Phogrin Regulates High-Fat Diet-Induced Compensatory Pancreatic ß-Cell Growth by Switching Binding Partners.

The receptor protein tyrosine phosphatase phogrin primarily localizes to hormone secretory granules in neuroendocrine cells. Concurrent with glucose-stimulated insulin secretion, phogrin translocates to pancreatic ß-cell plasma membranes, where it interacts with insulin receptors (IRs) to stabilize insulin receptor substrate 2 (IRS2) that, in turn, contributes to glucose-responsive ß-cell growth. Pancreatic ß-cell development was not altered in ß-cell-specific, phogrin-deficient mice, but the thymidine incorporation rate decreased in phogrin-deficient islets with a moderate reduction in IRS2 protein expression. In this study, we analyzed the ß-cell response to high-fat diet stress and found that the compensatory expansion in ß-cell mass was significantly suppressed in phogrin-deficient mice. Phogrin-IR interactions occurred only in high-fat diet murine islets and proliferating ß-cell lines, whereas they were inhibited by the intercellular binding of surface phogrin under confluent cell culture conditions. Thus, phogrin could regulate glucose-stimulated compensatory ß-cell growth by changing its binding partner from another ß-cell phogrin to IR in the same ß-cells.

Insulin granule morphology and crinosome formation in mice lacking the pancreatic ß cell-specific phogrin (PTPRN2) gene.

We recently reported that phogrin, also known as IA-2ß or PTPRN2, forms a complex with the insulin receptor in pancreatic ß cells upon glucose stimulation and stabilizes insulin receptor substrate 2. In ß cells of systemic phogrin gene knockout (IA-2ß-/-) mice, impaired glucose-induced insulin secretion, decreased insulin granule density, and an increase in the number and size of lysosomes have been reported. Since phogrin is expressed not only in ß cells but also in various neuroendocrine cells, the precise impact of phogrin expressed in ß cells on these cells remains unclear. In this study, we performed a comprehensive analysis of morphological changes in RIP-Cre+/-Phogrinflox/flox (ßKO) mice with ß cell-specific phogrin gene knockout. Compared to control RIP-Cre+/- Phogrin+/+ (Ctrl) mice, aged ßKO mice exhibited a decreased density of insulin granules, which can be categorized into three subtypes. While no differences were observed in the density and size of lysosomes and crinosomes, organelles involved in insulin granule reduction, significant alterations in the regions of lysosomes responding positively to carbohydrate labeling were evident in young ßKO mice. These alterations differed from those in Ctrl mice and continued to change with age. These electron microscopic findings suggest that phogrin expression in pancreatic ß cells plays a role in insulin granule homeostasis and crinophagy during aging, potentially through insulin autocrine signaling and other mechanisms.

2,2,6,6-Tetramethylpiperidine-1-oxyl acts as a volatile inhibitor of ferroptosis and neurological injury.

Ferroptosis, a type of oxidative stress cell death, has been implicated in cell injury in several diseases, and treatments with specific inhibitors have been shown to protect cells and tissues. Here we demonstrated that a treatment with the nitroxide radical, 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), prevented the ferroptotic cell death in an airborne manner. Other TEMPO derivatives and lipophilic antioxidants, such as Trolox and ferrostatin-1, also prevented cell death induced by erastin and RSL3; however, only TEMPO exhibited inhibitory activity from a physically distant location. TEMPO vaporized without decomposing and then dissolved again into a nearby water solution. Volatilized TEMPO inhibited glutamate-induced cell death in mouse hippocampal cell lines and also reduced neuronal cell death in a mouse ischemia model. These results suggest that TEMPO is a unique cell protective agent that acts in a volatility-mediated manner.

Differential Expression of Secretogranins II and III in Canine Adrenal Chromaffin Cells and Pheochromocytomas.

Secretogranin II (SgII) and III (SgIII) function within peptide hormone-producing cells and are involved in secretory granule formation. However, their function in active amine-producing cells is not fully understood. In this study, we analyzed the expression profiles of SgII and SgIII in canine adrenal medulla and pheochromocytomas by immunohistochemical staining. In normal adrenal tissues, the intensity of coexpression of these two secretogranins (Sgs) differed from each chromaffin cell, although a complete match was not observed. The coexpression of vesicular monoamine transporter 2 (VMAT2) with SgIII was similar to that with chromogranin A, but there was a subpopulation of VMAT2-expressing cells that were negative or hardly detectable for SgII. These results are the first to indicate that there are distinct expression patterns for SgII and SgIII in adrenal chromaffin cells. Furthermore, the expression of these two Sgs varied in intensity among pheochromocytomas and did not necessarily correlate with clinical plasma catecholamine levels in patients. However, compared with SgIII, the expression of SgII was shown to be strong at the single-cell level in some tumor tissues. These findings provide a fundamental understanding of the expression differences between SgII and SgIII in normal adrenal chromaffin cells and pheochromocytomas.

MiR-7-5p Is Involved in Ferroptosis Signaling and Radioresistance Thru the Generation of ROS in Radioresistant HeLa and SAS Cell Lines.

In cancer therapy, radioresistance or chemoresistance cells are major problems. We established clinically relevant radioresistant (CRR) cells that can survive over 30 days after 2 Gy/day X-ray exposures. These cells also show resistance to anticancer agents and hydrogen peroxide (H2O2). We have previously demonstrated that all the CRR cells examined had up-regulated miR-7-5p and after miR-7-5p knockdown, they lost radioresistance. However, the mechanism of losing radioresistance remains to be elucidated. Therefore, we investigated the role of miR-7-5p in radioresistance by knockdown of miR-7-5p using CRR cells. As a result, knockdown of miR-7-5p increased reactive oxygen species (ROS), mitochondrial membrane potential, and intracellular Fe2+ amount. Furthermore, miR-7-5p knockdown results in the down-regulation of the iron storage gene expression such as ferritin, up-regulation of the ferroptosis marker ALOX12 gene expression, and increases of Liperfluo amount. H2O2 treatment after ALOX12 overexpression led to the enhancement of intracellular H2O2 amount and lipid peroxidation. By contrast, miR-7-5p knockdown seemed not to be involved in COX-2 and glycolysis signaling but affected the morphology of CRR cells. These results indicate that miR-7-5p control radioresistance via ROS generation that leads to ferroptosis.

Expression Pattern of the LacZ Reporter in Secretogranin III Gene-trapped Mice.

Secretogranin III (SgIII) is a granin protein involved in secretory granule formation in peptide-hormone-producing endocrine cells. In this study, we analyzed the expression of the LacZ reporter in the SgIII knockout mice produced by gene trapping (SgIII-gtKO) for the purpose of comprehensively clarifying the expression patterns of SgIII at the cell and tissue levels. In the endocrine tissues of SgIII-gtKO mice, LacZ expression was observed in the pituitary gland, adrenal medulla, and pancreatic islets, where SgIII expression has been canonically revealed. LacZ expression was extensively observed in brain regions, especially in the cerebral cortex, hippocampus, hypothalamic nuclei, cerebellum, and spinal cord. In peripheral nervous tissues, LacZ expression was observed in the retina, optic nerve, and trigeminal ganglion. LacZ expression was particularly prominent in astrocytes, in addition to neurons and ependymal cells. In the cerebellum, at least four cell types expressed SgIII under basal conditions. The expression of SgIII in the glioma cell lines C6 and RGC-6 was enhanced by excitatory glutamate treatment. It also became clear that the expression level of SgIII varied among neuron and astrocyte subtypes. These results suggest that SgIII is involved in glial cell function, in addition to neuroendocrine functions, in the nervous system.

Botanical sulfane sulfur donors inhibit ferroptotic cell death caused by the depletion of cysteine.

Inhibitors against cystine-glutamate antiporter, including erastin, elicit ferroptotic cell death. The erastin-induced ferroptotic cell death appears to be caused by cysteine as well as glutathione depletion. Cysteine is an essential substrate for sulfane sulfur producing systems in cells, generating persulfides that function as intracellular antioxidants and intermediates in iron-sulfur cluster production. Therefore, we examined whether botanical sulfane sulfur donors such as diallyl trisulfide (DATS) and dimethyl trisulfide (DMTS) prevent ferroptotic cell death in HT1080 cells treated with erastin. As a result, DMTS (20 µM) and DATS (10 µM) rescued the erastin-treated HT1080 cells by 69.6% and 91.6%, respectively. Furthermore, DMTS-containing squeeze of cabbage (2.0 g/L) and DATS-containing squeeze of garlic (0.07 g/L) rescued the erastin-treated HT1080 cells by 76.5% and almost 100%, respectively. In conclusion, the ingestion of trisulfides may bring about increased resistance to ferroptotic cell death in vivo.

Antipsychotic olanzapine-induced misfolding of proinsulin in the endoplasmic reticulum accounts for atypical development of diabetes.

Second-generation antipsychotics are widely used to medicate patients with schizophrenia, but may cause metabolic side effects such as diabetes, which has been considered to result from obesity-associated insulin resistance. Olanzapine is particularly well known for this effect. However, clinical studies have suggested that olanzapine-induced hyperglycemia in certain patients cannot be explained by such a generalized mechanism. Here, we focused on the effects of olanzapine on insulin biosynthesis and secretion by mouse insulinoma MIN6 cells. Olanzapine reduced maturation of proinsulin, and thereby inhibited secretion of insulin; and specifically shifted the primary localization of proinsulin from insulin granules to the endoplasmic reticulum. This was due to olanzapine's impairment of proper disulfide bond formation in proinsulin, although direct targets of olanzapine remain undetermined. Olanzapine-induced proinsulin misfolding and subsequent decrease also occurred at the mouse level. This mechanism of olanzapine-induced ß-cell dysfunction should be considered, together with weight gain, when patients are administered olanzapine.

Culture in 10% O2 enhances the production of active hormones in neuro-endocrine cells by up-regulating the expression of processing enzymes.

To closely mimic physiological conditions, low oxygen cultures have been employed in stem cell and cancer research. Although in vivo oxygen concentrations in tissues are often much lower than ambient 21% O2 (ranging from 3.6 to 12.8% O2), most cell cultures are maintained at 21% O2 To clarify the effects of the O2 culture concentration on the regulated secretion of peptide hormones in neuro-endocrine cells, we examined the changes in the storage and release of peptide hormones in neuro-endocrine cell lines and endocrine tissues cultured in a relatively lower O2 concentration. In both AtT-20 cells derived from the mouse anterior pituitary and freshly prepared mouse pituitaries cultured in 10% O2 for 24 h, the storage and regulated secretion of the mature peptide hormone adrenocorticotropic hormone were significantly increased compared with those in cells and pituitaries cultured in ambient 21% O2, whereas its precursor proopiomelanocortin was not increased in the cells and tissues after being cultured in 10% O2 Simultaneously, the prohormone-processing enzymes PC1/3 and carboxypeptidase E were up-regulated in cells cultured in 10% O2, thus facilitating the conversion of prohormones to their active form. Similarly, culturing the mouse ß-cell line MIN6 and islet tissue in 10% O2 also significantly increased the conversion of proinsulin into mature insulin, which was secreted in a regulated manner. These results suggest that culture under 10% O2 is more optimal for endocrine tissues/cells to efficiently generate and secrete active peptide hormones than ambient 21% O2.

VAMP7 Regulates Autophagosome Formation by Supporting Atg9a Functions in Pancreatic ß-Cells From Male Mice.

Dysfunctional mitochondria are observed in ß-cells of diabetic patients, which are eventually removed by autophagy. Vesicle-associated membrane protein (VAMP)7, a vesicular SNARE protein, regulates autophagosome formation to maintain mitochondrial homeostasis and control insulin secretion in pancreatic ß-cells. However, its molecular mechanism is largely unknown. In this study, we investigated the molecular mechanism of VAMP7-dependent autophagosome formation using VAMP7-deficient ß-cells and ß-cell-derived Min6 cells. VAMP7 localized in autophagy-related (Atg)9a-resident vesicles of recycling endosomes (REs), which contributed to autophagosome formation, and it interacted with Hrb, Syntaxin16, and SNAP-47. Hrb recruited VAMP7 and Atg9a from the plasma membrane to REs. Syntaxin16 and SNAP-47 mediated autophagosome formation at a step later than the proper localization of VAMP7 to Atg9a-resident vesicles. Knockdown of Hrb, Syntaxin16, and SNAP-47 resulted in defective autophagosome formation, accumulation of dysfunctional mitochondria, and impairment of glucose-stimulated insulin secretion. Our data indicate that VAMP7 and Atg9a are initially recruited to REs to organize VAMP7 and Atg9a-resident vesicles in an Hrb-dependent manner. Additionally, VAMP7 forms a SNARE complex with Syntaxin16 and SNAP-47, which may cause fusions of Atg9a-resident vesicles during autophagosome formation. Thus, VAMP7 participates in autophagosome formation by supporting Atg9a functions that contribute to maintenance of mitochondrial quality.

The pseudophosphatase phogrin enables glucose-stimulated insulin signaling in pancreatic ß cells.

Autocrine insulin signaling is critical for pancreatic ß-cell growth and activity and is at least partially controlled by protein-tyrosine phosphatases (PTPs) that act on insulin receptors (IRs). The receptor-type PTP phogrin primarily localizes on insulin secretory granules in pancreatic ß cells. We recently reported that phogrin knockdown decreases the protein levels of insulin receptor substrate 2 (IRS2), whereas high-glucose stimulation promotes formation of a phogrin-IR complex that stabilizes IRS2. However, the underlying molecular mechanisms by which phogrin affects IRS2 levels are unclear. Here, we found that relative to wildtype mice, IRS2 levels in phogrin-knockout mice islets decreased by 44%. When phogrin was silenced by shRNA in pancreatic ß-cell lines, glucose-induced insulin signaling led to proteasomal degradation of IRS2 via a negative feedback mechanism. Phogrin overexpression in a murine hepatocyte cell line consistently prevented chronic insulin treatment-induced IRS2 degradation. In vitro, phogrin directly bound the IR without the assistance of other proteins and protected recombinant PTP1B from oxidation to potentiate its activity toward the IR. Furthermore, phogrin expression suppressed insulin-induced local generation of hydrogen peroxide and subsequent PTP1B oxidation, which allowed progression of IR dephosphorylation. Together, these results suggest that a transient interaction of phogrin with the IR enables glucose-stimulated autocrine insulin signaling through the regulation of PTP1B activity, which is essential for suppressing feedback-mediated IRS2 degradation in pancreatic ß cells.

Impaired Processing of Prohormones in Secretogranin III-Null Mice Causes Maladaptation to an Inadequate Diet and Stress.

Secretogranin III (SgIII), a member of the granin family, binds both to another granin, chromogranin A (CgA), and to a cholesterol-rich membrane that is destined for secretory granules (SGs). The knockdown of SgIII in adrenocorticotropic hormone (ACTH)-producing AtT-20 cells largely impairs the regulated secretion of CgA and ACTH. To clarify the physiological roles of SgIII in vivo, we analyzed hormone secretion and SG biogenesis in newly established SgIII-knockout (KO) mice. Although the SgIII-KO mice were viable and fertile and exhibited no overt abnormalities under ordinary rearing conditions, a high-fat/high-sucrose diet caused pronounced obesity in the mice. Furthermore, in the SgIII-KO mice compared with wild-type (WT) mice, the stimulated secretion of active insulin decreased substantially, whereas the storage of proinsulin increased in the islets. The plasma ACTH was also less elevated in the SgIII-KO mice than in the WT mice after chronic restraint stress, whereas the storage level of the precursor proopiomelanocortin in the pituitary gland was somewhat increased. These findings suggest that the lack of SgIII causes maladaptation of endocrine cells to an inadequate diet and stress by impairing the proteolytic conversion of prohormones in SGs, whereas SG biogenesis and the basal secretion of peptide hormones under ordinary conditions are ensured by the compensatory upregulation of other residual granins or factors.

Canine Salivary Glands: Analysis of Rab and SNARE Protein Expression and SNARE Complex Formation With Diverse Tissue Properties.

The comparative structure and expression of salivary components and vesicular transport proteins in the canine major salivary glands were investigated. Histochemical analysis revealed that the morphology of the five major salivary glands-parotid, submandibular, polystomatic sublingual, monostomatic sublingual, and zygomatic glands-was greatly diverse. Immunoblot analysis revealed that expression levels of α-amylase and antimicrobial proteins, such as lysozyme, lactoperoxidase, and lactoferrin, differed among the different glands. Similarly, Rab proteins (Rab3d, Rab11a, Rab11b, Rab27a, and Rab27b) and soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins VAMP4, VAMP8, syntaxin-2, syntaxin-3, syntaxin-4, and syntaxin-6 were expressed at various levels in individual glands. mmunohistochemistry of Rab3d, Rab11b, Rab27b, VAMP4, VAMP8, syntaxin-4, and syntaxin-6 revealed their predominant expression in serous acinar cells, demilunes, and ductal cells. The VAMP4/syntaxin-6 SNARE complex, which is thought to be involved in the maturation of secretory granules in the Golgi field, was found more predominantly in the monostomatic sublingual gland than in the parotid gland. These results suggest that protein expression profiles in canine salivary glands differ among individual glands and reflect the properties of their specialized functions.

Lipoxygenase-mediated generation of lipid peroxides enhances ferroptosis induced by erastin and RSL3.

In cancer cells the small compounds erastin and RSL3 promote a novel type of cell death called ferroptosis, which requires iron-dependent accumulation of lipid reactive oxygen species. Here we assessed the contribution of lipid peroxidation activity of lipoxygenases (LOX) to ferroptosis in oncogenic Ras-expressing cancer cells. Several 12/15-LOX inhibitors prevented cell death induced by erastin and RSL3. Furthermore, siRNA-mediated silencing of ALOX15 significantly decreased both erastin-induced and RSL3-induced ferroptotic cell death, whereas exogenous overexpression of ALOX15 enhanced the effect of these compounds. Immunofluorescence analyses revealed that the ALOX15 protein consistently localizes to cell membrane during the course of ferroptosis. Importantly, treatments of cells with ALOX15-activating compounds accelerated cell death at low, but not high doses of erastin and RSL3. These observations suggest that tumor ferroptosis is promoted by LOX-catalyzed lipid hydroperoxide generation in cellular membranes.

ATAT1 is essential for regulation of homeostasis-retaining cellular responses in corticotrophs along hypothalamic-pituitary-adrenal axis.

The production and secretion of adrenocorticotropin, a proopiomelanocortin (POMC)-derived hormone, by corticotrophs in the anterior pituitary, is regulated by corticotrophin-releasing hormone (CRH) and glucocorticoids. We have previously demonstrated that adrenalectomy induces α-tubulin N-acetyltransferase 1 (ATAT1) expression and α-tubulin acetylation in corticotrophs. However, the regulatory mechanism of ATAT1 expression and the function of acetylated microtubules in corticotrophs are unclear. Here, we analyze the effect of CRH or dexamethasone on Atat1 expression in the mouse corticotroph AtT20 cell line. The expression of Atat1 was increased by CRH and decreased by dexamethasone in AtT20 cells. We examined the effect of Atat1 knockdown on the expression of POMC-associated genes and the dexamethasone-induced nuclear translocation of glucocorticoid receptor (GR) by real-time polymerase chain reaction and Western blot analysis, respectively. Atat1 knockdown resulted in a significant increase in the expression of ACTH-producing genes and decreased the dexamethasone-induced nuclear translocation of GR accompanied with a reduction in α-tubulin acetylation. Atat1 overexpression resulted in a significant increase in α-tubulin acetylation and the dexamethasone-induced nuclear translocation of GR. These results suggest that the acetylated microtubules function as the rail-line for the transportation of GR into the nucleus. We conclude that ATAT1 finely tunes the cellular responses of corticotrophs to hormonal stimulation through an intracellular feedback circuit.

VAMP7 Regulates Autophagy to Maintain Mitochondrial Homeostasis and to Control Insulin Secretion in Pancreatic ß-Cells.

VAMP7 is a SNARE protein that mediates specific membrane fusions in intracellular trafficking and was recently reported to regulate autophagosome formation. However, its function in pancreatic ß-cells is largely unknown. To elucidate the physiological role of VAMP7 in ß-cells, we generated pancreatic ß-cell-specific VAMP7 knockout (Vamp7(flox/Y);Cre) mice. VAMP7 deletion impaired glucose-stimulated ATP production and insulin secretion, though VAMP7 was not localized to insulin granules. VAMP7-deficient ß-cells showed defective autophagosome formation and reduced mitochondrial function. p62/SQSTM1, a marker protein for defective autophagy, was selectively accumulated on mitochondria in VAMP7-deficient ß-cells. These findings suggest that accumulation of dysfunctional mitochondria that are degraded by autophagy caused impairment of glucose-stimulated ATP production and insulin secretion in Vamp7(flox/Y);Cre ß-cells. Feeding a high-fat diet to Vamp7(flox/Y);Cre mice exacerbated mitochondrial dysfunction, further decreased ATP production and insulin secretion, and consequently induced glucose intolerance. Moreover, we found upregulated VAMP7 expression in wild-type mice fed a high-fat diet and in db/db mice, a model for diabetes. Thus our data indicate that VAMP7 regulates autophagy to maintain mitochondrial quality and insulin secretion in response to pathological stress in ß-cells.

An essential role for functional lysosomes in ferroptosis of cancer cells.

Pharmacological challenges to oncogenic Ras-expressing cancer cells have shown a novel type of cell death, ferroptosis, which requires intracellular iron. In the present study, we assessed ferroptosis following treatment of human fibrosarcoma HT1080 cells with several inhibitors of lysosomal activity and found that they prevented cell death induced by the ferroptosis-inducing compounds erastin and RSL3. Fluorescent analyses with a reactive oxygen species (ROS) sensor revealed constitutive generation of ROS in lysosomes, and treatment with lysosome inhibitors decreased both lysosomal ROS and a ferroptotic cell-death-associated ROS burst. These inhibitors partially prevented intracellular iron provision by attenuating intracellular transport of transferrin or autophagic degradation of ferritin. Furthermore, analyses with a fluorescent sensor that detects oxidative changes in cell membranes revealed that formation of lipid ROS in perinuclear compartments probably represented an early event in ferroptosis. These results suggest that lysosomal activity is involved in lipid ROS-mediated ferroptotic cell death through regulation of cellular iron equilibria and ROS generation.

Expression of Secretogranin III in Chicken Endocrine Cells: Its Relevance to the Secretory Granule Properties of Peptide Prohormone Processing and Bioactive Amine Content.

The expression of secretogranin III (SgIII) in chicken endocrine cells has not been investigated. There is limited data available for the immunohistochemical localization of SgIII in the brain, pituitary, and pancreatic islets of humans and rodents. In the present study, we used immunoblotting to reveal the similarities between the expression patterns of SgIII in the common endocrine glands of chickens and rats. The protein-protein interactions between SgIII and chromogranin A (CgA) mediate the sorting of CgA/prohormone core aggregates to the secretory granule membrane. We examined these interactions using co-immunoprecipitation in chicken endocrine tissues. Using immunohistochemistry, we also examined the expression of SgIII in a wide range of chicken endocrine glands and gastrointestinal endocrine cells (GECs). SgIII was expressed in the pituitary, pineal, adrenal (medullary parts), parathyroid, and ultimobranchial glands, but not in the thyroid gland. It was also expressed in GECs of the stomach (proventriculus and gizzard), small and large intestines, and pancreatic islet cells. These SgIII-expressing cells co-expressed serotonin, somatostatin, gastric inhibitory polypeptide, glucagon-like peptide-1, glucagon, or insulin. These results suggest that SgIII is expressed in the endocrine cells that secrete peptide hormones, which mature via the intragranular enzymatic processing of prohormones and physiologically active amines in chickens.

Immunohistochemical analysis of IA-2 family of protein tyrosine phosphatases in rat gastrointestinal endocrine cells.

Islet-associated protein-2 (IA-2) and IA-2ß (also known as phogrin) are unique neuroendocrine-specific protein tyrosine phosphatases (PTPs). The IA-2 family of PTPs was originally identified from insulinoma cells and discovered to be major autoantigens in type 1 diabetes. Despite its expression in the neural and canonical endocrine tissues, data on expression of the IA-2 family of PTPs in gastrointestinal endocrine cells (GECs) are limited. Therefore, we immunohistochemically investigated the expression of the IA-2 family of PTPs in the rat gastrointestinal tract. In the stomach, IA-2 and IA-2ß were expressed in GECs that secrete serotonin, somatostatin, and cholecystokinin/gastrin-1. In addition to these hormones, secretin, gastric inhibitory polypeptide (also known as the glucose-dependent insulinotropic peptide), glucagon-like peptide-1, and glucagon, but not ghrelin were coexpressed with IA-2 or IA-2ß in duodenal GECs. Pancreatic islet cells that secrete gut hormones expressed the IA-2 family of PTPs. The expression patterns of IA-2 and IA-2ß were comparable. These results reveal that the IA-2 family of PTPs is expressed in a cell type-specific manner in rat GECs. The extensive expression of the IA-2 family of PTPs in pancreo-gastrointestinal endocrine cells and in the enteric plexus suggests their systemic contribution to nutritional control through a neuroendocrine signaling network.

Multiple sorting systems for secretory granules ensure the regulated secretion of peptide hormones.

Prior to secretion, regulated peptide hormones are selectively sorted to secretory granules (SGs) at the trans-Golgi network (TGN) in endocrine cells. Secretogranin III (SgIII) appears to facilitate SG sorting process by tethering of protein aggregates containing chromogranin A (CgA) and peptide hormones to the cholesterol-rich SG membrane (SGM). Here, we evaluated the role of SgIII in SG sorting in AtT-20 cells transfected with small interfering RNA targeting SgIII. In the SgIII-knockdown cells, the intracellular retention of CgA was greatly impaired, and only a trace amount of CgA was localized within the vacuoles formed in the TGN, confirming the significance of SgIII in both the tethering of CgA-containing aggregates and the establishment of the proper SG morphology. Although the intracellular retention of proopiomelanocortin (POMC) was considerably impaired in SgIII-knockdown cells, residual adrenocorticotropic hormone (ACTH)/POMC was still localized to some few remaining SGs together with another granin protein, secretogranin II (SgII), and was secreted in a regulated manner. Biochemical analyses indicated that SgII bound directly to the SGM in a cholesterol-dependent manner and was able to retain the aggregated form of POMC, revealing a latent redundancy in the SG sorting and retention mechanisms, that ensures the regulated secretion of bioactive peptides.