Yoshio Fujitani

Understanding the mechanisms that regulate cell identity acquisition and cell proliferation is crucial, not only for elucidating cellular functions but also for clarifying the pathogenesis of various diseases, including neoplasms. Pancreatic endocrine cells typically express a single hormone, and their numbers are tightly regulated. Contrary to this general principle, in this study, we found that proglucagon-deficient mice exhibit a significant increase in the number of glucagon (GCG) and pancreatic polypeptide (PP) double-positive cells (GCG⁺ PP⁺ cells), together with the hyperplasia of both PP and α cells. Similarly, glucagon receptor-deficient mice displayed PP-cell hyperplasia and an increased number of GCG⁺ PP⁺ cells, with PP-cell replication implicated in this process. mTOR signaling was activated in GCG⁺ PP⁺ cells, suggesting its involvement in endocrine differentiation. Furthermore, impaired hepatic GCG signaling led to elevated plasma amino acid levels, which in turn promoted pancreatic endocrine cell proliferation and disrupted the maintenance of cellular identity in mice. Moreover, we found that increased glutamine levels promote GCG⁺ PP⁺ cell formation via mTOR signaling, revealing a novel regulatory mechanism underlying pancreatic endocrine cell plasticity. These findings provide new insights into endocrine cell regulation, and may contribute to a better understanding of pancreatic neuroendocrine tumor development, as well as the identification of novel therapeutic strategies.

Understanding the mechanisms that regulate cellular identity and proliferation is crucial for elucidating cellular functions. Under normal conditions, pancreatic endocrine cells express only a single hormone, and their numbers are tightly regulated. Contrary to this general principle, our study revealed a significant increase in glucagon (GCG) and pancreatic polypeptide (PP) double-positive cells (GCG ⁺ PP ⁺ double-positive cells), along with hyperplasia of both PP and α cells in proglucagon-deficient mice. Similarly, systemic glucagon receptor-deficient mice exhibited PP-cell hyperplasia and an increase in GCG ⁺ PP ⁺ double-positive cells, with enhanced PP-cell self-replication observed at 4 weeks and the appearance of GCG ⁺ PP ⁺ double-positive cells at 10 weeks. Liver-specific glucagon receptor-deficient mice induced similar effects, which were linked to hyperaminoacidemia. Elevated glutamine levels were found to promote GCG ⁺ PP ⁺ double-positive cell formation via mTOR signaling, suggesting a molecular mechanism driving pancreatic endocrine cell plasticity. Collectively, these findings indicate that increased plasma amino acid levels caused by impaired glucagon action in the liver promotes the proliferation of α cells and PP cells, and disturbs their cellular identity maintenance.

Metabolic diseases are caused by a prolonged energy imbalance, and adipose tissue is known to be the main contributor. We previously reported that ZIP13, an Slc39a transporter whose deficiency causes Ehlers-Danlos syndrome spondylocheirodysplastic type 3 associated with lipoatrophy, inhibits the adipocyte browning pathway by modulating intracellular zinc status. The precise mechanisms of how ZIP13 regulates the homeostasis of adipose tissue remain unclear and therefore, we investigated the role of ZIP13 in mature adipocytes using adipocyte-specific Zip13 -deficient mice. We herein demonstrate that these mice show accelerated lipolysis and reduced respiratory exchange ratio. In addition, abundance of iron and zinc balance were altered during differentiation in normal adipocytes, whereas iron distribution was substantially affected in Zip13 -deficient adipocytes, which downregulated PDE activity and enhanced β-adrenergic receptor signaling pathways. Importantly, we confirmed that ZIP13 could transport both zinc and iron, using the Xenopus oocyte transport system and in silico structural dynamics simulations, and that the defect in iron distribution perturbs proper lipolysis. Together, these results illustrate that ZIP13 acts as a key regulator for lipolysis in adipocytes via the proper use of metals, and that the ZIP13-iron axis plays an important role in regulation of lipid metabolism.

The Ppy gene encodes pancreatic polypeptide (PP) secreted by PP‐ or γ‐cells, which are a subtype of endocrine cells localised mainly in the islet periphery. For a detailed characterisation of PP cells, we aimed to establish PP cell lines. To this end, we generated a mouse model harbouring the SV40 large T antigen (TAg) in the Rosa26 locus, which is expressed upon Ppy‐promoter‐mediated Cre–loxP recombination. Whereas Insulin1‐CreERT‐mediated TAg expression in beta cells resulted in insulinoma, surprisingly, Ppy‐Cre‐mediated TAg expression resulted in the malignant transformation of Ppy‐lineage cells. These mice showed distorted islet structural integrity at 5 days of age compared with normal islets. CK19⁺ duct‐like lesions contiguous with the islets were observed at 2 weeks of age, and mice developed aggressive pancreatic ductal adenocarcinoma (PDAC) at 4 weeks of age, suggesting that PDAC can originate from the islet/endocrine pancreas. This was unexpected as PDAC is believed to originate from the exocrine pancreas. RNA‐sequencing analysis of Ppy‐lineage islet cells from 7‐day‐old TAg⁺ mice showed a downregulation and an upregulation of endocrine and exocrine genes, respectively, in addition to the upregulation of genes and pathways associated with PDAC. These results suggest that the expression of an oncogene in Ppy‐lineage cells induces a switch from endocrine cell fate to PDAC. Our findings demonstrate that Ppy‐lineage cells may be an origin of PDAC and may provide novel insights into the pathogenesis of pancreatic cancer, as well as possible therapeutic strategies. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Aims/hypothesis Glucagon-expressing pancreatic alpha cells have attracted much attention for their plasticity to transdifferentiate into insulin-producing beta cells; however, it remains unclear precisely when, and from where, alpha cells emerge and what regulates alpha cell fate. We therefore explored the spatial and transcriptional heterogeneity of alpha cell differentiation using a novel time-resolved reporter system. Methods We established the mouse model, ‘Gcg-Timer’, in which newly generated alpha cells can be distinguished from more-differentiated cells by their fluorescence. Fluorescence imaging and transcriptome analysis were performed with Gcg-Timer mice during the embryonic and postnatal stages. Results Fluorescence imaging and flow cytometry demonstrated that green fluorescence-dominant cells were present in Gcg-Timer mice at the embryonic and neonatal stages but not after 1 week of age, suggesting that alpha cell neogenesis occurs during embryogenesis and early neonatal stages under physiological conditions. Transcriptome analysis of Gcg-Timer embryos revealed that the mRNAs related to angiogenesis were enriched in newly generated alpha cells. Histological analysis revealed that some alpha cells arise close to the pancreatic ducts, whereas the others arise away from the ducts and adjacent to the blood vessels. Notably, when the glucagon signal was suppressed by genetic ablation or by chemicals, such as neutralising glucagon antibody, green-dominant cells emerged again in adult mice. Conclusions/interpretation Novel time-resolved analysis with Gcg-Timer reporter mice uncovered spatiotemporal features of alpha cell neogenesis that will enhance our understanding of cellular identity and plasticity within the islets. Data availability Raw and processed RNA sequencing data for this study has been deposited in the Gene Expression Omnibus under accession number GSE229090. Graphical Abstract

Metal homeostasis is tightly regulated in cells and organisms, and its disturbance is frequently observed in some diseases such as neurodegenerative diseases and metabolic disorders. Previous studies suggest that zinc and iron are necessary for the normal functions of pancreatic β cells. However, the distribution of elements in normal conditions and the pathophysiological significance of dysregulated elements in the islet in diabetic conditions have remained unclear. In this study, to investigate the dynamics of elements in the pancreatic islets of a diabetic mouse model expressing human islet amyloid polypeptide (hIAPP): hIAPP transgenic (hIAPP-Tg) mice, we performed imaging analysis of elements using synchrotron scanning X-ray fluorescence microscopy and quantitative analysis of elements using inductively coupled plasma mass spectrometry. We found that in the islets, zinc significantly decreased in the early stage of diabetes, while iron gradually decreased concurrently with the increase in blood glucose levels of hIAPP-Tg mice. Notably, when zinc and/or iron were decreased in the islets of hIAPP-Tg mice, dysregulation of glucose-stimulated mitochondrial respiration was observed. Our findings may contribute to clarifying the roles of zinc and iron in islet functions under pathophysiological diabetic conditions.

Pancreatic polypeptide (PP), secreted from γ cells of the islets of Langerhans, is a 36 amino-acid peptide encoded by the Ppy gene. Although previous studies have reported that PP causes a decrease in appetite, the molecular mechanism that regulates PP secretion has not been fully elucidated. Lack of understanding of the regulatory mechanism of PP secretion may be partially owing to the lack of assay systems that can specifically detect PP. We recently developed the mouse monoclonal antibody 23-2D3 that specifically recognizes PP. In the present study, we developed a sandwich enzyme-linked immunosorbent assay for the measurement of mouse PP, and directly monitored intracellular Ca²⁺ concentrations in Ppy-expressing cells from a newly developed reporter mouse. Using these systems, we identified agonists, such as carbachol and glucose-dependent insulinotropic polypeptide (GIP), which stimulate PP secretion. We further demonstrated that, unlike the case of GIP-induced insulin secretion from β cells, there is a unique mechanism by which PP secretion is triggered by an increase in intracellular Ca²⁺ concentrations via voltage-dependent calcium channels even in low-glucose conditions.

Aims/Introduction: Several research groups have reported methods for quantifying pancreatic beta cell (β‐cell) injury by measuring β‐cell‐specific CpG unmethylation of the insulin gene in circulation using digital droplet PCR or next‐generation sequencing. However, these methods have certain disadvantages, such as the need to consider the background signal owing to the small number of target CpG sites and the need for unique equipment.Materials and Methods: We established a novel method for detecting four CpG unmethylations of the insulin gene using two‐step amplification refractory mutation system PCR. We applied it to type 1 diabetes (T1D) patients with a wide range of disease durations and to healthy adults.Results: The assay showed high linearity and could detect a single copy of unmethylated insulin DNA in experiments using methylated and unmethylated plasmid DNA. The unmethylated insulin DNA level in the type 1 diabetes group, whose β‐cell mass was considerably reduced, was similar to that of healthy adults. An inverse correlation was observed between copy number and disease duration in patients with unmethylated insulin DNA‐positive type 1 diabetes.Conclusions: We developed a novel method for detecting unmethylated insulin DNA in circulation that can be performed using a conventional real‐time PCR system. This method would be useful for analyzing dynamic profiles of β‐cells in human disease such as type 1 diabetes.