely dense matrices in which cristae were barely visible. A second group of mitochondria had light matrices with prominent cristae. The combined quick-freeze fixation and freeze-drying was applied to reevaluate the ultrastructural localization of inorganic phosphate that had been precipitated with lead in the beta cells of pancreatic islets. Accumulation of inorganic phosphate adjacent to the plasma membrane and over the nucleolus of the beta cell in nonstimulated islets was documented with better detail than heretofore possible.

Species dependent pancreatic islet toxicity produced by cyproheptadine: Alterations in beta cell structure and function

Cyproheptadine (CPH) produced marked cytoplasmic vacuolization of pancreatic islet tissue after repeated oral administration of 22.5–90 mg/kg to the rat. No islet cell degeneration was observed in tissue from mice, rabbits, or hamsters after po administration. Intraperitoneal administration of CPH to rats caused only a slight degree of vacuolization. Examination of aldehyde-fuchsin-stained rat islet tissue after administration of CPH, 45 mg/kg for 2 wk, indicated a marked degranulation of beta cells. Electron microscopic examination of islet tissue from CPH-treated rats (45 mg/kg) showed vesiculation of the rough endoplasmic reticulum and loss of secretory granules in beta cells. These high doses of CPH produced a marked glucose intolerance in the rat as well as a diminished reponse of plasma insulin levels to glucagon stimulation.

Electron microscopic studies on the secretory mechanism of pancreatic islet cells, with particular reference to beta cells. I. Normal secretion in mice, rats, rabbits and chickens, and secretion stimulated by a single intravenous injection of glucose or chlorpropamide in rats.

The secretory cycle of pancreatic islet cells of mice (C3H), Wistar rats, white rabbits and chickens has been indicated by ultrastructural changes of the roughsurfaced endoplasmic reticulum and Golgi body, both of which represent cellular activity, ranging from the granular to hypogranular appearance of the cytoplasm of pancreatic islet cells. The ergastoplasm seems to be a primary synthetic site of insulin or glucagon molecules and appears to be carried by numerous microvesicles in moderate electron density toward the Golgi zone. These microcarriers then, constitute the lamellar component of the Golgi apparatus. Irregular swelling and shortening of the above lamellae occur and the vacuoles with amorphous raw substance seem to be formed by pinching off at their terminal dilated portions. At the Golgi zone of beta cells of the animals, microfibrils measuring approximately 60A in width attach on the outer surface of some of the above vacuoles, and occur both inside and through it, whereas in rats, microvesicles may be frequently engulfed into sacs via their incomplete or partially interrupted surrounding membrance. Thereafter, immature granules may develop and gain electron density gradually, followed by a molding of the storage form of characteristic crystallization of mature granules. Moreover, even after leaving the Golgi area the microfilamentous structures also may fall into the sacs of secretory granules in beta cells, although less frequently. The developed granules are released by an emiocytotic process into the perisinusoidal space. A single intravenous injection of glucose or chlorpropamide in the rat was performed and the ultrastructural alterations of beta cell organelles were observed. The primary effect of these two beta cell stimulants were assumed to be different. The granule synthesis, storage and discharge in the stimulated conditions of beta cells were almost the same process as found in the non-stimulated beta cells, except in the maximally effected state of glucose administration.