Immunoreactivities for chromogranin A and B, and secretogranin II in the guinea-pig endocrine pancreas

The chromogranins are acidic proteins present in various endocrine cells and organs. They consist of chromogranin A (CgA), chromogranin B (CgB) and secretogranin II (SgII). In the pancreas, these proteins or their breakdown products are possibly involved in the regulation of pancreatic hormone secretion. The guinea-pig endocrine pancreas was now investigated immunohistochemically for the presence of the chromogranins in five endocrine cell types. CgA is a regular constituent of insulin (B-), pancreatic polypeptide (PP-) and enterochromaffin (EC-) cells. In addition, a minority of somatostatin (D-) cells were immunoreactive for CgA. CgB immunoreactivities were very faint and exclusively observed in B-cells. SgII was found in B- and PP-cells; a faint immunostaining for SgII was also seen in a few glucagon (A-) cells. Typically, the densities of CgA or SgII immunoreactivities varied among the members of a given cell population, e.g. among individual B- or PP-cells. The present findings about the heterogeneities of immunoreactivities for the chromogranins are in line with findings obtained in pancreatic endocrine cells of other species. The true reasons for these heterogeneities are enigmatic. It seems probable, however, that the corresponding immunoreactivities depend on the intracellular processing of the chromogranins which in turn might be related to the metabolic state of endocrine cells. This has to be examined in future by experimental investigations.     

Immunoreactivities for chromogranin A and B,and secretogranin II in the guinea-pig endocrine pancreas

Summary The chromogranins are acidic proteins present in various endocrine cells and organs. They consist of chromogranin A (CgA), chromogranin B (CgB) and secretogranin II (SgII). In the pancreas, these proteins or their breakdown products are possibly involved in the regulation of pancreatic hormone secretion. The guinea-pig endocrine pancreas was now investigated immunohistochemically for the presence of the chromogranins in five endocrine cell types. CgA is a regular constituent of insulin (B-), pancreatic polypeptide (PP-) and enterochromaffin (EC-) cells. In addition, a minority of somatostatin (D-) cells were immunoreactive for CgA. CgB immunoreactivities were very faint and exclusively observed in B-cells. SgII was found in B- and PP-cells; a faint immunostaining for SgII was also seen in a few glucagon (A-) cells. Typically, the densities of CgA or SgII immunoreactivities varied among the members of a given cell population, e.g. among individual B- or PP-cells. The present findings about the heterogeneities of immunoreactivities for the chromogranins are in line with findings obtained in pancreatic endocrine cells of other species. The true reasons for these heterogeneities are enigmatic. It seems probable, however, that the corresponding immunoreactivities depend on the intracellular processing of the chromogranins which in turn might be related to the metabolic state of endocrine cells. This has to be examined in future by experimental investigations.     

Topology of chromogranin A and secretogranin II in the rat anterior pituitary: potential marker proteins for distinct secretory pathways in gonadotrophs

Summary Chromogranins (Cg)/secretogranins (Sg) are representative acidic glycoproteins in secretory granules of many endocrine cells where they are co-stored and co-released with resident amines or peptides. The exact distribution of these proteins in the rat anterior pituitary is unknown. Therefore, pituitaries from untreated male rats were investigated by light- and electron-microscopical immunocytochemistry for the cellular and subcellular localization of CgA, CgB, and SgII. Endocrine cells, identified light-microscopically as gonadotrophs in adjacent semithin sections immunostained for follicle-stimulating hormone (FSH) and luteinizing hormone (LH), concomitantly were immunoreactive for CgA, CgB, and SgII. Ultrastructurally, gonadotrophs exhibited two types of secretory granules which varied in their immunoreactivities for gonadotropins and Cg/Sg. Large-sized (500 nm), moderately electron-dense granules showed antigenicities for FSH, LH, and CgA. Smaller-sized (200 nm), electron-dense granules were immunoreactive exclusively for LH and SgII. The distinct localization of CgA and SgII to morphologically and hormonally different secretory granules indicates the existence of two regulated secretory pathways in rat pituitary gonadotrophs. Hence, these proteins are considered as valuable tools to analyze the intracellular trafficking during granule biogenesis and the possible different regulation of FSH and LH secretion.     

Topology of chromogranin A and secretogranin II in the rat anterior pituitary: potential marker proteins for distinct secretory pathways in gonadotrophs.

Chromogranins (Cg)/secretogranins (Sg) are representative acidic glycoproteins in secretory granules of many endocrine cells where they are co-stored and co-released with resident amines or peptides. The exact distribution of these proteins in the rat anterior pituitary is unknown. Therefore, pituitaries from untreated male rats were investigated by light- and electron-microscopical immunocytochemistry for the cellular and subcellular localization of CgA, CgB, and SgII. Endocrine cells, identified light-microscopically as gonadotrophs in adjacent semithin sections immunostained for follicle-stimulating hormone (FSH) and luteinizing hormone (LH), concomitantly were immunoreactive for CgA, CgB, and SgII. Ultrastructurally, gonadotrophs exhibited two types of secretory granules which varied in their immunoreactivities for gonadotropins and Cg/Sg. Large-sized (500 nm), moderately electron-dense granules showed antigenicities for FSH, LH, and CgA. Smaller-sized (200 nm), electron-dense granules were immunoreactive exclusively for LH and SgII. The distinct localization of CgA and SgII to morphologically and hormonally different secretory granules indicates the existence of two regulated secretory pathways in rat pituitary gonadotrophs. Hence, these proteins are considered as valuable tools to analyze the intracellular trafficking during granule biogenesis and the possible different regulation of FSH and LH secretion.     

Chromogranin B: isolation from pheochromocytoma, N-terminal sequence, tissue distribution and secretory vesicle processing

The chromogranins/secretogranins are a family of neuroendocrine vesicle secretory proteins. Immunohistology and immunoblotting have suggested that a major soluble protein in human chromaffin granules may be chromogranin B (CgB). We purified from pheochromocytoma chromaffin granules an SDS-PAGE 110-120 kDa protein whose N-terminal sequence matched that previously deduced from a human CgB cDNA. An antibody directed against a synthetic human CgB N-terminal region specifically recognized the CgB N-terminus, though not the chromogranin A (CgA) N-terminus or the CgB C-terminus on immunoblots. An antiserum directed against CgB's C-terminus also visualized CgB but not CgA. By immunoblotting, CgB was a quantitatively major protein in human pheochromocytoma chromaffin granules, but a relatively minor in normal bovine adrenal medullary chromaffin granules. In a variety of normal bovine neuroendocrine tissues, the relative abundance of CgB immunoreactivity on immunoblots was: adrenal medulla greater than anterior pituitary greater than pancreas greater than small intestine, hypothalamus. Immunoblotting of neuroendocrine tissues (or their hormone storage vesicle cores) with both anti N-terminal and anti C-terminal CgB antisera suggested bidirectional cleavage or processing of CgB; in the anterior pituitary, a unique 40 kDa C-terminal fragment was observed. Bidirectional CgB cleavage was also suggested on immunoblots of chromaffin tissue from three species (human, bovine, rat). C-terminal processing of CgB was also confirmed by amino acid sequencing of SDS-PAGE-separated, polyvinylidene difluoride membrane-immobilized CgB fragments from pheochromocytoma chromaffin granules. Whether such fragments possess biological activity remains to be investigated.     

Proteolytic processing of chromogranin A by the prohormone convertase PC2

The neuroendocrine secretory protein chromogranin A (CgA) is a precursor for various biologically active peptides. Several single and paired basic residues are present within its primary amino acid sequence comprising cleavage sites for prohormone convertases. In this study, SH-SY5Y human neuroblastoma cells were stably transfected with the prohormone convertase PC2 to analyse the proteolytic processing of endogenous chromogranin A and, in particular, the formation of the chromogranin-A-derived peptide GE-25. Our analyses revealed a significant change in the pattern of proteolytic conversion of chromogranin A in cells expressing PC2. Mock-transfected control cells contained mainly the intact chromogranin A molecule and hardly any shorter products were found. On the other hand, PC2-transfected cells showed extensive processing of chromogranin A, resulting in significantly lower amounts of the intact precursor and especially high levels of the free peptide GE-25.     

The granin family of uniquely acidic proteins of the diffuse neuroendocrine system: comparative and functional aspects

The chromogranins A (CgA) and B (CgB) and secretogranin II (SgII) constitute the main members of a family of uniquely acidic secretory proteins in elements of the diffuse neuroendocrine system. These genetically distinct proteins, CgA, CgB, SgII and the less well known secretogranins III-VII are collectively referred to as 'granins' and characterised by numerous pairs of basic amino acids as potential cleavage sites for processing by the co-stored prohormone converting enzymes PC 1/3 and PC2. This review is directed towards comparative and functional aspects of the granins with emphasis on their phylogenetically conserved sequences. Recent developments provide ample evidence of widely different effects and targets for the intact granins and their derived peptides, intracellularly in the directed trafficking of storage components during granule maturation and extracellularly in autocrine, paracrine and endocrine interactions. Most of the effects assigned to the granin derived peptides fit into patterns of direct or indirect inhibitory modulations of major functions. So far, peptides derived from CgA (vasostatins, chromacin, pancreastatin, WE-14, catestatin and parastatin), CgB (secretolytin) and SgII (secretoneurin) are the most likely candidates for granin-derived regulatory peptides, of postulated relevance not only for homeostatic processes, but also for tissue assembly and repair, inflammatory responses and the first line of defence against invading microorganisms.     

Measurements of secretogranins II, III, V and proconvertases 1/3 and 2 in plasma from patients with neuroendocrine tumours

INTRODUCTION: Chromogranin (Cg) and secretogranin (Sg) are members of the granin family of proteins, which are expressed in neuroendocrine and nervous tissue. In recent publications we have presented generation of region-specific antibodies against CgA and CgB and also development of several region-specific radioimmunoassays for measurements of specific parts of the Cgs. In this study we describe generation of antibodies against SgII, SgIII, SgV and the proconvertases PC1/3 and PC2 and development of radioimmunoassays for measurements of these proteins. MATERIALS AND METHODS: Peptides homologous to defined parts of the secretogranin and proconvertase molecules were selected and synthesised. Antibodies were raised, radioimmunoassays were developed and circulating levels of the proteins in plasma samples from 22 patients with neuroendocrine tumours were measured in the assays. RESULTS: Increased plasma concentrations were recorded in 11, 4 and 3 of the patients with the SgII 154-165 (N-terminal secretoneurin), the SgII 172-186 (C-terminal Secretoneurin) and the SgII 225-242 assays respectively. The SgIII, SgV, PC1/3 and PC2 assays failed to detect increased concentrations in any of the patients. CONCLUSION: Increased concentrations of SgII, especially the N-terminal part of secretoneurin could be measured in plasma from patients with endocrine pancreatic tumours and in this case this assay was quite comparable to measurements of CgA and CgB. Even though secretoneurin was not as frequently increased as CgA and CgB in patients with carcinoid tumours or pheochromocytoma it may be a useful marker for endocrine pancreatic tumours.     

Measurements of chromogranin B can serve as a complement to chromogranin A

OBJECTIVE: CgA has been shown to be an excellent marker for neuroendocrine tumours. However, there are two major drawbacks with CgA measurements; elevated levels are common in patients with decreased renal function and in patients on treatment with proton pump inhibitors. These problems are not seen with CgB measurements. We have recently presented the development of 13 region-specific radioimmunoassays for measurements of CgB. A region-specific assay was identified, which measured higher concentrations of CgB than the other assays and seemed to be very useful as a marker for neuroendocrine tumours. The aim of the present study was therefore to further explore the diagnostic potential of this assay in the clinical management of patients with neuroendocrine tumours. METHODS: Measurements of CgB with two methods were compared with CgA in plasma samples from patients investigated for neuroendocrine tumours (N=86), patients with decreased renal function (N=35) and patients on treatment with proton pump inhibitors (N=29). RESULTS: The diagnostic sensitivity for the new CgB assay was almost as good as that for CgA. Furthermore, with CgB measurements we could avoid the falsely elevated levels of CgA found in patients with decreased renal function and treatment with proton pump inhibitors. CONCLUSIONS: We conclude that the new CgB assay can serve as a complement to CgA measurements as an important tumour marker for neuroendocrine tumours.     

A panel of 13 region-specific radioimmunoassays for measurements of human chromogranin B

INTRODUCTION: The primary structure of human chromogranin B (CgB) contains 15 pairs of basic amino acids, which are potential cleavage sites for specific endogenous proteases, but also other sites in the molecule can be subjected to cleavage. Several CgB-related peptides have been identified in tissue extracts. MATERIALS AND METHODS: Peptides homologous to defined parts of the human CgB molecule were selected and synthesized. Antibodies were raised and 13 specific radioimmunoassays were developed. Plasma samples from 19 patients with neuroendocrine tumors were collected and measured in all assays. RESULTS: All region-specific assays measured circulating levels of CgB-related peptides. Only five of the assays measured high concentrations of circulating CgB and two of them correlated with that of intact chromogranin A (CgA). CONCLUSION: The assays presented allow measurements of defined regions of CgB and will thus become important tools for further studies of the processing of CgB. One of the assays merit further investigations as a new marker for neuroendocrine tumors.     

Differential localization of prohormone convertases PC1 and PC2 in two distinct types of secretory granules in rat pituitary gonadotrophs

Prohormone convertases PC1 and PC2 are endoproteases involved in prohormone cleavage at pairs of basic amino acids. There is a report that prohormone convertase exists in the rat anterior pituitary gonadotrophs, where it had previously been considered that proprotein processing does not take place. In addition to luteinizing hormone and follicle-stimulating hormone, rat pituitary gonadotrophs contain chromogranin A (CgA) and secretogranin II (SgII), two members of the family of granin proteins, which have proteolytic sites in their molecules. In the present study we examined whether there is a close correlation between subcellular localization of prohormone convertases and granin proteins. Ultrathin sections of rat anterior pituitary were immunolabeled with anti-PC1 or -PC2 antisera and then stained with immunogold. Immunogold particles for PC1 were exclusively found in large, lucent secretory granules, whereas those for PC2 were seen in both large, lucent and small, dense granules. The double-immunolabeling also demonstrated colocalization of PC2 and SgII in small, dense granules and of PC1, PC2, and CgA in large, lucent granules. These immunocytochemical results suggest that PC2 may be involved in the proteolytic processing of SgII and that both PC1 and PC2 may be necessary to process CgA.     

Evaluation of chromogranin A expression in serum and tissues of breast cancer patients

Human chromogranin A (CgA) is a member of the granin family and is widely distributed in large dense core granules of endocrine and neuroendocrine cells. A variety of non-neuroendocrine carcinomas arising in various tissues show patterns of neuroendocrine differentiation. Expression of CgA has been documented in epithelial cells of normal mammary gland as well as in breast cancers, and elevation of serum CgA has been detected in patients with breast cancer. Our study was undertaken to evaluate the relationship between serum CgA levels and neuroendocrine features in breast cancer. In addition, we evaluated the expression of serum CgA in patients affected by breast cancer compared to controls and the relationship between serum CgA and tumor histology, extent of disease, lymph node status, tumor stage and serum CA 15.3 levels. We enrolled 266 patients with infiltrating ductal or lobular breast carcinoma and a group of 100 age-matched healthy women serving as controls. Serum CgA and CA 15.3 were assayed by specific immunoradiometric methods. The overall sensitivity of CgA and CA 15.3 was 0.06 and 0.34, respectively (chi2 19.1, p<0.0005). No relationship was found between serum levels of CgA and tumor histology, extent of disease, lymph node status or tumor stage while serum levels of CA 15.3 were strongly correlated with all these variables but tumor histology. No relationship was found between serum levels of CgA and CA 15.3. Immunostaining against CgA, CgB, NSE and synaptophysin was performed on primary tumor tissue of 14 serum CgA-positive and 24 serum CgA-negative patients and was negative in all cases. We also evaluated eight cases of pathologically-proven neuroendocrine breast cancer: only four and two of these showed positive CgA immunostaining and increased serum CgA concentration, respectively. In conclusion, serum CgA assay offers no additional information regarding the presence, the extent and the histology of breast cancer compared to the CA 15.3 assay. Moreover, serum CgA was not an accurate marker to identify or exclude the rare neuroendocrine differentiation of breast cancer. We therefore conclude that CgA is not useful as a serum marker in breast cancer.     

Immunohistochemical and Biochemical Studies with Region-Specific Antibodies to Chromogranins A and B and Secretogranins II and III in Neuroendocrine Tumors

This short review deals with our investigations in neuroendocrine tumors (NETs) with antibodies against defined epitopes of chromogranins (Cgs) A and B and secretogranins (Sgs) II and III. The immunohistochemical expression of different epitopes of the granin family of proteins varies in NE cells in normal human endocrine and non-endocrine organs and in NETs, suggesting post-translational processing. In most NETs one or more epitopes of the granins were lacking, but variations in the expression pattern occurred both in benign and malignant NETs. A few epitopes displayed patterns that may be valuable in differentiating between benign and malignant NET types, e.g., well-differentiated NET types expressed more CgA epitopes than the poorly differentiated ones and C-terminal secretoneurin visualized a cell type related to malignancy in pheochromocytomas. Plasma concentrations of different epitopes of CgA and CgB varied. In patients suffering from carcinoid tumors or endocrine pancreatic tumors the highest concentrations were found with epitopes from the mid-portion of CgA. For CgB the highest plasma concentrations were recorded for the epitope 439–451. Measurements of SgII showed that patients with endocrine pancreatic tumors had higher concentrations than patients with carcinoid tumors or pheochromocytomas. SgIII was not detectable in patients with NETs.     

Determinants for chromogranin A sorting into the regulated secretory pathway are also sufficient to generate granule-like structures in non-endocrine cells

In endocrine cells, prohormones and granins are segregated in the TGN (trans-Golgi network) from constitutively secreted proteins, stored in concentrated form in dense-core secretory granules, and released in a regulated manner on specific stimulation. The mechanism of granule formation is only partially understood. Expression of regulated secretory proteins, both peptide hormone precursors and granins, had been found to be sufficient to generate structures that resemble secretory granules in the background of constitutively secreting, non-endocrine cells. To identify which segment of CgA (chromogranin A) is important to induce the formation of such granule-like structures, a series of deletion constructs fused to either GFP (green fluorescent protein) or a short epitope tag was expressed in COS-1 fibroblast cells and analysed by fluorescence and electron microscopy and pulse-chase labelling. Full-length CgA as well as deletion constructs containing the N-terminal 77 residues generated granule-like structures in the cell periphery that co-localized with co-expressed SgII (secretogranin II). These are essentially the same segments of the protein that were previously shown to be required for granule sorting in wild-type PC12 (pheochromocytoma cells) cells and for rescuing a regulated secretory pathway in A35C cells, a variant PC12 line deficient in granule formation. The results support the notion that self-aggregation is at the core of granule formation and sorting into the regulated pathway.     

The disulfide-bonded loop of chromogranin B mediates membrane binding and directs sorting from the trans-Golgi network to secretory granules

The disulfide-bonded loop of chromogranin B (CgB), a regulated secretory protein with widespread distribution in neuroendocrine cells, is known to be essential for the sorting of CgB from the trans-Golgi network (TGN) to immature secretory granules. Here we show that this loop, when fused to the constitutively secreted protein alpha1-antitrypsin (AT), is sufficient to direct the fusion protein to secretory granules. Importantly, the sorting efficiency of the AT reporter protein bearing two loops (E2/3-AT-E2/3) is much higher compared with that of AT with a single disulfide-bonded loop. In contrast to endogenous CgB, E2/3-AT-E2/3 does not undergo Ca2+/pH-dependent aggregation in the TGN. Furthermore, the disulfide-bonded loop of CgB mediates membrane binding in the TGN and does so with 5-fold higher efficiency if two loops are present on the reporter protein. The latter finding supports the concept that under physiological conditions, aggregates of CgB are the sorted units of cargo which have multiple loops on their surface leading to high membrane binding and sorting efficiency of CgB in the TGN.     

Carboxypeptidase E,an essential element of the regulated secretory pathway,is expressed and partially co-localized with chromogranin A in chicken thymus

Although the functions of hormones and neuropeptides in the thymus have been extensively studied, we still do not know whether these intra-thymic humoral elements are released in a stimulated manner via the regulated secretory pathway or in a constitutive manner. Carboxypeptidase E (CpE) and chromogranin A (CgA) are functional and structural hallmarks of the regulated secretory pathway in (neuro)endocrine cells. Whereas we have previously shown a CgA-positive neuroendocrine population in the chicken thymus, the current study assesses the expression of CpE in the thymus, both at the mRNA and the protein level. Our immunohistochemical studies provide evidence for the co-existence of CgA and CpE in identical neuroendocrine cells in the thymus. CpE and CgA dual-positive cells have primarily been found in the transition zone between the cortex and medulla of the thymus, an area known to contain numerous arterioles and to be innervated by the autonomic nervous system. Our findings suggest that the diffuse neuroendocrine system serves as a relay for nervous stimuli delivered by the sympathetic and/or parasympathetic nervous system. Thus, these newly defined neuroendocrine cells might play an important role in the immuno-neuro-endocrine cross-talk in the thymus, potentially enabling thymopoiesis to be fine-tuned via the regulated secretory pathway by a variety of physical and environmental factors.     

Secretogranin III binds to cholesterol in the secretory granule membrane as an adapter for chromogranin A

Granin-family proteins, including chromogranin A (CgA) and secretogranin III (SgIII), are transported to secretory granules (SGs) in neuroendocrine cells. We previously showed that SgIII binds strongly to CgA in an intragranular milieu and targets CgA to SGs in pituitary and pancreatic endocrine cells. In this study, we demonstrated that with a sucrose density gradient of rat insulinoma-derived INS-1 cell homogenates, SgIII was localized to the SG fraction and was fractionated to the SG membrane (SGM) despite lacking the transmembrane region. With depletion of cholesterol from the SGM using methyl-beta-cyclodextrin, SgIII was impaired to bind to the SGM. Both SgIII and CgA were solubilized from the SGM by Triton X-100 in contrast to the Triton X-100 insolubility of carboxypeptidase E. SgIII and carboxypeptidase E strongly bound to the SGM-type liposome in intragranular conditions, but CgA did not. Instead, CgA bound to the SGM-type liposome only in the presence of SgIII. Immunocytochemical and pulse-chase experiments revealed that SgIII deleting the N-terminal lipid-binding region missorted to the constitutive pathway in mouse corticotroph-derived AtT-20 cells. Thus, we suggest that SgIII directly binds to cholesterol components of the SGM and targets CgA to SGs in pituitary and pancreatic endocrine cells.     

Purification, pH-dependent conformational change, aggregation, and secretory granule membrane binding property of secretogranin II (chromogranin C).

Secretogranin II (SgII) is one of the three major proteins, the other two being chromogranins A (CGA) and B (CGB), of secretory granules of neuroendocrine cells. The Ca(2+) storage proteins CGA and CGB not only are coupled to the IP(3) receptor (IP(3)R)/Ca(2+) channels that exist on the secretory granule membrane but also are known to play key roles in secretory granule biogenesis. Unlike the better studied CGA and CGB, secretogranin II has never been completely purified in the native state and studied. We have therefore purified SgII in native form from bovine adrenal medulla and subjected it to biochemical characterization. Secretogranin II consisted of largely beta-sheet and random coil structures with a low level of alpha-helicity. Like CGA and CGB, it also underwent pH-dependent conformational changes, showing 9.5% alpha-helicity at pH 7.5 and 17.0% alpha-helicity at pH 5.5. Secretogranin II also underwent acidic pH- and Ca(2+)-dependent aggregation, and it was approximately 8-fold more sensitive than CGA to Ca(2+) in its pH-dependent aggregation but was 8-fold less sensitive than CGB. Further, similar to CGA and CGB that had interacted with the secretory granule membrane at the intragranular pH 5.5, SgII also interacted with the secretory granule membrane at pH 5.5 and dissociated from it at near-physiological pH 7.5, implying similar roles of SgII in the cell as those of CGA and CGB. Secretogranin II hence appeared to actively participate in secretory granule biogenesis as has been proposed for CGA and CGB.     

Immunocytochemical localization of secretogranin III in the anterior lobe of male rat pituitary glands.

Secretogranin III (SgIII) is one of the acidic secretory proteins, designated as granins, which are specifically expressed in neuronal and endocrine cells. To clarify its precise distribution in the anterior lobe of the rat pituitary gland, we raised a polyclonal antiserum against rat SgIII for immunocytochemical analyses. By immunohistochemistry using semithin sections, positive signals for SgIII were detected intensely in mammotropes and thyrotropes, moderately in gonadotropes and corticotropes, but not in somatotropes. The distribution pattern of SgIII in the pituitary gland was similar to that of chromogranin B (CgB), also of the granin protein family, suggesting that the expressions of these two granins are regulated by common mechanisms. The localization of SgIII in endocrine cells was confirmed by immunoelectron microscopy. In particular, secretory granules of mammotropes and thyrotropes were densely and preferentially co-labeled for SgIII and CgB in their periphery. Moreover, positive signals for SgIII were occasionally found in cells containing both prolactin and TSH in secretory granules. These lines of evidence suggest that SgIII and CgB are closely associated with the secretory granule membrane and that this membrane association might contribute to gathering and anchoring of other soluble constituents to the secretory granule membrane.     

Defective Secretion of Islet Hormones in Chromogranin-B Deficient Mice

Granins are major constituents of dense-core secretory granules in neuroendocrine cells, but their function is still a matter of debate. Work in cell lines has suggested that the most abundant and ubiquitously expressed granins, chromogranin A and B (CgA and CgB), are involved in granulogenesis and protein sorting. Here we report the generation and characterization of mice lacking chromogranin B (CgB-ko), which were viable and fertile. Unlike neuroendocrine tissues, pancreatic islets of these animals lacked compensatory changes in other granins and were therefore analyzed in detail. Stimulated secretion of insulin, glucagon and somatostatin was reduced in CgB-ko islets, in parallel with somewhat impaired glucose clearance and reduced insulin release, but normal insulin sensitivity in vivo. CgB-ko islets lacked specifically the rapid initial phase of stimulated secretion, had elevated basal insulin release, and stored and released twice as much proinsulin as wildtype (wt) islets. Stimulated release of glucagon and somatostatin was reduced as well. Surprisingly, biogenesis, morphology and function of insulin granules were normal, and no differences were found with regard to β-cell stimulus-secretion coupling. We conclude that CgB is not required for normal insulin granule biogenesis or maintenance in vivo, but is essential for adequate secretion of islet hormones. Consequentially CgB-ko animals display some, but not all, hallmarks of human type-2 diabetes. However, the molecular mechanisms underlying this defect remain to be determined.