Language：English   Publishing type：Research paper (scientific journal)  

Patients with permanent hypoparathyroidism require lifelong replacement therapy to avoid life-threatening complications, The benefits of conventional treatment are limited, however. Transplanting a functional parathyroid gland (PTG) would yield better results. Parathyroid gland cells generated from pluripotent stem cells in vitro to date cannot mimic the physiological responses to extracellular calcium that are essential for calcium homeostasis. We thus hypothesized that blastocyst complementation (BC) could be a better strategy for generating functional PTG cells and compensating loss of parathyroid function. We here describe generation of fully functional PTGs from mouse embryonic stem cells (mESCs) with single-step BC. Using CRISPR-Cas9 knockout of Glial cells missing2 (Gcm2), we efficiently produced aparathyroid embryos for BC. In these embryos, mESCs differentiated into endocrinologically mature PTGs that rescued Gcm2-/- mice from neonatal death. The mESC-derived PTGs responded to extracellular calcium, restoring calcium homeostasis on transplantation into mice surgically rendered hypoparathyroid. We also successfully generated functional interspecies PTGs in Gcm2-/- rat neonates, an accomplishment with potential for future human PTG therapy using xenogeneic animal BC. Our results demonstrate that BC can produce functional endocrine organs and constitute a concept in treatment of hypoparathyroidism.

DOI： 10.1073/pnas.2216564120

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Hyperglycemia impairs immune response; however, it remains unknown whether the anti-tumor effects of anti-programmed cell death-1 antibody (PD-1-Ab) treatment are changed in hyperglycemic conditions. We analyzed the effect of PD-1-Ab on tumor growth in streptozotocin-induced diabetic mice (STZ-mice) subcutaneously inoculated with MC38 (a colon carcinoma cell line). Furthermore, we assessed the expression of chemokines by polymerase chain reaction (PCR) array in tumor-draining lymph nodes (dLNs) of these mice and MC38 cells cultured in different glucose concentrations. The suppressive effect of PD-1-Ab on tumor growth was attenuated. This was accompanied by fewer tumor-infiltrating CD8+ T cells, and STZ-mice had fewer tumor-infiltrating CD11c+ dendritic cells (DCs) than normoglycemic mice. mRNA expression levels of CXCL9, a chemokine recruiting CD8+ T cells, were lower in dLNs of STZ-mice than in normoglycemic mice after PD-1-Ab treatment, and its protein was expressed in DCs. In MC38 cells cultured with 25 mM glucose, mRNA expression of CCL7, a chemokine recruiting DCs, was decreased compared to cells cultured with 5 mM glucose. These results suggest that the STZ-induced hyperglycemia impairs the effect of PD-1-Ab treatment on MC38 tumor growth, and is accompanied by reduced infiltration of DCs and CD8+ T cells and decreased expression of CCL7 and CXCL9.

DOI： 10.1038/s41598-023-33049-7

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

When damaged, restoring the function of the hypothalamus is currently impossible. It is unclear whether neural stem cells exist in the hypothalamus. Studies have reported that adult rodent tanycytes around the third ventricle function as hypothalamic neural stem cell-like cells. However, it is currently impossible to collect periventricular cells from humans. We attempted to generate hypothalamic neural stem cell-like cells from human embryonic stem cells (ESCs). We focused on retina and anterior neural fold homeobox (RAX) because its expression is gradually restricted to tanycytes during the late embryonic stage. We differentiated RAX::VENUS knockin human ESCs (hESCs) into hypothalamic organoids and sorted RAX+ cells from mature organoids. The isolated RAX+ cells formed neurospheres and exhibited self-renewal and multipotency. Neurogenesis was observed when neurospheres were transplanted into the mouse hypothalamus. We isolated RAX+ hypothalamic neural stem cell-like cells from wild-type human ES organoids. This is the first study to differentiate human hypothalamic neural stem cell-like cells from pluripotent stem cells.

DOI： 10.1016/j.stemcr.2023.02.006

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DOI： 10.1507/endocrj.EJ22-0339

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media SA  

Introduction

The pituitary gland, regulating various hormones, is central in the endocrine system. As spontaneous recovery from hypopituitarism is rare, and exogenous-hormone substitution is clumsy, pituitary replacement via regenerative medicine, using pluripotent stem cells, is desirable. We have developed a differentiation method that in mice yields pituitary organoids (POs) derived from human embryonic stem cells (hESC). Efficacy of these POs, transplanted subcutaneously into hypopituitary mice, in reversing hypopituitarism was studied.

Methods

hESC-derived POs were transplanted into inguinal subcutaneous white adipose tissue (ISWAT) and beneath dorsal skin, a relatively avascular region (AR), of hypophysectomized severe combined immunodeficient (SCID) mice. Pituitary function was evaluated thereafter for ¾ 6mo, assaying basal plasma ACTH and ACTH response to corticotropin-releasing hormone (CRH) stimulation. Histopathologic examination of organoids 150d after transplantation assessed engraftment. Some mice received an inhibitor of vascular endothelial growth factor (VEGF) to permit assessment of how angiogenesis contributed to subcutaneous engraftment.

Results

During follow-up, both basal and CRH-stimulated plasma ACTH levels were significantly higher in the ISWAT group (p < 0.001 – 0.05 and 0.001 – 0.005, respectively) than in a sham-operated group. ACTH secretion also was higher in the ISWAT group than in the AR group. Histopathologic study found ACTH-producing human pituitary-cell clusters in both groups of allografts, which had acquired a microvasculature. POs qPCR showed expression of angiogenetic factors. Plasma ACTH levels decreased with VEGF-inhibitor administration.

Conclusions

Subcutaneous transplantation of hESC-derived POs into hypopituitary SCID mice efficaciously renders recipients ACTH-sufficient.

DOI： 10.3389/fendo.2023.1130465

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OBJECTIVE: Resting energy expenditure (REE) decreases if there is reduced energy intake and body weight (BW). The decrease in REE could make it difficult for patients with obesity to maintain decreased BW. This study aimed to investigate the correlation among changes in REE, energy intake, and BW during the weight loss process in patients with obesity. MATERIALS AND METHODS: We conducted a retrospective cohort study of patients hospitalized for the treatment of obesity in Japan. Patients received fully controlled diet during hospitalization and performed exercises if able. REE was measured once a week using a hand-held indirect calorimetry. Energy intake was determined by actual dietary intake. RESULTS: Of 44 inpatients with obesity, 17 were included in the analysis. Their BW decreased significantly after 1 week (-4.7 ± 2.0 kg, P < 0.001) and 2 weeks (-5.7 ± 2.2 kg, P < 0.001). The change in REE after 1 and 2 weeks was positively correlated with the energy intake/energy expenditure ratio (r = 0.66, P = 0.004 at 1 week, r = 0.71, P = 0.002 at 2 weeks). Using a regression equation (y = 0.5257x - 43.579), if the energy intake/energy expenditure ratio within the second week was 82.9%, the REE after 2 weeks was similar to the baseline level. There was no significant correlation between the change in REE and BW. CONCLUSION: Our data suggest that changes in REE depend on energy intake/energy expenditure ratio and that the decrease in REE can be minimized by matching energy intake to energy expenditure, even during the weight loss process.

DOI： 10.20945/2359-3997000000532

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)  

Generation of a variety of organs and tissues from human pluripotent stem cells (hPSCs) has been attempted in vitro. We here present a simple and efficient method for induction of hypothalamic and pituitary tissues from hPSCs. On provision of exogenous agents important for early hypothalamus-pituitary organogenesis, including bone morphogenetic protein 4 and activators of sonic hedgehog, in three-dimensional culture, hPSCs spontaneously form spherical organoids with two distinct tissues, hypothalamus and adenohypophysis. The pituitary tissues derived from hPSCs not only secrete adenocorticotropic hormone, but also retain both positive and negative feedback mechanisms, recapitulating mature endocrine organs in vivo. Furthermore, the results of ectopic transplantation with mouse models of hypopituitarism suggest that these hypothalamus-pituitary organoids have potential as engraftment organs. In addition to their use in transplantation for patients with hypopituitarism they will allow establishment of disease models in vitro and enable research impossible in humans. Hypothalamus-pituitary organoids promise to be a powerful tool in regenerative medicine, drug discovery, and basic research into pituitary development.

DOI： 10.3389/fendo.2022.1025825

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DOI： 10.1111/jne.13223

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DOI： 10.1016/j.pestbp.2022.105291

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The hypothalamus is comprised of heterogenous cell populations and includes highly complex neural circuits that regulate the autonomic nerve system. Its dysfunction therefore results in severe endocrine disorders. Although recent experiments have been conducted for in vitro organogenesis of hypothalamic neurons from embryonic stem (ES) or induced pluripotent stem (iPS) cells, whether these stem cell-derived hypothalamic neurons can be useful for regenerative medicine remains unclear. We therefore performed orthotopic transplantation of mouse ES cell (mESC)-derived hypothalamic neurons into adult mouse brains. We generated electrophysiologically functional hypothalamic neurons from mESCs and transplanted them into the supraoptic nucleus of mice. Grafts extended their axons along hypothalamic nerve bundles in host brain, and some of them even projected into the posterior pituitary (PPit), which consists of distal axons of the magnocellular neurons located in hypothalamic supraoptic and paraventricular nuclei. The axonal projections to the PPit were not observed when the mESC-derived hypothalamic neurons were ectopically transplanted into the substantia nigra reticular part. These findings suggest that our stem cell-based orthotopic transplantation approach might contribute to the establishment of regenerative medicine for hypothalamic and pituitary disorders.

DOI： 10.1371/journal.pone.0276694

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Familial neurohypophyseal diabetes insipidus (FNDI) is a degenerative disease of vasopressin (AVP) neurons. Studies in mouse in vivo models indicate that accumulation of mutant AVP prehormone is associated with FNDI pathology. However, studying human FNDI pathology in vivo is technically challenging. Therefore, an in vitro human model needs to be developed. When exogenous signals are minimized in the early phase of differentiation in vitro, mouse embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs) differentiate into AVP neurons, whereas human ESCs/iPSCs die. Human ESCs/iPSCs are generally more similar to mouse epiblast stem cells (mEpiSCs) compared to mouse ESCs. In this study, we converted human FNDI-specific iPSCs by the naive conversion kit. Although the conversion was partial, we found improved cell survival under minimal exogenous signals and differentiation into rostral hypothalamic organoids. Overall, this method provides a simple and straightforward differentiation direction, which may improve the efficiency of hypothalamic differentiation.

DOI： 10.1038/s41598-022-22405-8

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BACKGROUND: Thyroid dysfunction is frequently caused by treatment with anti-programmed cell death-1 ligand 1 antibodies (PD-L1-Abs) as well as anti-cancer drugs, including ramucirumab (RAM) and multi-targeted tyrosine kinase inhibitors (multi-TKIs), which are often used prior to PD-L1-Ab treatment in cancer patients. METHODS: A total of 148 patients treated with PD-L1-Abs were evaluated for anti-thyroid antibodies at baseline and for thyroid function every six weeks for 24 weeks after treatment initiation and then were observed until the visits stopped. RESULTS: Of the 148 patients, 15 (10.1%) developed thyroid dysfunction after PD-L1-Ab treatment (destructive thyroiditis in eight and hypothyroidism without preceding thyrotoxicosis in seven). The prevalences of an elevated thyroid-stimulating hormone (TSH) level at baseline (3/15 [20.0%] vs. 4/133 [3.0%], p < 0.05), positive anti-thyroglobulin antibodies (TgAb) at baseline (4/15 [26.7%] vs. 5/133 [3.8%], p < 0.05) and prior treatment with RAM or multi-TKIs (3/15 [20.0%] vs. 5/133 [3.8%], p < 0.05) were significantly higher in patients with versus without thyroid dysfunction. In a multivariate analysis, elevated TSH level at baseline, TgAb positivity at baseline and prior treatment with RAM or multi-TKIs were significantly associated with the development of thyroid dysfunction, with odds ratios of 7.098 (95% confidence interval [CI], 1.154-43.638), 11.927 (95% CI, 2.526-56.316) and 8.476 (95% CI, 1.592-45.115), respectively. CONCLUSIONS: The results of this real-world study suggest that the risk of thyroid dysfunction induced by PD-L1-Abs can be predicted by the TSH level at baseline, TgAb positivity at baseline and prior treatment with RAM or multi-TKIs.

DOI： 10.1210/clinem/dgac467

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Leptin, a hormone secreted by adipocytes, exhibits therapeutic potential for the treatment of insulin-dependent diabetes mellitus (IDDM). Protein tyrosine phosphatase 1B (PTP1B) is a key enzyme that negatively regulates leptin receptor signaling. Here, the role of PTP1B in the treatment of IDDM was investigated using PTP1B deficient (KO) mice and a PTP1B inhibitor. IDDM wild-type (WT) mice induced by streptozotocin showed marked hyperglycemia compared to non-IDDM WT mice. KO mice displayed significantly improved glucose metabolism equivalent to non-IDDM WT mice, whereas peripheral or central administration of leptin partially improved glucose metabolism in IDDM WT mice. Peripheral combination therapy of leptin and a PTP1B inhibitor in IDDM WT mice improved glucose metabolism to the same level as non-IDDM WT mice. Leptin was shown to act on the arcuate nucleus in the hypothalamus to suppress gluconeogenesis in liver and enhance glucose uptake in both brown adipose tissue and soleus muscle via the sympathetic nervous system. These effects were enhanced by PTP1B deficiency. Thus, treatment of IDDM with leptin, PTP1B deficiency or a PTP1B inhibitor was shown to enhance leptin activity in the hypothalamus to improve glucose metabolism. These findings suggest a potential alternative therapy for IDDM.

DOI： 10.2337/db21-0953

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Binge eating is a characteristic symptom observed in obese individuals that is related to dysfunction of dopaminergic neurons (DNs). Intermittent administration of a high-fat diet (HFD) is reported to induce binge-like eating, but the underlying mechanisms remain unclear. We generated dopaminergic neuron specific IKKβ deficient mice (KO) to examine the effects of inflammation in DNs on binge-like eating under inflammatory conditions associated with HFD. After administration of HFD for 4 weeks, mice were fasted for 24 h, and then the consumption of HFD was measured for 2 h. We also evaluated that the mRNA expressions of inflammatory cytokines, glial markers, and dopamine signaling-related genes in the ventral tegmental area (VTA) and striatum. Moreover, insulin was administered intraventricularly to assess downstream signaling. The consumption of HFD was significantly reduced, and the phosphorylation of AKT in the VTA was significantly increased in female KO compared to wild-type (WT) mice. Analyses of mRNA expressions revealed that DNs activity and inflammation in the VTA were significantly decreased in female KO mice. Thus, our data suggest that HFD-induced inflammation with glial cell activation in the VTA affects DNs function and causes abnormal eating behaviors accompanied by insulin resistance in the VTA of female mice.

DOI： 10.3390/nu14183835

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Pituitary adenomas are characterized by abnormal growth in the pituitary gland. Surgical excision is the first-line treatment for functional (hormone-producing) pituitary adenomas, except for prolactin-producing adenomas; however, complete excision is technically challenging, and many patients require long-term medication after the treatment. In addition, the pathophysiology of pituitary adenomas, such as tumorigenesis, has not been fully understood. Pituitary adenoma pathophysiology has mainly been studied using animal models and animal tumor-derived cell lines. Nevertheless, experimental studies on human pituitary adenomas are difficult because of the significant differences among species and the lack of reliable cell lines. Recently, several methods have been established to differentiate pituitary cells from human pluripotent stem cells (hPSCs). The induced pituitary hormone-producing cells retain the physiological properties already lost in tumor-derived cell lines. Moreover, CRISPR/Cas9 systems have expedited the introduction of causative gene mutations in various malignant tumors into hPSCs. Therefore, hPSC-derived pituitary cells have great potential as a novel platform for studying the pathophysiology of human-specific pituitary adenomas and developing novel drugs. This review presents an overview of the recent progresses in hPSC applications for pituitary research, functional pituitary adenoma pathogenesis, and genome-editing techniques for introducing causative mutations. We also discuss future applications of hPSCs for studying pituitary adenomas.

DOI： 10.3390/cancers14153660

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media SA  

Human stem cell-derived organoid culture enables the in vitro analysis of the cellular function in three-dimensional aggregates mimicking native organs, and also provides a valuable source of specific cell types in the human body. We previously established organoid models of the hypothalamic-pituitary (HP) complex using human pluripotent stem cells. Although the models are suitable for investigating developmental and functional HP interactions, we consider that isolated pituitary cells are also useful for basic and translational research on the pituitary gland, such as stem cell biology and regenerative medicine. To develop a method for the purification of pituitary cells in HP organoids, we performed surface marker profiling of organoid cells derived from human induced pluripotent stem cells (iPSCs). Screening of 332 human cell surface markers and a subsequent immunohistochemical analysis identified epithelial cell adhesion molecule (EpCAM) as a surface marker of anterior pituitary cells, as well as their ectodermal precursors. EpCAM was not expressed on hypothalamic lineages; thus, anterior pituitary cells were successfully enriched by magnetic separation of EpCAM⁺ cells from iPSC-derived HP organoids. The enriched pituitary population contained functional corticotrophs and their progenitors; the former responded normally to a corticotropin-releasing hormone stimulus. Our findings would extend the applicability of organoid culture as a novel source of human anterior pituitary cells, including stem/progenitor cells and their endocrine descendants.

DOI： 10.3389/fendo.2022.941166

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BACKGROUND: Previous studies showed that although the risk of thyroid dysfunction [thyroid immune-related adverse events (irAEs)] induced by anti-programmed cell death-1 antibodies (PD-1-Ab) was as low as 2% to 7% in patients negative for anti-thyroid antibodies (ATAs) at baseline, it was much higher (30%-50%) in patients positive for ATAs. However, whether a similar increase occurs with combination therapy using PD-1-Ab plus anti-cytotoxic T-lymphocyte antigen-4 antibody (CTLA-4-Ab) is unknown. METHODS: A total of 451 patients with malignancies treated with PD-1-Ab, CTLA-4-Ab, or a combination of PD-1-Ab and CTLA-4-Ab (PD-1/CTLA-4-Abs) were evaluated for ATAs at baseline and for thyroid function every 6 weeks for 24 weeks after treatment initiation and then observed until the last clinical visit. RESULTS: Of the 451 patients, 51 developed thyroid irAEs after immunotherapy [41 of 416 (9.9%) treated with PD-1-Ab, 0 of 8 (0%) treated with CTLA-4-Ab, and 10 of 27 (37.0%) treated with PD-1/CTLA-4-Abs]. The cumulative incidence of thyroid irAEs was significantly higher in patients who were positive vs negative for ATAs at baseline after both PD-1-Ab [28/87 (32.2%) vs 13/329 (4.0%), P < 0.001] and PD-1/CTLA-4-Abs [6/10 (60.0%) vs 4/17 (23.5%), P < 0.05] treatments. The risk of thyroid irAEs induced by PD-1/CTLA-4Abs, which was significantly higher than that induced by PD-1-Ab, in patients negative for ATAs at baseline was not statistically different from that induced by PD-1-Ab in patients positive for ATAs at baseline. CONCLUSIONS: This study showed that the incidence of thyroid irAEs was high and not negligible after PD-1/CTLA-4-Abs treatment even in patients negative for ATAs at baseline.

DOI： 10.1210/clinem/dgab829

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Prolactin (PRL), a hormone involved in lactation, is mainly produced and secreted by the lactotrophs of the anterior pituitary (AP) gland. We previously reported a method to generate functional adrenocorticotropic hormone-producing cells by differentiating the AP and hypothalamus simultaneously from human induced pluripotent stem cells (iPSCs). However, PRL-producing cells in the induced AP have not been investigated. Here, we confirmed the presence of PRL-producing cells and evaluated their endocrine functions. We differentiated pituitary cells from human iPSCs using serum-free floating culture of embryoid-like aggregates with quick reaggregation (SFEB-q) method and evaluated the appearance and function of PRL-producing cells. Secretion of PRL from the differentiated aggregates was confirmed, which increased with further culture. Fluorescence immunostaining and immunoelectron microscopy revealed PRL-producing cells and PRL-positive secretory granules, respectively. PRL secretion was promoted by various prolactin secretagogues such as thyrotropin-releasing hormone, vasoactive intestinal peptide, and prolactin-releasing peptide, and inhibited by bromocriptine. Moreover, the presence of tyrosine hydroxylase-positive dopaminergic nerves in the hypothalamic tissue area around the center of the aggregates connecting to PRL-producing cells indicated the possibility of recapitulating PRL regulatory mechanisms through the hypothalamus. In conclusion, we generated pituitary lactotrophs from human iPSCs; these displayed similar secretory responsiveness as human pituitary cells in vivo. In the future, this is expected to be used as a model of human PRL-producing cells for various studies, such as drug discovery, prediction of side effects, and elucidation of tumorigenic mechanisms using disease-specific iPSCs. Furthermore, it may help to develop regenerative medicine for the pituitary gland.

DOI： 10.1210/endocr/bqac004

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Society for Neuroscience  

Hypothalamic melanin-concentrating hormone (MCH) neurons are important regulators of multiple physiological processes, such as sleep, feeding, and memory. Despite the increasing interest in their neuronal functions, the molecular mechanism underlying MCH neuron development remains poorly understood. We report that a three-dimensional culture of mouse embryonic stem cells (mESCs) can generate hypothalamic-like tissues containing MCH-positive neurons, which reproduce morphologic maturation, neuronal connectivity, and neuropeptide/neurotransmitter phenotype of native MCH neurons. Using this in vitro system, we demonstrate that Hedgehog (Hh) signaling serves to produce major neurochemical subtypes of MCH neurons characterized by the presence or absence of cocaine- and amphetamine-regulated transcript (CART). Without exogenous Hh signals, mESCs initially differentiated into dorsal hypothalamic/prethalamic progenitors and finally into MCH+CART+ neurons through a specific intermediate progenitor state. Conversely, activation of the Hh pathway specified ventral hypothalamic progenitors that generate both MCH+CART- and MCH+CART+ neurons. These results suggest that in vivo MCH neurons may originate from multiple cell lineages that arise through early dorsoventral patterning of the hypothalamus. Additionally, we found that Hh signaling supports the differentiation of mESCs into orexin/hypocretin neurons, a well-defined cell group intermingled with MCH neurons in the lateral hypothalamic area (LHA). The present study highlights and improves the utility of mESC culture in the analysis of the developmental programs of specific hypothalamic cell types.Significance StatementA growing body of literature has revealed the importance of hypothalamic melanin-concentrating hormone (MCH) neurons in energy homeostasis and the cognitive function, but their developmental biology remains relatively unknown. To establish a new approach for addressing this issue, we tested the ability of an in vitro differentiation system of mouse embryonic stem cells (mESCs) to recapitulate the development of MCH neurons. The mESC culture robustly generated MCH-positive neurons resembling native neurons in several aspects and provided evidence that Hedgehog (Hh) signaling is a key factor to produce neurochemical subtypes of MCH neurons. Our results demonstrate the suitability of mESC culture as a platform to study the molecular mechanisms underlying the development of MCH neurons and possibly of other hypothalamic cell types.

DOI： 10.1523/ENEURO.0442-21.2022

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：(一社)日本内分泌学会  

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DOI： 10.1007/s00125-021-05610-4

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DOI： 10.1007/s00125-021-05516-1

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Previous studies suggest that signaling by the gamma-aminobutyric acid (GABA) type B receptor (GABABR) is involved in the regulation of binge eating, a disorder which might contribute to the development of obesity. Here, we show that intermittent access to a high fat diet (HFD) induced binge-like eating behavior with activation of dopamine receptor d1 (drd1)-expressing neurons in the caudate putamen (CPu) and nucleus accumbens (NAc) in wild-type (WT) mice. The activation of drd1-expressing neurons during binge-like eating was substantially increased in the CPu, but not in the NAc, in corticostriatal neuron-specific GABABR-deficient knockout (KO) mice compared to WT mice. Treatment with the GABABR agonist, baclofen, suppressed binge-like eating behavior in WT mice, but not in KO mice, as reported previously. Baclofen also suppressed the activation of drd1-expressing neurons in the CPu, but not in the NAc, during binge-like eating in WT mice. Thus, our data suggest that GABABR signaling in CPu neurons expressing drd1 suppresses binge-like consumption during a HFD in mice.

DOI： 10.1038/s41598-021-98590-9

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Wolfram syndrome (WS) is mainly caused by mutations in the WFS1 gene and characterized by diabetes mellitus, optic atrophy, hearing loss, and central diabetes insipidus (CDI). WFS1 is an endoplasmic reticulum (ER)-resident transmembrane protein, and Wfs1 knockout (Wfs1-/-) mice, which have been used as a mouse model for WS, reportedly manifested impairment of glucose tolerance due to pancreatic β-cell loss. In the present study, we examined water balance, arginine vasopressin (AVP) secretion, and ER stress in AVP neurons of the hypothalamus in Wfs1-/- mice. There were no differences in urine volumes between Wfs1-/- and wild-type mice with free access to water. Conversely, when mice were subjected to intermittent water deprivation (WD) for 20 weeks, during which water was unavailable for 2 days a week, urine volumes were larger in Wfs1-/- mice, accompanied by lower urine AVP concentrations and urine osmolality, compared to wild-type mice. The mRNA expression of immunoglobulin heavy chain binding protein, a marker of ER stress, was significantly increased in the supraoptic nucleus and paraventricular nuclei in Wfs1-/- mice compared to wild-type mice after WD. Our results thus showed that Wfs1 knockout leads to a decrease in AVP secretion during dehydration, which could explain in part the mechanisms by which Wfs1 mutations cause CDI in humans.

DOI： 10.1007/s11102-021-01135-6

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Other Link： http://link.springer.com/article/10.1007/s11102-021-01135-6/fulltext.html

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DOI： 10.1038/s41598-021-92386-7

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

<title>Abstract</title>For pituitary regenerative medicine, the creation of a hypophyseal model in monkeys is necessary to conduct future preclinical studies; however, previous studies reported that hypophysectomy in monkeys is not always safe or satisfactory. This study aimed to create a hypophyseal dysfunction model in a cynomolgus monkey using a safer surgical technique and establish the protocol of pituitary hormone replacement therapy for this model. Surgical resection of the pituitary gland of a 7.8-year-old healthy adult cynomolgus male monkey weighing 5.45 kg was performed to create a hypophyseal dysfunction model for future regenerative studies. Endoscopic transoral transsphenoidal surgery was used to perform hypophysectomy under navigation support. These procedures were useful for confirming total removal of the pituitary gland without additional bone removal and preventing complications such as cerebrospinal fluid leakage. Total removal was confirmed by pathological examination and computed tomography. Hypopituitarism was verified with endocrinological examinations including stimulation tests. Postoperatively, the monkey’s general condition of hypopituitarism was treated with hormone replacement therapy, resulting in long-term survival. The success of a minimally invasive and safe surgical method and long-term survival indicate the creation of a hypophyseal dysfunction model in a cynomolgus monkey; hence, this protocol can be employed in the future.

DOI： 10.1038/s41598-021-90209-3

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Other Link： http://www.nature.com/articles/s41598-021-90209-3

Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.1126/scitranslmed.abb7495

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The reward system plays an important role in the pathogenesis of not only drug addiction, but also diet-induced obesity. Recent studies have shown that insulin and leptin receptor signaling in the ventral tegmental area (VTA) regulate energy homeostasis and that their dysregulation is responsible for obesity and altered food preferences. Although a high-fat diet (HFD) induces inflammation that leads to insulin and leptin resistance in the brain, it remains unclear whether HFD induces inflammation in the VTA. In the present study, we placed male mice on a chow diet or HFD for 3, 7, and 28 days and evaluated the mRNA expression of inflammatory cytokines and microglial activation markers in the VTA. The HFD group showed significantly elevated mRNA expressions of IL1β at 3 days; tumor necrosis factor-alpha (TNFα), IL1β, IL6, Iba1, and CD11b at 7 days; and TNFα, IL1β, Iba1, and CD11b at 28 days. The changes in TNFα were also confirmed in immunohistochemical analysis. Next, after administration of chow or HFD for 7 days, we selected mice with equal weights in both groups. In experiments using these mice, Akt phosphorylation in the VTA was significantly decreased after intracerebroventricular injection of insulin, whereas no change in STAT3 phosphorylation was found with leptin. Taken together, these results suggest that HFD induces inflammation at least partly associated with microglial activation in the VTA leading to insulin resistance, independently of the energy balance. Our data provide new insight into the pathophysiology of obesity caused by a dysfunctional reward system under HFD conditions.

DOI： 10.1016/j.neuroscience.2021.02.009

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Arginine vasopressin (AVP) synthesized in the magnocellular neurons of the hypothalamus is transported through their axons and released from the posterior pituitary into the systemic circulation to act as an antidiuretic hormone. AVP synthesis and release are precisely regulated by changes in plasma osmolality. Magnocellular AVP neurons receive innervation from osmosensory and sodium-sensing neurons, but previous studies showed that AVP neurons per se are osmosensitive as well. In the current study, we made AVP-Venus reporter mice and showed that Venus was expressed exclusively in AVP neurons and was upregulated under water deprivation. In hypothalamic organotypic cultures from the AVP-Venus mice, Venus-labeled AVP neurons in the supraoptic and paraventricular nuclei survived for 1 month, and Venus expression was upregulated by forskolin. Furthermore, in dissociated Venus-labeled magnocellular neurons, treatment with NaCl, but not with mannitol, decreased Venus fluorescence in the soma of the AVP neurons. Thus, Venus expression in AVP-Venus transgenic mice, as well as in primary cultures, faithfully showed the properties of intrinsic AVP expression. These findings indicate that AVP-Venus mice as well as the primary hypothalamic cultures could be useful for studying magnocellular AVP neurons.

DOI： 10.1016/j.peptides.2021.170517

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BMJ  

Background

Pituitary dysfunction is a life-threatening immune-related adverse event (irAE) induced by immune checkpoint inhibitors (ICIs). To date, it is not possible to identify patients who may develop pituitary irAEs prior to ICI treatment. The aim of this study was to characterize the predisposition for ICI-induced pituitary irAEs by analyzing anti-pituitary antibodies (APAs) and human leukocyte antigens (HLAs).

Methods

In this case–control study, APAs and HLA alleles were analyzed in 62 patients (17 who developed ICI-induced isolated adrenocorticotropic hormone deficiency (ICI-IAD), 5 who developed ICI-induced hypophysitis (ICI-H) and 40 who did not develop pituitary irAEs) treated with ICIs between November 2, 2015, and March 31, 2020, at Nagoya University Hospital. The main outcome measures in this study were the association between the development of pituitary irAEs with APAs at baseline and after treatment and HLA alleles.

Results

Eleven of 17 (64.7%) patients who developed ICI-IAD had APAs at baseline, whereas APAs were positive only in 1 of 40 (2.5%) control patients. Although APAs were negative at baseline in all patients who developed ICI-H, they had become positive before the onset of ICI-H in 3 of 4 patients several weeks after ipilimumab administration. At the onset of ICI-IAD and ICI-H, APAs were positive in 15 of 17 (88.2%) and 4 of 5 (80%) patients, respectively. The prevalence of HLA-Cw12, HLA-DR15, HLA-DQ7, and HLA-DPw9 was significantly higher in patients with ICI-IAD, whereas that of HLA-Cw12 and HLA-DR15 was significantly higher in patients with ICI-H than in controls.

Conclusions

This study showed distinct and overlapped patterns of APAs and HLA alleles between ICI-IAD and ICI-H. Our findings also showed that positive APAs at baseline and after treatment, together with susceptible HLA alleles, could become predictive biomarkers for ICI-IAD and ICI-H, respectively.

Trial registration number

UMIN000019024.

DOI： 10.1136/jitc-2021-002493

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Language：English   Publishing type：Research paper (scientific journal)  

Excessive sodium salt (NaCl) or fat intake is associated with a variety of increased health risks. However, whether excessive NaCl intake accompanied by a high-fat diet (HFD) affects glucose metabolism has not been elucidated. In this study, C57BL/6J male mice were fed a normal chow diet (NCD), a NCD plus high-NaCl diet (NCD plus NaCl), a HFD, or a HFD plus high-NaCl diet (HFD plus NaCl) for 30 weeks. No significant differences in body weight gain, insulin sensitivity, and glucose tolerance were observed between NCD-fed and NCD plus NaCl-fed mice. In contrast, body and liver weights were decreased, but the weight of epididymal white adipose tissue was increased in HFD plus NaCl-fed compared to HFD-fed mice. HFD plus NaCl-fed mice had lower plasma glucose levels in an insulin tolerance test, and showed higher plasma glucose and lower plasma insulin levels in an intraperitoneal glucose tolerance test compared to HFD-fed mice. The β-cell area and number of islets were decreased in HFD plus NaCl-fed compared to HFD-fed mice. Increased Ki67-positive β-cells, and increased expression levels of Ki67, CyclinB1, and CyclinD1 mRNA in islets were observed in HFD-fed but not HFD plus NaCl-fed mice when compared to NCD-fed mice. Our data suggest that excessive NaCl intake accompanied by a HFD exacerbates glucose intolerance, with impairment in insulin secretion caused by the attenuation of expansion of β-cell mass in the pancreas.

DOI： 10.1371/journal.pone.0248065

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

The hypothalamus and pituitary have been identified to play essential roles in maintaining homeostasis. Various diseases can disrupt the functions of these systems, which can often result in serious lifelong symptoms. The current treatment for hypopituitarism involves hormone replacement therapy. However, exogenous drug administration cannot mimic the physiological changes that are a result of hormone requirements. Therefore, patients are at a high risk of severe hormone deficiency, including adrenal crisis. Pluripotent stem cells (PSCs) self-proliferate and differentiate into all types of cells. The generation of endocrine tissues from PSCs has been considered as another new treatment for hypopituitarism. Our colleagues established a 3-dimensional (3D) culture method for embryonic stem cells (ESCs). In this culture, the ESC-derived aggregates exhibit self-organization and spontaneous formation of highly ordered patterning. Recent results have shown that strict removal of exogenous patterning factors during early differentiation efficiently induces rostral hypothalamic progenitors from mouse ESCs. These hypothalamic progenitors generate vasopressinergic neurons, which release neuropeptides upon exogenous stimulation. Subsequently, we reported adenohypophysis tissue self-formation in 3D cultures of mouse ESCs. The ESCs were found to differentiate into both nonneural oral ectoderm and hypothalamic neuroectoderm in adjacent layers. Interactions between the 2 tissues appear to be critically important for in vitro induction of a Rathke's pouch-like developing embryo. Various endocrine cells were differentiated from nonneural ectoderm. The induced corticotrophs efficiently secreted adrenocorticotropic hormone when engrafted in vivo, which rescued hypopituitary hosts. For future regenerative medicine, generation of hypothalamic and pituitary tissues from human PSCs is necessary. We and other groups succeeded in establishing a differentiation method with the use of human PSCs. Researchers could use these methods for models of human diseases to elucidate disease pathology or screen potential therapeutics.

DOI： 10.1210/jendso/bvaa188

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

This paper overviews the development and differentiation of the hypothalamus and pituitary gland from embryonic stem (ES) and induced pluripotent stem (iPS) cells. It is important to replicate the developmental process in vivo to create specific cells/organoids from ES/iPS cells. We also introduce the latest findings and discuss future issues for clinical application. Neuroectodermal progenitors are induced from pluripotent stem cells by strictly removing exogenous patterning factors during the early differentiation period. The induced progenitors differentiate into rostral hypothalamic neurons, in particular magnocellular vasopressin+ neurons. In three-dimensional cultures, the ES/iPS cells differentiate into hypothalamic neuroectoderm as well as non-neural head ectoderm back to back. Rathke's pouch-like structures self-organize at the interface between the two layers and generated various endocrine cells, including corticotrophs and somatotrophs from the Rathke's pouch-like structures. Our next objective is to sophisticate our stepwise methodology to establish the novel transplantation treatment for hypopituitarism and to apply it to developmental disease models.

DOI： 10.1111/dgd.12719

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE PHARMACOLOGICAL SOC  

We investigated whether peripheral combination treatment of a sodium-glucose cotransporter 2 (SGLT2) inhibitor and leptin improves glucose metabolism in insulin-dependent diabetes mellitus (IDDM) model mice. Twelve-week-old male C57BL6 mice were intraperitoneally administered a high dose of streptozotocin to produce IDDM. IDDM mice were then divided into five groups: SGLT2 inhibitor treatment alone, leptin treatment alone, leptin and SGLT2 inhibitor co-treatment, untreated IDDM mice, and healthy mice groups. The blood glucose (BG) level at the end of the dark cycle was measured, and a glucose tolerance test (GTT) was performed and compared between the five groups. Leptin was peripherally administered at 20 mu g/day using an osmotic pump, and an SGLT2 inhibitor, ipragliflozin, was orally administered at 3 mg/kg/day. Monotherapy with SGLT2 inhibitor or leptin significantly improved glucose metabolism in mice as evaluated by BG and GTT compared with the untreated group, whereas the co-treatment group with SGLT2 inhibitor and leptin further improved glucose metabolism as compared with the monotherapy group. Notably, glucose metabolism in the co-treatment group improved to the same level as that in the healthy mice group. Thus, peripheral combination treatment with leptin and SGLT2 inhibitor improved glucose metabolism in IDDM mice without the use of insulin. (C) 2021 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society.

DOI： 10.1016/j.jphs.2021.08.010

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

<title>Abstract</title>The immunoglobulin heavy chain binding protein (BiP), also referred to as 78-kDa glucose-regulated protein (GRP78), is a pivotal endoplasmic reticulum (ER) chaperone which modulates the unfolded protein response under ER stress. Our previous studies showed that BiP is expressed in arginine vasopressin (AVP) neurons under non-stress conditions and that BiP expression is upregulated in proportion to the increased AVP expression under dehydration. To clarify the role of BiP in AVP neurons, we used a viral approach in combination with shRNA interference for BiP knockdown in mouse AVP neurons. Injection of a recombinant adeno-associated virus equipped with a mouse AVP promoter and BiP shRNA cassette provided specific BiP knockdown in AVP neurons of the supraoptic (SON) and paraventricular nuclei (PVN) in mice. AVP neuron-specific BiP knockdown led to ER stress and AVP neuronal loss in the SON and PVN, resulting in increased urine volume due to lack of AVP secretion. Immunoelectron microscopy of AVP neurons revealed that autophagy was activated through the process of AVP neuronal loss, whereas no obvious features characteristic of apoptosis were observed. Pharmacological inhibition of autophagy by chloroquine exacerbated the AVP neuronal loss due to BiP knockdown, indicating a protective role of autophagy in AVP neurons under ER stress. In summary, our results demonstrate that BiP is essential for the AVP neuron system.

DOI： 10.1038/s41598-020-76839-z

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Other Link： http://www.nature.com/articles/s41598-020-76839-z

Language：English   Publishing type：Research paper (scientific journal)  

Misfolded or unfolded proteins in the ER are said to be degraded only after translocation or isolation from the ER. Here, we describe a mechanism by which mutant proteins are degraded within the ER. Aggregates of mutant arginine vasopressin (AVP) precursor were confined to ER-associated compartments (ERACs) connected to the ER in AVP neurons of a mouse model of familial neurohypophysial diabetes insipidus. The ERACs were enclosed by membranes, an ER chaperone and marker protein of phagophores and autophagosomes were expressed around the aggregates, and lysosomes fused with the ERACs. Moreover, lysosome-related molecules were present within the ERACs, and aggregate degradation within the ERACs was dependent on autophagic-lysosomal activity. Thus, we demonstrate that protein aggregates can be degraded by autophagic-lysosomal machinery within specialized compartments of the ER.

DOI： 10.1016/j.isci.2020.101648

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Four disease-specific induced pluripotent stem cell (iPSC) lines were respectively derived from peripheral blood mononuclear cells of two affected individuals in a family affected by familial neurohypophyseal diabetes insipidus carrying the c.314G>C mutation. The expression of pluripotency markers (NANOG, OCT4, and SOX2), maintenance of a normal karyotype, absence of episomal vectors used for iPSC generation, and presence of the original pathogenic mutation were confirmed for each iPSC line. The ability to differentiate into three germ layers was confirmed by a teratoma formation assay. These iPSC lines can help in disease recapitulation in vitro using organoids and elucidation of disease mechanisms.

DOI： 10.1016/j.scr.2020.101960

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

We generated three disease-specific iPSC lines from a Multiple endocrine neoplasia type 1 (MEN1) patient and three control iPSC lines from an unaffected blood relative of the patient using unutilized lymphoblastoid B cell lines (LCLs) as a cell resource. The expression of pluripotency markers, retaining of normal karyotype of chromosome, absence of episomal vectors used for generating the iPSCs and EBV used for generating LCLs, and the potential to differentiate into three germ layers, were confirmed for each iPSC line. These iPSC lines can be useful for construction of the disease models in vitro, and elucidation of the disease mechanisms.

DOI： 10.1016/j.scr.2020.101846

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Glial cells can mediate hypothalamic inflammatory processes induced in response to a high-fat diet (HFD). We used magnetic-activated cell sorting (MACS) to isolate microglia and astrocytes from hypothalamus of mice fed HFD and examined changes in expression of inflammation-related cytokines and markers related to glial cell activation status. Hypothalamus from male C57BL6 mice fed a chow diet (chow) or HFD for 1, 3, or 28 days were collected and microglia and astrocytes were isolated by MACS. After confirming cell viability by fluorescence activated cell sorting, mRNA expression levels of inflammation-related cytokines and markers of glial cell activation status were examined by qRT-PCR, which revealed that both glial cell types isolated by MACS retained specificity. On day 3 of HFD, both CD86 and TNFα mRNA expression was significantly increased in microglia relative to the chow group. In astrocytes, TNFα mRNA expression levels were similar between the chow and HFD groups on day 3, but anti-inflammatory cytokine IL-10 levels were significantly increased. On day 7 of HFD, TNFα expression in microglia decreased to levels comparable to the chow group while that in astrocytes remained unchanged. On day 28 of HFD, TNFα levels were significantly increased in both microglia and astrocytes, which had increased mRNA expression of CD86 and MAO-B, respectively. For both glial cell types, results for TNFα expression assessed by RT-PCR and immunohistochemical analysis were similar. These results indicate that the role of microglia and astrocytes in hypothalamic inflammation under HFD conditions changed with time and these changes were accompanied by changes in the activation status of glial cells. Our data suggest that early after initiating HFD, hypothalamic astrocytes suppress diet-induced inflammation at least in part by secreting IL-10, whereas continued HFD feeding impairs this suppressive function such that both microglia and astrocytes promote hypothalamic inflammation.

DOI： 10.1016/j.neuint.2020.104733

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

BACKGROUND: Anti-programmed cell death-1 (PD-1) antibodies can cause thyroid dysfunction. However, no predictive biomarkers enabling stratification of thyroid dysfunction risk have been identified. METHODS: A total of 209 patients treated with an anti-PD-1 antibody were evaluated for anti-thyroid antibodies at baseline and prospectively for thyroid function every 6 weeks for 24 weeks after treatment initiation, and then observed until the visits stopped. Thyroid ultrasonography was performed if the patient was positive for anti-thyroid antibodies at baseline. RESULTS: Of the 209 patients, 19 (9.1%) developed thyroid dysfunction (destructive thyroiditis or hypothyroidism). The cumulative incidence of thyroid dysfunction was significantly higher in patients who were positive vs. negative for anti-thyroid antibodies (15/44 [34.1%] vs. 4/165 [2.4%], p < 0.001). Forty-two patients positive for anti-thyroid antibodies at baseline were divided into two groups according to the presence of an irregular echo pattern. The cumulative incidence of thyroid dysfunction was significantly higher in those with an irregular vs. a regular echo pattern (13/23 [56.5%] vs. 1/19 [5.3%], p = 0.001). None of the patients developed thyroid dysfunction after the initial 24-week period. CONCLUSIONS: The risk of thyroid dysfunction induced by anti-PD-1 antibodies can be predicted by evaluation of anti-thyroid antibodies and the thyroid echo pattern at baseline. TRIAL REGISTRATION: UMIN000019024.

DOI： 10.1038/s41416-020-0736-7

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Other Link： http://www.nature.com/articles/s41416-020-0736-7

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Pituitary develops from oral ectoderm in contact with adjacent ventral hypothalamus. Impairment in this process results in congenital pituitary hypoplasia (CPH); however, there have been no human disease models for CPH thus far, prohibiting the elucidation of the underlying mechanisms. In this study, we established a disease model of CPH using patient-derived induced pluripotent stem cells (iPSCs) and 3D organoid technique, in which oral ectoderm and hypothalamus develop simultaneously. Interestingly, patient iPSCs with a heterozygous mutation in the orthodenticle homeobox 2 (OTX2) gene showed increased apoptosis in the pituitary progenitor cells, and the differentiation into pituitary hormone-producing cells was severely impaired. As an underlying mechanism, OTX2 in hypothalamus, not in oral ectoderm, was essential for progenitor cell maintenance by regulating LHX3 expression in oral ectoderm via FGF10 expression in the hypothalamus. Convincingly, the phenotype was reversed by the correction of the mutation, and the haploinsufficiency of OTX2 in control iPSCs revealed a similar phenotype, demonstrating that this mutation was responsible. Thus, we established an iPSC-based congenital pituitary disease model, which recapitulated interaction between hypothalamus and oral ectoderm and demonstrated the essential role of hypothalamic OTX2.

DOI： 10.1172/JCI127378

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Type 2 diabetes and dyslipidemia are diseases that collectively increase the risk of patients developing cardiovascular complications. Several incretin-based drugs are reported to improve lipid metabolism, and one of these medications, anagliptin, is a dipeptidyl peptidase-4 (DPP-4) inhibitor that has been shown to decrease serum triglyceride and low-density lipoproteins cholesterol. This study aimed to conduct an investigation into the effects of anagliptin on serum lipid profiles. This multicenter, open-label, randomized (1:1), parallel group study was designed to evaluate the effects of anagliptin on serum lipid profiles (triglycerides, lipoproteins, apolipoproteins, and cholesterol fractions). The study involved 24 patients with type 2 diabetes at two participating hospitals for a period of 24 weeks. Patients were randomly assigned to the anagliptin (n = 12) or control (n = 12) groups. Patients in the anagliptin group were treated with 200 mg of the drug twice daily. Patients in the control group did not receive anagliptin, but continued with their previous treatment schedules. Lipid metabolism was examined under fasting conditions at baseline and 24 weeks. Patients treated with anagliptin for 24 weeks exhibited significantly reduced levels of serum apolipoprotein B-48, a marker for lipid transport from the intestine, compared with the control group patients (P < 0.05). After 24 weeks of treatment, serum adiponectin levels were significantly raised, whereas glycated hemoglobin (HbA1c) levels were significantly lower compared with the baseline in the anagliptin group (P < 0.05), but not in the control group. This study showed that the DPP-4 inhibitor anagliptin reduces fasting apolipoprotein B-48 levels, suggesting that this drug may have beneficial effects on lipid metabolism possibly mediated by the inhibition of intestinal lipid transport.

DOI： 10.1371/journal.pone.0228004

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Language：English   Publishing type：Research paper (scientific journal)  

The pituitary is a major hormone center that secretes systemic hormones responding to hypothalamus-derived-releasing hormones. Previously, we reported the independent pituitary induction and hypothalamic differentiation of human embryonic stem cells (ESCs). Here, a functional hypothalamic-pituitary unit is generated using human induced pluripotent stem (iPS) cells in vitro. The adrenocorticotropic hormone (ACTH) secretion capacity of the induced pituitary reached a comparable level to that of adult mouse pituitary because of the simultaneous maturation with hypothalamic neurons within the same aggregates. Corticotropin-releasing hormone (CRH) from the hypothalamic area regulates ACTH cells similarly to our hypothalamic-pituitary axis. Our induced hypothalamic-pituitary units respond to environmental hypoglycemic condition in vitro, which mimics a life-threatening situation in vivo, through the CRH-ACTH pathway, and succeed in increasing ACTH secretion. Thus, we generated powerful hybrid organoids by recapitulating hypothalamic-pituitary development, showing autonomous maturation on the basis of interactions between developing tissues.

DOI： 10.1016/j.celrep.2019.12.009

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INTRODUCTION: The present study aimed to evaluate the effects of flash glucose monitoring (FGM) and conventional self-monitoring of blood glucose (SMBG) on glycemic control in patients with non-insulin-treated type 2 diabetes. RESEARCH DESIGN AND METHODS: In this 24-week, multicenter, open-label, randomized (1:1), parallel-group study, patients with non-insulin-treated type 2 diabetes at five hospitals in Japan were randomly assigned to the FGM (n=49) or SMBG (n=51) groups and were provided each device for 12 weeks. The primary outcome was change in glycated hemoglobin (HbA1c) level, and was compared using analysis of covariance model that included baseline values and group as covariates. RESULTS: Forty-eight participants in the FGM group and 45 in the SMBG group completed the study. The mean HbA1c levels were 7.83% (62.1 mmol/mol) in the FGM group and 7.84% (62.2 mmol/mol) in the SMBG group at baseline, and the values were reduced in both FGM (-0.43% (-4.7 mmol/mol), p<0.001) and SMBG groups (-0.30% (-3.3 mmol/mol), p=0.001) at 12 weeks. On the other hand, HbA1c was significantly decreased from baseline values in the FGM group, but not in the SMBG group at 24 weeks (FGM: -0.46% (-5.0 mmol/mol), p<0.001; SMBG: -0.17% (-1.8 mmol/mol), p=0.124); a significant between-group difference was also observed (difference -0.29% (-3.2 mmol/mol), p=0.022). Diabetes Treatment Satisfaction Questionnaire score was significantly improved, and the mean glucose levels, SD of glucose, mean amplitude of glycemic excursions and time in hyperglycemia were significantly decreased in the FGM group compared with the SMBG group. CONCLUSIONS: Glycemic control was better with FGM than with SMBG after cessation of glucose monitoring in patients with non-insulin-treated type 2 diabetes. TRIAL REGISTRATION NUMBER: UMIN000026452, jRCTs041180082.

DOI： 10.1136/bmjdrc-2019-001115

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japan Endocrine Society  

DOI： 10.1507/endocrj.EJ19-0224

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Language：English   Publishing type：Research paper (scientific journal)  

BACKGROUND: Several immune-related adverse events (irAEs) are reported to be associated with therapeutic efficacy of immune checkpoint inhibitors, yet whether pituitary dysfunction, a life-threatening irAE, affects overall survival (OS) in patients with malignancies is unclear. This prospective study examined the association of pituitary dysfunction (pituitary-irAE) with OS of patients with non-small cell lung carcinoma (NSCLC) or malignant melanoma (MM). METHODS: A total of 174 patients (NSCLC, 108; MM, 66) treated with ipilimumab, nivolumab, pembrolizumab, or atezolizumab at Nagoya University Hospital were evaluated for OS and the development of pituitary-irAE. Kaplan-Meier curves of OS as a function of the development of pituitary-irAE were produced with the log-rank test as a primary endpoint. RESULTS: Pituitary-irAE was observed in 16 patients (4 (3.7%) with NSCLC, 12 (18.2%) with MM) having two different disease types: hypophysitis with deficiency of multiple anterior pituitary hormones accompanied by pituitary enlargement, and isolated adrenocorticotropic hormone (ACTH) deficiency without pituitary enlargement. Among these patients, 6 developed pituitary-irAE while being treated with ipilimumab (6/25 patients (24.0%) treated with ipilimumab) and 10 developed pituitary-irAE during treatment with nivolumab or pembrolizumab (10/167 (6.0%)). All 16 patients had ACTH deficiency and were treated with physiological doses of hydrocortisone. The development of pituitary-irAE was associated with better OS in patients with NSCLC (not reached vs 441 (95% CI not calculated) days, p<0.05) and MM (885 (95% CI 434 to 1336) vs 298 (95% CI 84 to 512) days, p<0.05). CONCLUSIONS: In our study cohort, the incidence of pituitary-irAE was higher than previously reported and the development of pituitary-irAE predicted better prognosis for both NSCLC and MM when patients were treated with physiological doses of hydrocortisone. CLINICAL TRIALS REGISTRATION: UMIN000019024.

DOI： 10.1136/jitc-2020-000779

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Language：English   Publishing type：Research paper (scientific journal)  

Binge eating could contribute to the development of obesity, and previous studies suggest that gamma-aminobutyric acid (GABA) type B receptor (GABABR) signaling is involved in the regulation of binge eating. Here, we show that time-restricted access to a high-fat diet (HFD) induces binge-like eating behavior in wild-type mice. HFD consumption during restricted time was significantly increased in corticostriatal neuron-specific GABABR-deficient mice compared with wild-type mice. Furthermore, the GABABR agonist baclofen suppressed HFD intake during restricted time in wild-type mice but not in corticostriatal or dopaminergic neuron-specific GABABR-deficient mice. In contrast, there were no significant differences in food consumption among genotypes under ad libitum access to HFD. Thus, our data show that the mesolimbic system regulates food consumption under time-restricted but not ad libitum access to HFD and have identified a mechanism by which GABABR signaling suppresses binge-like eating of HFD.

DOI： 10.1016/j.isci.2019.09.032

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

High differentiation efficiency is one of the most important factors in developing an in vitro model from pluripotent stem cells. In this report, we improved the handling technique applied to mouse-induced pluripotent stem (iPS) cells, resulting in better differentiation into hypothalamic vasopressin (AVP) neurons. We modified the culture procedure to make the maintenance of iPS cells in an undifferentiated state much easier. Three-dimensional floating culture was demonstrated to be effective for mouse iPS cells. We also improved the differentiation method with regards to embryology, resulting in a greater number of bigger colonies of AVP neurons differentiating from mouse iPS cells. Fgf8, which was not used in the original differentiation method, increased iPS differentiation into AVP neurons. These refinements will be useful as a valuable tool for the modeling of degenerative disease in AVP neurons in vitro using disease-specific iPS cells in future studies.

DOI： 10.1016/j.scr.2019.101572

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Tanycytes have recently been accepted as neural stem/progenitor cells in the postnatal hypothalamus. Persistent retina and anterior neural fold homeobox (Rax) expression is characteristic of tanycytes in contrast to its transient expression of whole hypothalamic precursors. In this study, we found that Rax+ residual cells in the maturation phase of hypothalamic differentiation in mouse embryonic stem cell (mESC) cultures had similar characteristics to ventral tanycytes. They expressed typical neural stem/progenitor cell markers, including Sox2, vimentin, and nestin, and differentiated into mature neurons and glial cells. Quantitative RT-PCR analysis showed that Rax+ residual cells expressed Fgf-10, Fgf-18, and Lhx2, which are expressed by ventral tanycytes. They highly expressed tanycyte-specific genes Dio2 and Gpr50 compared with Rax+ early hypothalamic progenitor cells. Therefore, Rax+ residual cells in the maturation phase of hypothalamic differentiation were considered to be more differentiated and similar to late progenitor cells and tanycytes. They self-renewed and formed neurospheres when cultured with exogenous FGF-2. Additionally, these Rax+ neurospheres differentiated into three neuronal lineages (neurons, astrocytes, and oligodendrocytes), including neuropeptide Y+ neuron, that are reported to be differentiated from ventral tanycytes toward the arcuate nuclei. Thus, Rax+ residual cells were multipotent neural stem/progenitor cells. Rax+ neurospheres were stably passaged and retained high Sox2 expression even after multiple passages. These results suggest the successful induction of Rax+ tanycyte-like cells from mESCs [induced tanycyte-like (iTan) cells]. These hypothalamic neural stem/progenitor cells may have potential in regenerative medicine and as a research tool.

DOI： 10.1210/en.2019-00105

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/s00441-018-2880-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier Ireland Ltd  

Familial neurohypophysial diabetes insipidus (FNDI), characterized by progressive polyuria and loss of arginine vasopressin (AVP) neurons, is an autosomal dominant disorder caused by AVP gene mutations. Our previous studies with FNDI model mice demonstrated that mutant proteins accumulated in the endoplasmic reticulum (ER) of AVP neurons. Here, we examined therapeutic effects of the chemical chaperone 4-phenylbutylate (4-PBA) in FNDI mice. Treatment with 4-PBA reduced mutant protein accumulation in the ER of FNDI mice and increased AVP release, leading to reduced urine volumes. Furthermore, AVP neuron loss under salt loading was attenuated by 4-PBA treatment. These data suggest that 4-PBA ameliorated mutant protein accumulation in the ER of AVP neurons and thereby prevented FNDI phenotype progression.

DOI： 10.1016/j.neulet.2018.06.013

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nagoya University  

Recently, the effects of stem cell supernatants or exosomes, such as skin wounds, have attracted attention. However, the effects of the induced pluripotent stem (iPS) cell-derived exosomes (iPS-Exos) have not been investigated in detail. Here, we investigated the effects of iPS-Exos on skin wound healing using an animal model. We isolated iPS-Exos from the iPS cell culture media. Control exosomes were isolated from unused iPS cell culture media (M-Exos). We first observed the morphologic characteristics of the isolated exosomes and examined the expression of surface antigens. The effects of these exosomes on the migratory response and proliferation of fibroblasts were analyzed as well. Additionally, using a diabetic ulcer model, the effects of iPS-Exos and M-Exos on skin wound healing were investigated. Transmission electron microscope analysis demonstrated that the size of iPS-Exos (120 ± 25 nm) was significantly larger than that of M-Exos (≤ 100 nm). Flow cytometry analyses showed that iPS-Exos were positive for CD9, CD63, and CD81, whereas they were negative for HLA-ABC and -DR expression. The migratory ability of fibroblasts cocultured with iPS-Exos was shown to be higher than that of the cells cocultured with M-Exos, as demonstrated using scratch assay. Skin wound healing model results showed that the administration of iPS-Exos results in a faster wound closure compared with that observed in the M-Exo group. In conclusion, the results obtained here indicate that iPS-Exos may promote the migration of fibroblasts in vitro and in vivo, suggesting the possibility of using iPS-Exos for the treatment of diabetic ulcer.

DOI： 10.18999/nagjms.80.2.141

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DOI： 10.18999/nagjms.80.2.141

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：John Wiley and Sons Ltd  

Autoimmune hypophysitis (AH) is thought to be an autoimmune disease characterized by lymphocytic infiltration of the pituitary gland. Among AH pathologies, lymphocytic infundibulo-neurohypophysitis (LINH) involves infiltration of the neurohypophysis and/or the hypothalamic infundibulum, causing central diabetes insipidus resulting from insufficiency of arginine vasopressin secretion. The pathophysiological and pathogenetic mechanisms underlying LINH are largely unknown. Clinically, differentiating LINH from other pituitary diseases accompanied by mass lesions, including tumours, has often been difficult, because of similar clinical manifestations. We recently reported that rabphilin-3A is an autoantigen and that anti-rabphilin-3A antibodies constitute a possible diagnostic marker for LINH. However, the involvement of rabphilin-3A in the pathogenesis of LINH remains to be elucidated. This study was undertaken to explore the role of rabphilin-3A in lymphocytic neurohypophysitis and to investigate the mechanism. We found that immunization of mice with rabphilin-3A led to neurohypophysitis. Lymphocytic infiltration was observed in the neurohypophysis and supraoptic nucleus 1 month after the first immunization. Mice immunized with rabphilin-3A showed an increase in the volume of urine that was hypotonic as compared with control mice. Administration of a cocktail of monoclonal anti-rabphilin-3A antibodies did not induce neurohypophysitis. However, abatacept, which is a chimeric protein that suppresses T-cell activation, decreased the number of T cells specific for rabphilin-3A in peripheral blood mononuclear cells (PBMCs). It ameliorated lymphocytic infiltration of CD3+ T cells in the neurohypophysis of mice that had been immunized with rabphilin-3A. Additionally, there was a linear association between the number of T cells specific for rabphilin-3A in PBMCs and the number of CD3+ T cells infiltrating the neurohypophysis. In conclusion, we suggest that rabphilin-3A is a pathogenic antigen, and that T cells specific for rabphilin-3A are involved in the pathogenesis of neurohypophysitis in mice. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley &amp
Sons, Ltd.

DOI： 10.1002/path.5046

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PubMed

Language：English   Publishing type：Research paper (scientific journal)  

The anterior pituitary gland produces several hormones essential for regulation of the somatic endocrine system. Deficiency of these hormones can cause life-threatening diseases, including adrenal crisis. Pituitary tissue generated from human pluripotent stem cells is expected to provide better treatment than current hormone replacement therapy. During early mammalian development, the pituitary anlage (Rathke's pouch) develops from non-neural ectoderm adjacent to the developing ventral hypothalamus. The close interaction between these two tissues is crucial for Rathke's pouch development and involves several signaling molecules. Early exposure of human embryonic stem cells in 3D floating culture to sonic hedgehog and bone morphogenetic protein 4 promoted the cells' differentiation into oral ectoderm and, subsequently, hormone-producing cells such as corticotrophs (adrenocorticotropic hormone-producing cells). The differentiation approach described herein, which induces the formation of pituitary tissue in contact with hypothalamic neural tissue, mimics mammalian pituitary development. The differentiated corticotrophs are functional, responding normally to both release and feedback signals. © 2018 by John Wiley & Sons, Inc.

DOI： 10.1002/cpns.48

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Context: Immune checkpoint inhibitors, including anti-programmed cell death-1 (PD-1) antibodies, have become promising treatments for a variety of advanced malignancies. However, these medicines can cause immune-related adverse events (irAEs), including endocrinopathies. Objective: This study examined the incidence of endocrine irAEs induced by nivolumab. Patients and Main Outcome Measured: Sixty-six patients treated with nivolumab at Nagoya University Hospital were prospectively evaluated for pituitary hormones, thyroid function, antithyroid antibodies (Abs), and glucose levels every 6 weeks after the initiation of nivolumab for 24 weeks. Results: Four out of 66 patients developed destructive thyroiditis, and three patients developed hypothyroidism requiring levothyroxine replacement. The prevalence of positive anti-thyroglobulin Abs (TgAbs) and/or anti-thyroid peroxidase Abs (TPOAbs) at baseline was significantly higher in the group that developed destructive thyroiditis (3/4) compared with the group that did not develop thyroiditis (3/62; P = 0.002). There were no significant differences in other clinical variables between the groups. There were no endocrine irAEs other than destructive thyroiditis during the 24 weeks. The prevalence of TgAbs and/or TPOAbs at baseline was not associated with the development of other irAEs, including pneumonitis, colitis, or skin reactions. Conclusions: Our real-world data showed that destructive thyroiditis was an endocrine irAE that was frequently induced by nivolumab and was significantly associated with positive TgAbs and/or TPOAbs before treatment. Our findings indicate that evaluating these Abs before treatment may help identify patients with a high risk of thyroidal irAEs and may have important clinical benefit.

DOI： 10.1210/js.2017-00432

Web of Science

PubMed

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nature Publishing Group  

Arginine-vasopressin (AVP) neurons exist in the hypothalamus, a major region of the diencephalon, and play an essential role in water balance. Here, we established the differentiation method for AVP-secreting neurons from human embryonic stem cells (hESCs) by recapitulating in vitro the in vivo embryonic developmental processes of AVP neurons. At first, the differentiation efficiency was improved. That was achieved through the optimization of the culture condition for obtaining dorsal hypothalamic progenitors. Secondly, the induced AVP neurons were identified by immunohistochemistry and these neurons secreted AVP after potassium chloride stimulation. Additionally, other hypothalamic neuropeptides were also detected, such as oxytocin, corticotropin-releasing hormone, thyrotropin-releasing hormone, pro-opiomelanocortin, agouti-related peptide, orexin, and melanin-concentrating hormone. This is the first report describing the generation of secretory AVP neurons derived from hESCs. This method will be applicable to research using disease models and, potentially, for regenerative medicine of the hypothalamus.

DOI： 10.1038/s41598-018-22053-x

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

Sequestosome 1 (SQSTM1) also known as ubiquitin-binding protein p62 (p62) is a cargo protein involved in the degradation of misfolded proteins via selective autophagy. Disruption of autophagy and resulting accumulation of misfolded proteins in the endoplasmic reticulum (ER) leads to ER stress. ER stress is implicated in several neurodegenerative diseases and obesity. As knockout of p62 (p62KO) reportedly induces obesity in mice, we examined how p62 contributes to ER stress and the ensuing unfolded protein response (UPR) in hypothalamus using mouse organotypic cultures in the present study. Cultures from p62KO mice showed significantly reduced formation of LC3-GFP puncta, an index of autophagosome formation, in response to the chemical ER stressor thapsigargin compared to wild-type (WT) cultures. Hypothalamic cultures from p62KO mice exhibited higher basal expression of the UPR/ER stress markers CHOP mRNA and ATF4 mRNA than WT cultures. Thapsigargin enhanced CHOP, ATF4, and BiP mRNA as well as p-elF2 alpha protein expression in both NWT and p62KO cultures, but all peak values were greater in p621(0 cultures. A proteasome inhibitor increased p62 expression in WT cultures and upregulated the UPR/ER stress markers CHOP mRNA and ATF4 mRNA in both genotypes, but to a greater extent in p62KO cultures. Therefore, p62 deficiency disturbed autophagosome formation and enhanced both basal and chemically induced ER stress, suggesting that p62 serves to prevent ER stress in mouse hypothalamus by maintaining protein folding capacity. (C) 2017 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.neulet.2017.06.014

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Hypothalamic insulin receptor signaling regulates energy balance and glucose homeostasis via agouti related protein (AgRP). While protein tyrosine phosphatase 1B (PTP1B) is classically known to be a negative regulator of peripheral insulin signaling by dephosphorylating both insulin receptor beta (IR beta) and insulin receptor substrate, the role of PTP1B in hypothalamic insulin signaling remains to be fully elucidated. In the present study, we investigated the role of PTP1B in hypothalamic insulin signaling using PTP1B deficient (KO) mice in vivo and ex vivo. For the in vivo study, hypothalamic insulin resistance induced by a high-fat diet (HFD) improved in KO mice compared to wild-type (WT) mice. Hypothalamic AgRP mRNA expression levels were also significantly decreased in KO mice independent of body weight changes. In an ex vivo study using hypothalamic organotypic cultures, insulin treatment significantly increased the phosphorylation of both IR beta and Akt in the hypothalamus of KO mice compared to WT mice, and also significantly decreased AgRP mRNA expression levels in KO mice. While incubation with inhibitors of phosphatidylinositol-3 kinase (PI3K) had no effect on basal levels of Akt phosphorylation, these suppressed insulin induction of Akt phosphorylation to almost basal levels in WT and KO mice. The inhibition of the PI3K-Akt pathway blocked the downregulation of AgRP mRNA expression in KO mice treated with insulin. These data suggest that PTP1B acts on the hypothalamic insulin signaling via the PI3K-Akt pathway. Together, our results suggest a deficiency of PTP1B improves hypothalamic insulin sensitivity resulting in the attenuation of AgRP mRNA expression under HFD conditions. (C) 2017 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2017.05.019

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Purpose IgG4-related disease is a systemic inflammatory disease characterized by infiltration of IgG4-positive plasma cells into multiple organs, including the pituitary gland. Autoimmunity is thought to be involved in the pathogenesis of IgG4-related disease. The diagnosis of IgG4-related hypophysitis (IgG4-RH) is difficult because its clinical features, such as pituitary swelling and hypopituitarism, are similar to those of other pituitary diseases, including lymphocytic hypophysitis and sellar/suprasellar tumors. The presence and significance of anti-pituitary antibodies (APA) in IgG4-RH is unclear.
Methods In this case-control study, we used single indirect immunofluorescence on human pituitary substrates to assess the prevalence of serum APA in 17 patients with IgG4-RH, 8 control patients with other pituitary diseases (lymphocytic infundibulo-neurohypophysitis, 3; craniopharyngioma, 2; germinoma, 3), and 9 healthy subjects.We further analyzed the endocrine cells targeted by the antibodies using double indirect immunofluorescence.
Results APA were found in 5 of 17 patients with IgG4-RH (29%), and in none of the pituitary controls or healthy subjects. The endocrine cells targeted by the antibodies in the 5 IgG4-RH cases were exclusively corticotrophs. Antibodies were of the IgG1 subclass, rather than IgG4, in all 5 cases, suggesting that IgG4 is not directly involved in the pathogenesis. Finally, antibodies recognized pro-opiomelanocortin in 2 of the cases.
Conclusions Our study suggests that autoimmunity is involved in the pathogenesis of IgG4-RH and that corticotrophs are the main antigenic target, highlighting a possible new diagnostic marker for this condition.

DOI： 10.1007/s11102-016-0780-8

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Protein tyrosine phosphatase 1B (PTP1B) regulates leptin signaling in hypothalamic neurons via the JAK2-STAT3 pathway. PTP1B has also been implicated in the regulation of inflammation in the periphery. However, the role of PTP1B in hypothalamic inflammation, which is induced by a high-fat diet (HFD), remains to be elucidated. Here, we showed that STAT3 phosphorylation (p-STAT3) was increased in microglia in the hypothalamic arcuate nucleus of PTP1B knock-out mice (KO) on a HFD, accompanied by decreased Tnf and increased Il10 mRNA expression in the hypothalamus compared to wild-type mice (WT). In hypothalamic organotypic cultures, incubation with TNF alpha led to increased p-STAT3, accompanied by decreased Tnf and increased Il10 mRNA expression, in KO compared to WT. Incubation with p-STAT3 inhibitors or microglial depletion eliminated the differences in inflammation between genotypes. These data indicate an important role of JAK2-STAT3 signaling negatively regulated by PTP1B in microglia, which attenuates hypothalamic inflammation under HFD conditions. (C) 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

DOI： 10.1016/j.ebiom.2017.01.007

Web of Science

PubMed

Language：English   Publishing type：Part of collection (book)   Publisher：Humana Press Inc.  

The adenohypophysis, which mainly consists of anterior pituitary, plays important roles for endocrine systems by secreting several hormones indispensable for maintaining homeostasis. During early mouse development, the pituitary primordium (called Rathke’s pouch) develops from oral ectoderm adjacent to ventral hypothalamus by interaction between these two tissues. By using mouse embryonic stem cells (ESCs), we recapitulated this in vivo micro-environment of the pituitary development and demonstrated that Rathke’s pouch-like structures were self-formed from three-dimensional (3D) floating culture. The mouse ESC-derived Rathke’s pouch-like structures subsequently differentiated into hormone-producing cells such as corticotrophs and somatotrophs. We have modified this technique for human pluripotent stem cells and recently reported that pituitary placodes can also be generated from human ESCs through a similar process. Here, we describe a protocol for human ESC culture for in vitro generation of 3D pituitary tissue.

DOI： 10.1007/978-1-4939-6949-4_5

Web of Science

Scopus

PubMed

Language：English  

DOI： 10.1507/endocrj.EJ16-0232

Language：English  

DOI： 10.1007/978-3-319-41603-8

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

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DOI： 10.1242/dev.079590.

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DOI： 10.1038/nature10637.

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Total pages：324   Language：Japanese Book type：Scholarly book

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J-GLOBAL

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：(一社)日本内分泌学会  

Language：Japanese   Publisher：(一社)日本内分泌学会  

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

J-GLOBAL

DOI： 10.1016/j.neulet.2018.06.013

PubMed

DOI： 10.1007/s00441-018-2880-4

PubMed

DOI： 10.1002/path.5046

PubMed

DOI： 10.1002/cpns.48

PubMed

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：(一社)日本内分泌学会  

DOI： 10.1210/js.2017-00432

PubMed

DOI： 10.1038/s41598-018-22053-x

PubMed

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

J-GLOBAL

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

J-GLOBAL

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：(一社)日本内分泌学会  

J-GLOBAL

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：(一社)日本内分泌学会  

DOI： 10.1016/j.neulet.2017.06.014

PubMed

DOI： 10.1007/s11102-016-0780-8

PubMed

DOI： 10.1016/j.bbrc.2017.05.019

PubMed

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：(一社)日本内分泌学会  

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Event date： 2008.6

Language：English   Presentation type：Oral presentation (general)  

Country：United States  

Event date： 2008.5

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2008.5

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Country：Japan  

Event date： 2007.11

Language：Japanese   Presentation type：Poster presentation  

Country：Japan  

Event date： 2007.11

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2007.11

Language：Japanese   Presentation type：Poster presentation  

Country：Japan  

Event date： 2007.11

Language：English   Presentation type：Oral presentation (general)  

Country：United States  

Event date： 2007.11

Language：English   Presentation type：Poster presentation  

Country：United States  

Event date： 2007.6

Language：Japanese   Presentation type：Poster presentation  

Country：Japan  

Event date： 2006.6

Language：English   Presentation type：Poster presentation  

Country：United States  

Event date： 2006.6

Language：English   Presentation type：Poster presentation  

Country：United States  

Event date： 2006.5

Language：Japanese   Presentation type：Poster presentation  

Country：Japan  

Event date： 2006.5

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2004.3

Language：Japanese   Presentation type：Poster presentation  

Country：Japan  

Event date： 2003.5

Language：Japanese   Presentation type：Poster presentation  

Country：Japan  

Event date： 2003.5

Language：Japanese   Presentation type：Poster presentation  

Country：Japan  

Event date： 2003.3

Language：Japanese   Presentation type：Poster presentation  

Country：Japan  

Event date： 2003.3

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2002.3

Language：Japanese   Presentation type：Poster presentation  

Country：Japan  

Language：Japanese   Presentation type：Oral presentation (general)  

Language：Japanese   Presentation type：Poster presentation  

Language：Japanese   Presentation type：Oral presentation (general)  

Language：Japanese   Presentation type：Oral presentation (general)  

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Language：English  

Language：Japanese   Presentation type：Oral presentation (general)  

Language：Japanese   Presentation type：Oral presentation (general)  

Language：Japanese   Presentation type：Oral presentation (general)  

Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Language：Japanese   Presentation type：Oral presentation (general)  

Language：Japanese   Presentation type：Poster presentation  

Language：Japanese   Presentation type：Oral presentation (general)  

Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Language：Japanese   Presentation type：Oral presentation (general)  

Language：Japanese   Presentation type：Poster presentation  

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Language：English   Presentation type：Oral presentation (general)  

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Language：Japanese   Presentation type：Poster presentation  

Language：English   Presentation type：Symposium, workshop panel (nominated)  

Language：Japanese   Presentation type：Oral presentation (general)  

Language：English   Presentation type：Oral presentation (general)  

Language：English   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Application no：特願2018-006810  Date applied：2018.1

Application no：特願2016-010940  Date applied：2016.1

Announcement no：特開WO2017126551  Date announced：2017.2

Patent/Registration no：特許7023496  Date issued：2022.2

Applicant：須賀英隆, 小川晃一郎, 笠井貴敏, 有馬寛

Application no：特願2016-010940  Date applied：2016.1

Country of applicant：Domestic  

Applicant：笹井芳樹, 大曽根親文, 須賀英隆

Application no：特願2014-152384  Date applied：2014.7

Country of applicant：Domestic  

Application no：特願2014-152384  Date applied：2014.7

Patent/Registration no：特許6789814  Date registered：2022.2  Date issued：2022.3

Application no：特願2011-239803  Date applied：2011.10

Patent/Registration no：特許6516810  Date registered：2019.4 

Application no：2011-239803．  Date applied：2011

Country of applicant：Domestic  

Application no：2007-232310  Date applied：2007

Country of applicant：Domestic  

Applicant：東海国立大学機構,住友ファーマ株式会社、住友化学株式会社

Application no：特願2023-021207 

Date announced：2023.2