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.

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.

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.

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.

Intracellular and in vivo oxygen sensing using phosphorescent Ir(III) complexes with a modified acetylacetonato ligand.

Small luminescent molecular probes based on the iridium(III) complex BTP, (btp)2Ir(acac) (btp = benzothienylpyridine, acac = acetylacetone) have been developed for sensing intracellular and in vivo O2. These compounds are BTPSA (containing an anionic carboxyl group), BTPNH2 (containing a cationic amino group), and BTPDM1 (containing a cationic dimethylamino group); all substituents are incorporated into the ancillary acetylacetonato ligand of BTP. Introduction of the cationic dimethylamino group resulted in an almost 20-fold increase in cellular uptake efficiency of BTPDM1 by HeLa cells compared with BTP. The phosphorescence intensity of BTPDM1 internalized in living cells provided a visual representation of the O2 gradient produced by placing a coverslip over cultured monolayer cells. The intracellular O2 levels (pO2) inside and outside the edge of the coverslip could be evaluated by measuring the phosphorescence lifetime of BTPDM1. Furthermore, intravenous administration of 25 nmol BTPDM1 to tumor-bearing mice allowed the tumor region to be visualized by BTPDM1 phosphorescence. The lifetime of BTPDM1 phosphorescence from tumor regions was much longer than that from extratumor regions, thereby demonstrating tumor hypoxia (pO2 = 6.1 mmHg for tumor and 50 mmHg for extratumor epidermal tissue). Tissue distribution studies showed that 2 h after injection of BTPDM1 into a mouse, the highest distribution was in liver and kidney, while after 24 h, BTPDM1 was excreted in the feces. These results demonstrate that BTPDM1 can be used as a small molecular probe for measuring intracellular O2 levels in both cultured cells and specific tissues and organs.

Silylation improves the photodynamic activity of tetraphenylporphyrin derivatives in vitro and in vivo.

The effects of silyl and hydrophilic groups on the photodynamic properties of tetraphenylporphyrin (TPP) derivatives have been studied in vitro and in vivo. Silylation led to an improvement in the quantum yield of singlet oxygen sensitization for both sulfo and carboxy derivatives, although the silylation did not affect other photophysical properties. Silylation also improved the cellular uptake efficiency for both sulfo and carboxy derivatives, enhancing the in vitro photodynamic activity of the photosensitizer in U251 human glioma cells. The carboxy derivative (SiTPPC4 ) was found to show higher cellular uptake efficiency and in vitro photodynamic activity than the corresponding sulfo derivative (SiTPPS4 ), which indicates that the carboxy group is a more promising hydrophilic group than the sulfo group in the silylated porphyrin. SiTPPC4 was found to show high selective accumulation efficiency in tumors, although almost no tumor selectivity was observed for the nonsilylated porphyrin. The concentration of SiTPPC4 in tumors was 13 times higher than that in muscle 12 h after drug administration. We also studied tumor response after treatment and found that silylation enhanced in vivo photodynamic activity significantly. SiTPPC4 shows higher photodynamic activity than NPe6 with white light irradiation.

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.

Luminal interaction of phogrin with carboxypeptidase E for effective targeting to secretory granules.

Phogrin, a receptor tyrosine phosphatase-like protein, is localized to dense-core secretory granules (SGs) in various neuroendocrine cells. A previous report showed that the N-terminal luminal domain mediates targeting of this protein to SGs in AtT-20 cells. Here, we show that the luminal domain specifically interacts with carboxypeptidase E (CPE), one of the key proteins involved in peptide hormone sorting, in a weakly acidic condition. The luminal domain consists of pro-sequence domain (pro) and subsequent N-side mature domain and the pro domain was preferentially required for phogrin interaction with CPE and for its targeting to SGs. Small interfering RNA-directed reduction of the CPE protein level resulted in an improper accumulation of phogrin at the trans-Golgi network in AtT-20 cells. This finding indicates that CPE is involved in the sorting process of phogrin to SGs. However, SG localization of CPE was hindered by overexpression of the phogrin mutants that lack the transport motif of binding to clathrin adaptor complexes. Phogrin-depleted AtT-20 cells also exhibited reduced CPE targeting and increased CPE degradation. Our results suggest that the luminal interaction between phogrin and CPE contributes to their targeting to SGs in a cooperative manner in neuroendocrine cells.

Chronic exercise enhances insulin secretion ability of pancreatic islets without change in insulin content in non-diabetic rats.

We evaluated the effect of chronic exercise on insulin secretion in response to high-glucose by using a perifusion method with isolated pancreatic islets from normal rats. Male Wistar rats were assigned to one of two groups: a sedentary group and a trained group. Running exercise was carried out on a treadmill for one hour per day, five days per week, for six, nine, or 12 weeks. The chronic exercise significantly enhanced the insulin secretion ability of pancreatic islets in response to the high-glucose stimulation upon nine and 12 weeks of exercise. The insulin content in the pancreas and the weight of the pancreas did not change upon nine weeks of exercise. Potassium-stimulated insulin secretion was also increased in the islets isolated from rats that trained for nine weeks compared with that in sedentary rats, suggesting that insulin secretion events downstream of membrane depolarization are involved in targets of the exercise effect. These findings suggest that chronic exercise could be a useful strategy not only for the maintenance of peripheral insulin sensitivity but also for the promotion of islet function to secrete insulin in non-diabetics.

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.

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.

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.

Author Correction: Emergence of unprecedented climate change in projected future precipitation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Emergence of unprecedented climate change in projected future precipitation.

The future time of emergence when precipitation changes due to anthropogenic influences begins to continuously exceed the previous maximum value is defined as the 'tipping year' Historical experiments and future experiments simulated by state-of-the-art climate models were utilized. A total of 510,000 time series from year 1856 to 2095 were generated by sampling the natural internal variability in precipitation. The time evolutions of internal variability in the whole time period were estimated from the combination of past and future experiments with preindustrial control experiments. A large ensemble size enabled an estimation of the probability density function of the tipping year at each grid point, providing precise information on the uncertainty of the projection. The tipping year of average precipitation emerges earlier in high latitudes than in lower latitudes. In some regions in lower latitudes and mid-latitudes, the tipping year of intense precipitation emerges faster than that of average precipitation. The tipping years of average and intense precipitation are earlier for higher anthropogenic forcing scenarios than for lower scenarios. The global average of the tipping year for intense precipitation might be attributed to the enhancement of the thermodynamic effect (moisture) rather than the dynamic effect (vertical motion).

A large form of secretogranin III functions as a sorting receptor for chromogranin A aggregates in PC12 cells.

Granin-family proteins, including chromogranin A and secretogranin III, are sorted to the secretory granules in neuroendocrine cells. We previously demonstrated that secretogranin III binds chromogranin A and targets it to the secretory granules in pituitary corticotrope-derived AtT-20 cells. However, secretogranin III has not been identified in adrenal chromaffin and PC12 cells, where chromogranin A is correctly sorted to the secretory granules. In this study, low levels of a large and noncleaved secretogranin III have been identified in PC12 cells and rat adrenal glands. Although the secretogranin III expression was limited in PC12 cells, when the FLAG-tagged secretogranin III lacking the secretory granule membrane-binding domain was expressed excessively, hemagglutinin-tagged chromogranin A was unable to target to the secretory granules at the tips and shifted to the constitutive secretory pathway. Secretogranin III was able to bind the aggregated form of chromogranin A, suggesting that a small quantity of secretogranin III is enough to carry a large quantity of chromogranin A. Furthermore, secretogranin III bound adrenomedullin, a major peptide hormone in chromaffin cells. Indeed, small interfering RNA-directed secretogranin III depletion impaired intracellular retention of chromogranin A and adrenomedullin, suggesting that they are constitutively released to the medium. We suggest that the sorting function of secretogranin III for chromogranin A is common in PC12 and chromaffin cells as well as in other endocrine cells, and a small amount of secretogranin III is able to sort chromogranin A aggregates together with adrenomedullin to secretory granules.

Reactive oxygen species-mediated pancreatic beta-cell death is regulated by interactions between stress-activated protein kinases, p38 and c-Jun N-terminal kinase, and mitogen-activated protein kinase phosphatases.

Pancreatic beta-cells are susceptible to reactive oxygen species (ROS), which are known to be generated by high or low glucose (LG), hypoxic, or cytokine-producing conditions. When we cultured mouse beta-cell-derived MIN6 cells in a LG condition, we detected a significant generation of ROS, including hydrogen peroxide, which was comparable to the ROS production in hypoxic or cytokine-treated conditions. ROS accumulation induced by the LG culture led to cell death, which was prevented by the ROS scavengers N-acetylcysteine and manganese(III)tetrakis(4-benzoic acid) porphyrin. We next investigated the mechanism of stress-activated protein kinases (SAPKs), c-jun N-terminal kinase (JNK) and p38, in ROS-induced MIN6 cell death. Activation of p38 occurred immediately after the LG culture, whereas JNK activation increased slowly 8 h later. Adenoviral p38 expression decreased MIN6 cell death, whereas the JNK expression increased it. Consistently, blocking p38 activation by inhibitors increased beta-cell death, whereas JNK inhibitors decreased it. We then examined the role of MAPK phosphatases (MKPs) specific for stress-activated protein kinases in beta-cell death. We found that MKP-1 presented an increase in its oxidized product after the LG culture. ROS scavengers prevented the appearance of this oxidized product and JNK activation. Thus, ROS-induced MKP inactivation causes sustained activation of JNK, which contributes to beta-cell death. Adenoviral overexpression of MKP-1 and MKP-7 prevented the phosphorylation of JNK at 36 h after the LG culture, and decreased MIN6 beta-cell death. We suggest that beta-cell death is regulated by interactions between JNK and its specific MKPs.

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.

Molecular probes for sensing the cholesterol composition of subcellular organelle membranes.

Neuroendocrine cells contain two types of secretagogue-regulated acidic compartments: secretory granules (SGs) and synaptic-like microvesicles (SLMVs), which can be identified by acidotropic probes such as acridine orange (AO) and DAMP. We investigated the accumulation of these probes in SGs and SLMVs as a function of glucose levels in the culture media using a pancreatic beta-cell line MIN6. AO was accumulated in the low-glucose condition, but not in the high-glucose condition. The AO accumulation correlated well with the SLMV dynamics by glucose and DAMP was localized in the SGs. Because SG membranes are reportedly high in cholesterol, we prepared liposomes with increasing cholesterol levels. AO is well incorporated into liposomes having a 20 to 40 mol% cholesterol composition, whereas DAMP was so in those having over 40 mol% cholesterol levels. Indeed, when cholesterol was depleted from MIN6 SG membranes, DAMP incorporation decreased, instead AO was incorporated. In PC12 cells, AO incorporation into SGs was significant but DAMP incorporation was limited. Consistently, the cholesterol composition was found 37 to 39 mol% in the SG membrane of PC12 cells. We suggest that cholesterol-sensing probes, AO and DAMP, are useful tools for investigating cholesterol compositions in acidic organelle membranes.

Higher plant RecA-like protein is homologous to RadA.

A novel RecA-like protein, differing from Dmc1 and Rad51, was characterized in Oryza sativa L. cv. Nipponbare. Because the protein is homologous to bacterial RadA, the gene was designated OsRadA. The open reading frame was predicted to encode a 66kDa protein of 619 amino acid residues and was found in plants but not animals or yeast. OsRadA showed D-loop and single-stranded DNA-dependent ATPase activities. Gene expression was found to be high in meristematic tissues, and was localized in the nucleus. An RNAi mutant of Arabidopsis thaliana RadA (AtRadA) was sensitive to mutagenic agents such as UV and MMC, suggesting that RadA functions in DNA repair.

A subset of p23 localized on secretory granules in pancreatic beta-cells.

Proteins on the membrane of secretory granules (SGs) involved in their biogenesis and exocytosis are poorly characterized compared with those of synaptic vesicle in neurons. Thus the secretory granule membrane was prepared from a mouse pancreatic beta-cell line MIN6 by subcellular fractionation, and protein constituents were analyzed by microscale two-dimensional liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Using this proteomics approach, one of the p24 family proteins, p23, was unexpectedly found in the granule fraction, although p24 proteins are generally regarded as functioning in the early secretory pathways between the endoplasmic reticulum and the Golgi apparatus. We further showed that p23 is expressed at high levels in endocrine cells. Furthermore, immunocytochemical analyses of pancreatic beta-cells at the light and electron microscopic levels demonstrated that a significant amount of p23 is localized on the insulin granule membrane, although it is most intensely concentrated at the cis-Golgi compartment as previously shown in non-endocrine cells. These findings suggest that a fraction of p23 enters post-Golgi compartments and may function in the biogenesis and/or quality control of SGs.