Secretory Granules Of Endocrine Cells Research Articles

Serum Secretogranin III Concentrations Were Increased in Subjects with Metabolic Syndrome and Independently Associated with Fasting Plasma Glucose Levels.

Secretogranin III (SCG3) plays a crucial role in the biogenesis of secretory granules in endocrine cells, and thus affects glucose homeostasis by regulating insulin secretion by pancreatic beta cells. Insulin resistance and compensatory hyperinsulinemia are hallmarks of metabolic syndrome (MetS). However, the role of SCG3 in MetS remains unclear. Therefore, we investigated the relationship between serum SCG3 levels and metabolic parameters in subjects with and without MetS. This was a case control study, and 295 subjects were recruited. Serum SCG3 concentrations were compared between groups. Associations between SCG3 levels and clinico-metabolic parameters were also examined. We found serum SCG3 levels were higher in the MetS group than non-MetS group (122.6 ± 79.2 vs. 90.6 ± 58.5 nmol/L, p = 0.009). Specifically, elevated SCG3 levels were found in subjects with high fasting plasma glucose (FPG) levels, central obesity, or hypertriglyceridemia. Additionally, MetS was an independent factor of serum SCG3 levels in multivariate linear regression analyses. Moreover, FPG, free fatty acids, and waist circumference were positively associated with serum SCG3 concentrations after adjusting for insulin levels, high-sensitivity C-reactive protein, and cardiovascular risk factors. In conclusion, serum SCG3 concentrations were higher in subjects with MetS and were independently associated with FPG levels.

Improved Serial Sectioning Techniques for Correlative Light-Electron Microscopy Mapping of Human Langerhans Islets.

Combined analysis of immunostaining for various biological molecules coupled with investigations of ultrastructural features of individual cells is a powerful approach for studies of cellular functions in normal and pathological conditions. However, weak antigenicity of tissues fixed by conventional methods poses a problem for immunoassays. This study introduces a method of correlative light and electron microscopy imaging of the same endocrine cells of compact and diffuse islets from human pancreatic tissue specimens. The method utilizes serial sections obtained from Epon-embedded specimens fixed with glutaraldehyde and osmium tetroxide. Double-immunofluorescence staining of thick Epon sections for endocrine hormones (insulin and glucagon) and regenerating islet-derived gene 1 α (REG1α) was performed following the removal of Epoxy resin with sodium ethoxide, antigen retrieval by autoclaving, and de-osmification treatment with hydrogen peroxide. The immunofluorescence images of endocrine cells were superimposed with the electron microscopy images of the same cells obtained from serial ultrathin sections. Immunofluorescence images showed well-preserved secretory granules in endocrine cells, whereas electron microscopy observations demonstrated corresponding secretory granules and intracellular organelles in the same cells. In conclusion, the correlative imaging approach developed by us may be useful for examining ultrastructural features in combination with immunolocalisation of endocrine hormones in the same human pancreatic islets.

Amyloid-like aggregation of provasopressin in diabetes insipidus and secretory granule sorting

BackgroundAggregation of peptide hormone precursors in the trans-Golgi network is an essential process in the biogenesis of secretory granules in endocrine cells. It has recently been proposed that this aggregation corresponds to the formation of functional amyloids. Our previous finding that dominant mutations in provasopressin, which cause cell degeneration and diabetes insipidus, prevent native folding and produce fibrillar aggregates in the endoplasmic reticulum (ER) might thus reflect mislocalized amyloid formation by sequences that evolved to mediate granule sorting.ResultsHere we identified two sequences responsible for fibrillar aggregation of mutant precursors in the ER: the N-terminal vasopressin nonapeptide and the C-terminal glycopeptide. To test their role in granule sorting, the glycopeptide was deleted and/or vasopressin mutated to inactivate ER aggregation while still permitting precursor folding and ER exit. These mutations strongly reduced sorting into granules and regulated secretion in endocrine AtT20 cells.ConclusionThe same sequences — vasopressin and the glycopeptide — mediate physiological aggregation of the wild-type hormone precursor into secretory granules and the pathological fibrillar aggregation of disease mutants in the ER. These findings support the amyloid hypothesis for secretory granule biogenesis.

Adrenalectomy facilitates ATAT1 expression and α-tubulin acetylation in ACTH-producing corticotrophs.

Microtubules play an important role in the intracellular transport of secretory granules in endocrine cells and in mitosis and the maintenance of cell morphology and are composed of heterodimers of α- and β-tubulin. α-Tubulin N-acetyltransferase 1 (ATAT1), which acetylates the lysine residue at position 40 of α-tubulin, functions not only in stabilizing microtubule structures and forming the primary cilium assembly but also in vesicular trafficking in neurons. However, the localization of ATAT1 and the role of α-tubulin acetylation in endocrine cells in the pituitary are still poorly understood. Corticotrophs in the anterior lobe of the pituitary produce and secrete adrenocorticotropin (ACTH). Although removal of the adrenal gland, a target organ of ACTH, is reported to promote the synthesis and secretion of ACTH in corticotrophs and to induce structural alterations in their organelles, uncertainty remains as to whether the acetylation of α-tubulin is involved in such intracellular events of corticotrophs. We investigate the expression and localization of ATAT1 and the acetylation of α-tubulin in the pituitary of normal and adrenalectomized rats. We find that ATAT1 is localized to the Golgi apparatus of endocrine cells in the anterior lobe of normal pituitary and that the expression levels of ATAT1 and acetylation levels of α-tubulin increase following adrenalectomy. These results agree with the hypothesis that the acetylation of α-tubulin by ATAT1 regulates the intracellular transport of secretory granules in corticotrophs.

Stimulatory effect of the secretogranin-ll derived peptide secretoneurin on food intake and locomotion in female goldfish (Carassius auratus)

Secretoneurin (SN) is a conserved peptide derived by proteolytic processing from the middle domain of the ∼600 amino acid precursor secretogranin-II (SgII). Secretoneurin is widely distributed in secretory granules of endocrine cells and neurons and has important roles in reproduction as it stimulates luteinizing hormone release from the pituitary. A potential new role of SN in goldfish feeding is the subject of this study. Firstly, we established that acute (26h; p<0.0001) and short-term (72h; p=0.016) fasting increased SgIIa precursor mRNA levels 1.25-fold in the telencephalon, implicating SN in the control of feeding. Secondly, we determined that intracerebroventricular injections of the type A SN (SNa; 0.2 and 1ng/g BW) increased food intake and locomotor behavior by 60min. Fish injected with the lower and higher doses of SNa (0.2 and 1ng/g) respectively exhibited significant 1.77- and 2.58-fold higher food intake (p<0.0001) than the saline-injected control fish. Locomotor behavior was increased by 1.35- and 2.26-fold for 0.2ng/g SNa (p=0.0001) and 1ng/g SNa (p<0.0001), respectively. Injection of 1ng/g SNa increased mRNA levels of hypothalamic neuropeptide Y 1.36-fold (p=0.038) and decreased hypothalamic cocaine-and amphetamine-regulated transcript by 33% (p=0.01) at 2h and 5h post-injection, respectively. These data suggest interactions of SNa with stimulatory and inhibitory pathways of food intake control in fish.

Aggregation chaperones enhance aggregation and storage of secretory proteins in endocrine cells.

Calcium-induced aggregation has been proposed to play a role in the sorting and storage of secretory proteins in secretory granules of endocrine cells. The regulation of this process is not known. Hexahistidine epitope tags were used to create aggregation chaperones that enhance the calcium-induced aggregation of secretory granule proteins in vitro. Indeed, 100% recovery of the aggregating target protein was achieved without any modification of the target protein. The aggregation chaperone is not trapped in the aggregates. Co-expression of His(6)-tagged secreted alkaline phosphatase and the regulated secretory protein chromogranin A resulted in an increased chromogranin storage in secretory granules, and stimulated secretion of chromogranin A increased 50%. However, secretion of secreted alkaline phosphatase was not affected by the hexahistidine epitope tag. Thus, calcium-induced aggregation is not a passive process; rather, aggregation and sorting of secretory proteins can be regulated by aggregation chaperones in the secretory pathway of endocrine cells.

Influence of Ca2+ and pH on the folding of the prourotensin II precursor

Proper folding is a crucial step for the trafficking of proteins through the secretory pathway. We hypothesized that the secretory granules of endocrine cells provide optimal folding conditions of prohormone precursors for cleavage. Here, using circular dichroism and in vitro processing on purified prourotensin II (ProUII), we show that the precursor undergoes pH- and Ca2+-dependent conformational and stability changes. ProUII has a stable tertiary structure at pH 5.5 in presence of Ca2+ and is correctly cleaved in these conditions by prohormone convertases. Taken together, our results support the notion that precursors may need to be optimally folded in the lumen of secretory granules for their processing.

Chromogranin A as a crucial factor in the sorting of peptide hormones to secretory granules.

Chromogranin A (CgA) is a soluble glycoprotein stored along with hormones and neuropeptides in secretory granules of endocrine cells. In the last four decades, intense efforts have been concentrated to characterize the structure and the biological function of CgA. Besides, CgA has been widely used as a diagnostic marker for tumors of endocrine origin, essential hypertension, various inflammatory diseases, and neurodegenerative disorders such as amyotrophic lateral sclerosis and Alzheimer's disease. CgA displays peculiar structural features, including numerous multibasic cleavage sites for prohormone convertases as well as a high proportion of acidic residues. Thus, it has been proposed that CgA represents a precursor of biologically active peptides, and a "granulogenic protein" that plays an important role as a chaperone for catecholamine storage in adrenal chromaffin cells. The widespread distribution of CgA throughout the neuroendocrine system prompted several groups to investigate the role of CgA in peptide hormone sorting to the regulated secretory pathway. This review summarizes the findings and theoretical concepts around the molecular machinery used by CgA to exert this putative intracellular function. Since CgA terminal regions exhibited strong sequence conservation through evolution, our work focused on the implication of these domains as potential functional determinants of CgA. Characterization of the molecular signals implicating CgA in the intracellular traffic of hormones represents a major biological issue that may contribute to unraveling the mechanisms defining the secretory competence of neuroendocrine cells.

Secretogranin III: a Bridge between Core Hormone Aggregates and the Secretory Granule Membrane

Secretory granules in endocrine cells selectively store bioactive peptide hormones and amines, which are secreted in a regulated manner upon appropriate stimulation. In addition to bioactive substances, various proteins and lipids characteristic of secretory granules are likely recruited to a restricted space at the trans-Golgi Network (TGN), and the space then matures to the secretory granule. Although experimental findings so far have strongly suggested that aggregation- and receptor-mediated processes are essential for the formation of secretory granules, the putative link between these two processes remains to be clarified. Recently, secretogranin III (SgIII) has been identified as a specific binding protein for chromogranin A (CgA), a representative constituent of the core aggregate within secretory granules, and it was later revealed that SgIII can also bind to the cholesterol-rich membrane domain at the TGN. Based on its multifaceted binding properties, SgIII may act as a central player in the formation of cholesterol-rich membrane platforms. Upon these platforms, essential processes for secretory granule biogenesis coordinately occur; that is, selective recruitment of prohormones, processing and modifying of prohormones, and condensation of mature hormones as an aggregate. This review summarizes the findings and theoretical concepts on the issue to date and then focuses on the putative role of SgIII in secretory granule biogenesis in endocrine cells.

First Signature of Islet β-Cell-Derived Naturally Processed Peptides Selected by Diabetogenic Class II MHC Molecules

The diversity of Ags targeted by T cells in autoimmune diabetes is unknown. In this study, we identify and characterize a limited number of naturally processed peptides from pancreatic islet beta-cells selected by diabetogenic I-A(g7) molecules of NOD mice. We used insulinomas transfected with the CIITA transactivator, which resulted in their expression of class II histocompatibility molecules and activation of diabetogenic CD4 T cells. Peptides bound to I-A(g7) were isolated and examined by mass spectrometry: some peptides derived from proteins present in secretory granules of endocrine cells, and a number were shared with cells of neuronal lineage. All proteins to which peptides were identified were expressed in beta cells from normal islets. Peptides bound to I-A(g7) molecules contained the favorable binding motif characterized by acidic amino acids at the P9 position. The draining pancreatic lymph nodes of prediabetic NOD mice contained CD4 T cells that recognized three different natural peptides. Furthermore, four different peptides elicited CD4 T cells, substantiating the presence of such self-reactive T cells. The overall strategy of identifying natural peptides from islet beta-cells opens up new avenues to evaluate the repertoire of self-reactive T cells and its role in onset of diabetes.

Induction of GABAergic Postsynaptic Differentiation by α-Neurexins

Beta-neurexin and neuroligin cell adhesion molecules contribute to synapse development in the brain. The longer alpha-neurexins function at both glutamate and gamma-aminobutyric acid (GABA) synapses in coupling to presynaptic calcium channels. Binding of alpha-neurexins to neuroligins was recently reported, but the role of the alpha-neurexins in synapse development has not been well studied. Here we report that in COS cell neuron coculture assays, all three alpha-neurexins induce clustering of the GABAergic postsynaptic scaffolding protein gephyrin and neuroligin 2 but not of the glutamatergic postsynaptic scaffolding protein PSD-95 or neuroligin 1/3/4. alpha-Neurexins also induce clustering of the GABA(A) receptor gamma2 subunit. This synapse promoting activity of alpha-neurexins is mediated by the sixth LNS (laminin neurexin sex hormone-binding protein) domain and negatively modulated by upstream sequences. Although inserts at splice site 4 (S4) in beta-neurexins promote greater clustering activity for GABA than glutamate proteins in coculture assay, alpha-neurexin-specific sequences confer complete specificity for GABA proteins. We further report a developmental increase in the ratio of -S4 to +S4 forms of neurexins correlating with an increase in glutamate relative to GABA synaptogenesis and activity regulation of splicing at S4. Thus, +S4 beta-neurexins and, even more selectively, alpha-neurexins may be mediators of GABAergic synaptic protein recruitment and stabilization.

Glutamatergic chemical transmission: look! Here, there, and anywhere.

Vesicular glutamate transporter (VGLUT) is responsible for the active transport of L-glutamate in synaptic vesicles and thus plays an essential role in the glutamatergic chemical transmission in the central nervous system. VGLUT comprises three isoforms, VGLUT1, 2, and 3, and is a potential marker for the glutamatergic phenotype. Recent studies indicated that VGLUT is also expressed in non-neuronal cells, and localized with various organelles such as synaptic-like microvesicles in the pineal gland, and hormone-containing secretory granules in endocrine cells. L-Glutamate is stored in these organelles, secreted upon various forms of stimulation, and then acts as a paracrine-like modulator. Thus, VGLUTs highlight a novel framework of glutamatergic signaling and reveal its diverse modes of action.

Disruption of a receptor-mediated mechanism for intracellular sorting of proinsulin in familial hyperproinsulinemia.

In familial hyperproinsulinemia, specific mutations in the proinsulin gene are linked with a profound increase in circulating plasma proinsulin levels. However, the molecular and cellular basis for this disease remains uncharacterized. Here we investigated how these mutations may disrupt the sorting signal required to target proinsulin to the secretory granules of the regulated secretory pathway, resulting in the unregulated release of proinsulin. Using a combination of molecular modeling and site-directed mutagenesis, we have identified structural molecular motifs in proinsulin that are necessary for correct sorting into secretory granules of endocrine cells. We show that membrane carboxypeptidase E (CPE), previously identified as a prohormone-sorting receptor, is essential for proinsulin sorting. This was demonstrated through short interfering RNA-mediated depletion of CPE and transfection with a dominant negative mutant of CPE in a beta-cell line. Mutant proinsulins found in familial hyperproinsulinemia failed to bind to CPE and were not sorted efficiently. These findings provide evidence that the elevation of plasma proinsulin levels found in patients with familial hyperproinsulinemia is caused by the disruption of CPE-mediated sorting of mutant proinsulins to the regulated secretory pathway.

Association of Caldendrin splice isoforms with secretory vesicles in neurohypophyseal axons and the pituitary

Caldendrin is a neuronal calcium-binding protein, which is highly enriched in the postsynaptic density fraction and exhibits a prominent somato-dendritic distribution in brain. Two additional splice variants derive from the caldendrin gene, which have unrelated N-termini and were previously only detected in the retina. We now show that these isoforms are present in neurohypophyseal axons and on secretory granules of endocrine cells. In light of the described interaction of the Caldendrin C-terminus with Q-type Ca v2.1 calcium channels these data suggest that this interaction takes place in neurohypophyseal axons and pituitary cells indicating functions of the short splice variants in triggering Ca 2+ transients to a vesicular target interaction.

Immature Granules Are Not Major Sites for Segregation of Constitutively Secreted Granule Content Proteins in NIT-1 Insulinoma Cells

Immature secretory granules (ISG's) are sites of segregation of proteins destined for secretion by unregulated pathways from those stored in mature secretory granules in endocrine cells. To determine whether significant soluble protein sorting occurs in ISG's, the secretion of soluble versions of the pancreatic protein GP2 (GP2-GPI−) and placental alkaline phosphatase (SEAP) was analyzed in NIT-1 cells. By immunofluorescence microscopy, neither protein localized to SG's in transfected cells. Their secretion was secretagogue-independent in pulse-chase radiolabeling experiments even at early times of chase, while a small increase in the secretion of amylase, which is known to enter ISG's, could be detected. Finally, in sucrose gradient fractionation experiments, SEAP was present in light density fractions. We conclude that while some proteins, such as amylase, have a limited intrinsic capacity to enter ISG's, the segregation of proteins secreted via the constitutive pathway from SG content proteins occurs primarily in the trans Golgi network.

Calpain inhibitor inhibits secretory granule maturation and secretion of GH.

Clathrin- and AP-1-coated buds are present on immature secretory granules of endocrine cells that mature into clathrin-uncoated granules. The mechanism of clathrin and adaptor protein uncoating has remained obscure. Benzyloxycarbonyl-L-leucyl-L-leucinal (ZLLal), a calpain inhibitor, reduced growth hormone (GH) secretion with intracellular accumulation, in a GH-secreting rat pituitary tumor cell. Pulse and chase demonstrated that ZLLal retarded the turnover of clathrin (Clt.H) and adaptins. ZLLal-treatment co-immunoprecipitated the increased amounts of GH with Clt.H and adaptins compared to control cells, suggesting the intracellular accumulation of immature secretory granules. Clt.H and adaptins were limited-proteolyzed by m-calpain in vitro, indicating that calpain may be involved partly in the maturation of secretory granules in endocrine cells via the process of clathrin uncoating.

Processing of chromogranin A in the parathyroid: generation of parastatin-related peptides

Chromogranin A (CgA) is a glycoprotein present in secretory granules of endocrine cells. In the parathyroid, it is costored and cosecreted with parathormone (PTH) in response to hypocalcemia. CgA is the precursor of several bioactive peptides including pancreastatin and betagranin. Parastatin (PARA, pCgA 347–419) is a novel peptide that we generated in vitro by enzymatic digestion of pCgA. In vitro, it inhibits low Ca 2+-stimulated parathyroid secretion. Full activity resides in its first 19 residues. In order to determine if PARA or PARA-derived peptides are natural products of the parathyroid, we generated an antiserum directed against pCgA 347–359 corresponding to the bioactive N-terminal sequence of pPARA (pPARA 1–13 antiserum), and developed a specific radioimmunoassay that we used in conjunction with various chromatographic separations. We identified small peptides carrying the pPARA 1–13 immunoactivity in extracts and secretion medium of porcine parathyroid glands. Continuous and pulse-chase radiolabeling studies, along with immunoprecipitation using PARA 1–13 antiserum demonstrate that a newly-synthesized PARA-related peptide fraction with a Mr of 11 kDa is secreted by the parathyroid cells and accumulates in the secretion medium. Edman degradation of the 11 kDa PARA-related peptide band by Edman degradation yielded three major N-terminal sequences: S-K-M-D-R-L-A-K-E-L—(residues 313–322), D-R-L-A-K-E-L-T-A-E—(residues 316–325), and A-K-E-L-T-A-E-K-R-L—(residues 319–329), in a molar ratio of approximately 1:2:1. The peptide bonds required to be cleaved to yield these peptides, Trp-Ser, Met-Asp and Leu-Ala, suggest that a chymotrypsin-like endopeptidase participated in their formation. The molecular size and the results of amino acid compositional analysis, indicate that the C-termini of these peptides extended variably to residues 384–401 of pCgA. These results demonstrate that processing of CgA by the parathyroid gland generates bioactive PARA-related peptides that could affect the gland’s secretory activity.

Vesicular monoamine transporter 2 as a marker of gastric enterochromaffin-like cell tumors.

The vesicular monoamine transporter 2 (VMAT2) facilitates the ATP-dependent accumulation of biogenic amine inside the secretory granules of endocrine cells and neurons and was demonstrated in the histamine-producing enterochromaffin-like (ECL) cells of the stomach. In the present investigation, VMAT2 immunohistochemistry was tested in 85 endocrine tumors, of which 60 were well differentiated gastrointestinal and pancreatic growths, 5 poorly differentiated (neuro)endocrine carcinomas (PDEC) and 1 mixed PDEC/ECL cell carcinoma of the stomach, 12 pheochromocytomas/paragangliomas, 3 adrenocortical lesions, 2 parathyroid and 2 lung neuroendocrine tumors. Extensive and intense VMAT2 immunoreactivity was observed in 16 of 16 gastric ECL cell tumors, 6 of 6 adrenal pheochromocytomas, 2 of 2 chromaffin paragangliomas and in 3 of the 4 carotid body paragangliomas investigated. Rare VMAT2-positive cells were observed in 12 of 21 intestinal enterochromaffin (EC) cell tumors, in 9 of 11 pancreatic neuroendocrine tumors, and in the mixed PDEC/ ECL cell carcinoma of the stomach (differentiated cells only). No VMAT2 immunoreactivity was observed in five gastrin, four somatostatin and three enteroglucagon/peptideYY tumors of the gastrointestinal tract, in six gastric PDECs, in three adrenocortical growths, and two parathyroid and two lung neuroendocrine tumors. These data support VMAT2 immunohistochemistry as being a useful tool for the diagnosis of gastric ECL cell tumors, separating them from all other endocrine tumors arising in the gastroduodenal area i.e., gastrin, somatostatin, EC cell and PDEC tumors, all of which proved essentially negative.

Immunohistochemical localization of chromogranin A in endocrine tissues and endocrine tumors of dogs.

Chromogranin A is present in the secretory granules of endocrine cells and functions in hormone packaging, secretory granule stabilization, and regulation of hormone secretion. Immunohistochemical identification of chromogranin A can facilitate diagnosis of endocrine neoplasia. Normal and neoplastic canine tissues were stained immunohistochemically for chromogranin A. Staining of normal endocrine tissues demonstrated chromogranin A in chromaffin cells of the adrenal medulla, C cells of the thyroid gland, and pancreatic islets. The parathyroid chief cells and anterior pituitary stained lightly positive for chromogranin A. Pheochromocytomas (7/7), chemodectomas (5/7), islet cell carcinomas (2/6), pituitary adenomas (4/6), parathyroid adenomas (3/7), and a C-cell carcinoma (1/1) stained positive for chromogranin A. The data indicate that chromogranin A is widely distributed in canine endocrine tissues, and immunohistochemical staining of chromogranin A can be used to confirm the presence of secretory granules in endocrine tumors.

A Leucine-based Motif Mediates the Endocytosis of Vesicular Monoamine and Acetylcholine Transporters

Specific transport proteins mediate the packaging of neurotransmitters into secretory vesicles and consequently require targeting to the appropriate intracellular compartment. To identify residues in the neuron-specific vesicular monoamine transporter (VMAT2) responsible for endocytosis, we examined the effect of amino (NH2-) and carboxyl (COOH-)-terminal mutations on steady state distribution and internalization. Deletion of a critical COOH-terminal domain sequence (AKEEKMAIL) results in accumulation of VMAT2 at the plasma membrane and a 50% reduction in endocytosis. Site-directed mutagenesis shows that replacement of the isoleucine-leucine pair within this sequence by alanine-alanine alone reduces endocytosis by 50% relative to wild type VMAT2. Furthermore, the KEEKMAIL sequence functions as an internalization signal when transferred to the plasma membrane protein Tac, and the mutation of the isoleucine-leucine pair also abolishes internalization of this protein. The closely related vesicular acetylcholine transporter (VAChT) contains a similar di-leucine sequence within the cytoplasmic COOH-terminal domain that when mutated results in accumulation of VAChT at the plasma membrane. The VAChT di-leucine sequence also confers internalization when appended to two other proteins and in one of these chimeras, conversion of the di-leucine sequence to di-alanine reduces the internalization rate by 50%. Both VMAT2 and VAChT thus use leucine-based signals for efficient endocytosis and as such are the first synaptic vesicle proteins known to use this motif for trafficking.