Possible involvement of zinc transporter ZIP13 in myogenic differentiation.

Ehlers-Danlos syndrome spondylodysplastic type 3 (EDSSPD3, OMIM 612350) is an inherited recessive connective tissue disorder that is caused by loss of function of SLC39A13/ZIP13, a zinc transporter belonging to the Slc39a/ZIP family. We previously reported that patients with EDSSPD3 harboring a homozygous loss of function mutation (c.221G > A, p.G64D) in ZIP13 exon 2 (ZIP13G64D) suffer from impaired development of bone and connective tissues, and muscular hypotonia. However, whether ZIP13 participates in the early differentiation of these cell types remains unclear. In the present study, we investigated the role of ZIP13 in myogenic differentiation using a murine myoblast cell line (C2C12) as well as patient-derived induced pluripotent stem cells (iPSCs). We found that ZIP13 gene expression was upregulated by myogenic stimulation in C2C12 cells, and its knockdown disrupted myotubular differentiation. Myocytes differentiated from iPSCs derived from patients with EDSSPD3 (EDSSPD3-iPSCs) also exhibited incomplete myogenic differentiation. Such phenotypic abnormalities of EDSSPD3-iPSC-derived myocytes were corrected by genomic editing of the pathogenic ZIP13G64D mutation. Collectively, our findings suggest the possible involvement of ZIP13 in myogenic differentiation, and that EDSSPD3-iPSCs established herein may be a promising tool to study the molecular basis underlying the clinical features caused by loss of ZIP13 function.

Zinc and iron dynamics in human islet amyloid polypeptide-induced diabetes mouse model.

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 ß 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.

Overexpression of Pdx1, reduction of p53, or deletion of CHOP attenuates pancreas hypoplasia in mice with pancreas-specific O-GlcNAc transferase deletion.

Deletion of O-GlcNAc transferase (Ogt) in pancreatic epithelial progenitor cells results in pancreatic hypoplasia at birth, partly due to increased apoptosis during embryonic development. Constitutive loss of Ogt in ß-cells results in increased ER stress and apoptosis, and in the Ogt-deficient pancreas, transcriptomic data previously revealed both tumor suppressor protein p53 and pancreatic duodenal homeobox 1 (Pdx1), key cell survival proteins in the developing pancreas, as upstream regulators of differentially expressed genes. However, the specific roles of these genes in pancreatic hypoplasia are unclear. In this study, we explored the independent roles of p53, ER stress protein CHOP, and Pdx1 in pancreas development and their use in the functional rescue of pancreatic hypoplasia in the context of Ogt loss. Using in vivo genetic manipulation and morphometric analysis, we show that Ogt plays a key regulatory role in pancreas development. Heterozygous, but not homozygous, loss of pancreatic p53 afforded a partial rescue of ß-cell, α-cell, and exocrine cell masses, while whole body loss of CHOP afforded a partial rescue in pancreas weight and a full rescue in exocrine cell mass. However, neither was sufficient to fully mitigate pancreatic hypoplasia at birth in the Ogt-deficient pancreas. Furthermore, overexpression of Pdx1 in the pancreatic epithelium resulted in partial rescues in pancreas weight and ß-cell mass in the Ogt loss background. These findings highlight the requirement of Ogt in pancreas development by targeting multiple proteins such as transcription factor Pdx1 and p53 in the developing pancreas.

Establishment of an enzyme-linked immunosorbent assay for mouse pancreatic polypeptide clarifies the regulatory mechanism of its secretion from pancreatic γ cells.

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 ß 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.

Characterisation of Ppy-lineage cells clarifies the functional heterogeneity of pancreatic beta cells in mice.

AIMS/HYPOTHESIS: Pancreatic polypeptide (PP) cells, which secrete PP (encoded by the Ppy gene), are a minor population of pancreatic endocrine cells. Although it has been reported that the loss of beta cell identity might be associated with beta-to-PP cell-fate conversion, at present, little is known regarding the characteristics of Ppy-lineage cells. METHODS: We used Ppy-Cre driver mice and a PP-specific monoclonal antibody to investigate the association between Ppy-lineage cells and beta cells. The molecular profiles of endocrine cells were investigated by single-cell transcriptome analysis and the glucose responsiveness of beta cells was assessed by Ca2+ imaging. Diabetic conditions were experimentally induced in mice by either streptozotocin or diphtheria toxin. RESULTS: Ppy-lineage cells were found to contribute to the four major types of endocrine cells, including beta cells. Ppy-lineage beta cells are a minor subpopulation, accounting for 12-15% of total beta cells, and are mostly (81.2%) localised at the islet periphery. Unbiased single-cell analysis with a Ppy-lineage tracer demonstrated that beta cells are composed of seven clusters, which are categorised into two groups (i.e. Ppy-lineage and non-Ppy-lineage beta cells). These subpopulations of beta cells demonstrated distinct characteristics regarding their functionality and gene expression profiles. Ppy-lineage beta cells had a reduced glucose-stimulated Ca2+ signalling response and were increased in number in experimental diabetes models. CONCLUSIONS/INTERPRETATION: Our results indicate that an unexpected degree of beta cell heterogeneity is defined by Ppy gene activation, providing valuable insight into the homeostatic regulation of pancreatic islets and future therapeutic strategies against diabetes. DATA AVAILABILITY: The single-cell RNA sequence (scRNA-seq) analysis datasets generated in this study have been deposited in the Gene Expression Omnibus (GEO) under the accession number GSE166164 ( www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE166164 ).

ATF4-mediated transcriptional regulation protects against ß-cell loss during endoplasmic reticulum stress in a mouse model.

OBJECTIVE: Activating transcription factor 4 (ATF4) is a transcriptional regulator of the unfolded protein response and integrated stress response (ISR) that promote the restoration of normal endoplasmic reticulum (ER) function. Previous reports demonstrated that dysregulation of the ISR led to development of severe diabetes. However, the contribution of ATF4 to pancreatic ß-cells remains poorly understood. In this study, we aimed to analyze the effect of ISR enhancer Sephin1 and ATF4-deficient ß-cells to clarify the role of ATF4 in ß-cells under ER stress conditions. METHODS: To examine the role of ATF4 in vivo, ISR enhancer Sephin1 (5 mg/kg body weight, p.o.) was administered daily for 21 days to Akita mice. We also established ß-cell-specific Atf4 knockout (ßAtf4-KO) mice that were further crossed with Akita mice. These mice were analyzed for characteristics of diabetes, ß-cell function, and morphology of the islets. To identify the downstream factors of ATF4 in ß-cells, the islets of ßAtf4-KO mice were subjected to cDNA microarray analyses. To examine the transcriptional regulation by ATF4, we also performed in situ PCR analysis of pancreatic sections from mice and ChIP-qPCR analysis of CT215 ß-cells. RESULTS: Administration of the ISR enhancer Sephin1 improved glucose metabolism in Akita mice. Sephin1 also increased the insulin-immunopositive area and ATF4 expression in the pancreatic islets. Akita/ßAtf4-KO mice exhibited dramatically exacerbated diabetes, shown by hyperglycemia at an early age, as well as a remarkably short lifespan owing to diabetic ketoacidosis. Moreover, the islets of Akita/ßAtf4-KO mice presented increased numbers of cells stained for glucagon, somatostatin, and pancreatic polypeptide and increased expression of aldehyde dehydrogenase 1 family member 3, a marker of dedifferentiation. Using microarray analysis, we identified atonal BHLH transcription factor 8 (ATOH8) as a downstream factor of ATF4. Deletion of ATF4 in ß-cells showed reduced Atoh8 expression and increased expression of undifferentiated markers, Nanog and Pou5f1. Atoh8 expression was also abolished in the islets of Akita/ßAtf4-KO mice. CONCLUSIONS: We conclude that transcriptional regulation by ATF4 maintains ß-cell identity via ISR modulation. This mechanism provides a promising target for the treatment of diabetes.

Pancreatic Ppy-expressing γ-cells display mixed phenotypic traits and the adaptive plasticity to engage insulin production.

The cellular identity of pancreatic polypeptide (Ppy)-expressing γ-cells, one of the rarest pancreatic islet cell-type, remains elusive. Within islets, glucagon and somatostatin, released respectively from α- and δ-cells, modulate the secretion of insulin by ß-cells. Dysregulation of insulin production raises blood glucose levels, leading to diabetes onset. Here, we present the genetic signature of human and mouse γ-cells. Using different approaches, we identified a set of genes and pathways defining their functional identity. We found that the γ-cell population is heterogeneous, with subsets of cells producing another hormone in addition to Ppy. These bihormonal cells share identity markers typical of the other islet cell-types. In mice, Ppy gene inactivation or conditional γ-cell ablation did not alter glycemia nor body weight. Interestingly, upon ß-cell injury induction, γ-cells exhibited gene expression changes and some of them engaged insulin production, like α- and δ-cells. In conclusion, we provide a comprehensive characterization of γ-cells and highlight their plasticity and therapeutic potential.

Single-Cell Transcriptome Analysis Dissects the Replicating Process of Pancreatic Beta Cells in Partial Pancreatectomy Model.

Heterogeneity of gene expression and rarity of replication hamper molecular analysis of ß-cell mass restoration in adult pancreas. Here, we show transcriptional dynamics in ß-cell replication process by single-cell RNA sequencing of murine pancreas with or without partial pancreatectomy. We observed heterogeneity of Ins1-expressing ß-cells and identified the one cluster as replicating ß-cells with high expression of cell proliferation markers Pcna and Mki67. We also recapitulated cell cycle transition accompanied with switching expression of cyclins and E2F transcription factors. Both transient activation of endoplasmic reticulum stress responders like Atf6 and Hspa5 and elevated expression of tumor suppressors like Trp53, Rb1, and Brca1 and DNA damage responders like Atm, Atr, Rad51, Chek1, and Chek2 during the transition to replication associated fine balance of cell cycle progression and protection from DNA damage. Taken together, these results provide a high-resolution map depicting a sophisticated genetic circuit for replication of the ß-cells.

Leukotriene A4 hydrolase deficiency protects mice from diet-induced obesity by increasing energy expenditure through neuroendocrine axis.

Obesity is a health problem worldwide, and brown adipose tissue (BAT) is important for energy expenditure. Here, we explored the role of leukotriene A4 hydrolase (LTA4 H), a key enzyme in the synthesis of the lipid mediator leukotriene B4 (LTB4 ), in diet-induced obesity. LTA4 H-deficient (LTA4 H-KO) mice fed a high-fat diet (HFD) showed a lean phenotype, and bone-marrow transplantation studies revealed that LTA4 H-deficiency in non-hematopoietic cells was responsible for this lean phenotype. LTA4 H-KO mice exhibited greater energy expenditure, but similar food intake and fecal energy loss. LTA4 H-KO BAT showed higher expression of thermogenesis-related genes. In addition, the plasma thyroid-stimulating hormone and thyroid hormone concentrations, as well as HFD-induced catecholamine secretion, were higher in LTA4 H-KO mice. In contrast, LTB4 receptor (BLT1)-deficient mice did not show a lean phenotype, implying that the phenotype of LTA4 H-KO mice is independent of the LTB4 /BLT1 axis. These results indicate that LTA4 H mediates the diet-induced obesity by reducing catecholamine and thyroid hormone secretion.

Rubicon in pancreatic beta cells plays a limited role in maintaining glucose homeostasis following increased insulin resistance.

Autophagy has been reported to play a crucial role in the maintenance of intracellular homeostasis, including in pancreatic beta cells. Rubicon, which interacts with the phosphoinositide 3-kinase (PI3K) complex, through autophagy-related 14 (ATG14), is among the few autophagy regulators that have been reported to inhibit autophagic flux to date and the deletion of Rubicon has been shown to increase autophagic flux. Based on previous results showing a causal relationship between autophagic dysfunction and pancreatic beta-cell impairment, we hypothesized that the deletion of Rubicon in pancreatic beta cells would improve cell integrity and confer protective effects. To test this hypothesis, we first confirmed that Rubicon knockdown (KD) promoted autophagic flux in ßTC3 pancreatic beta-cell line. Next, we generated pancreatic beta-cell-specific Rubicon knockout (ßKO) mice, by administering tamoxifen to Rubiconflox/flox:MIP-Cre-ERT mice, which showed normal glucose tolerance and insulin secretion under a normal chow diet, despite successful gene recombination. We also attempted to increase insulin resistance by feeding the mice with a high-fat diet for an additional 2 months to find little differences among the parameters evaluated for glucose metabolism. Finally, severe insulin resistance was induced with insulin receptor antagonist treatment, which resulted in comparable glucose homeostasis measurements between Rubicon ßKO and control mice. In summary, these results suggest that in pancreatic beta cells, Rubicon plays a limited role in the maintenance of systemic glucose homeostasis.

How do you make new ß-cells in humans? An attempt to create human ß-cells from α-cells of brain-dead donors.

Ex vivo reprogramming of human α-cells toward functional ß-cells by adenoviral transduction of key transcription factors.

Development of monoclonal mouse antibodies that specifically recognize pancreatic polypeptide.

Pancreatic polypeptide (PP) is a 36-amino acid peptide encoded by the Ppy gene, which is produced by a small population of cells located in the periphery of the islets of Langerhans. Owing to the high amino acid sequence similarity among neuropeptide Y family members, antibodies against PP that are currently available are not convincingly specific to PP. Here we report the development of mouse monoclonal antibodies that specifically bind to PP. We generated Ppy knockout (Ppy-KO) mice in which the Ppy-coding region was replaced by Cre recombinase. The Ppy-KO mice were immunized with mouse PP peptide, and stable hybridoma cell lines producing anti-PP antibodies were isolated. Firstly, positive clones were selected in an enzyme-linked immunosorbent assay for reactivity with PP coupled to bovine serum albumin. During the screening, hybridoma clones producing antibodies that cross-react to the peptide YY (PYY) were excluded. In the second screening, hybridoma clones in which their culture media produce no signal in Ppy-KO islets but detect specific cells in the peripheral region of wild-type islets, were selected. Further studies demonstrated that the selected monoclonal antibody (23-2D3) specifically recognizes PP-producing cells, not only in mouse, but also in human and rat islets. The monoclonal antibodies with high binding specificity for PP developed in this study will be fundamental for future studies towards elucidating the expression profiles and the physiological roles of PP.

Enhanced Expression of the Key Mitosis Regulator Cyclin B1 Is Mediated by PDZ-Binding Kinase in Islets of Pregnant Mice.

The proliferation of pancreatic ß cells is enhanced to enable an increase in ß-cell mass and to compensate for insulin resistance during pregnancy. To elucidate the mechanisms involved, we previously investigated islets from pregnant and nonpregnant mice by gene expression profiling and found that the expression of postsynaptic density-95/Discs large/zonula occludens-1 (PDZ)-binding kinase (Pbk), a member of the mitogen-activated protein kinase kinase family, is increased in pregnant mouse islets compared with control mouse islets. Among the pregnancy hormones, treatment with estradiol upregulated Pbk expression. Inhibition of Pbk expression using a small interfering RNA for Pbk reduced bromodeoxyuridine incorporation in mouse insulinoma 6 cells, which was accompanied by a decreased expression of Ccnb1, a regulatory gene involved in mitosis. Ccnb1 expression was augmented in mouse islets during pregnancy. The forced expression of Pbk using an adenovirus system in isolated mouse islets increased Ccnb1 expression, and the Pbk inhibitor HI-TOPK-032 suppressed Ccnb1 expression in islets isolated from pregnant mice. Our results suggest that Pbk contributes to the expansion of islets during pregnancy and that Ccnb1 may assist Pbk in its role in ß-cell proliferation.

Heterogeneity of autophagic status in pancreatic ß cells under metabolic stress.

Autophagy in ß cells has been demonstrated to play a pivotal role in cellular homeostasis and the progression of glucose intolerance. Although autophagic activity is affected by metabolic stress both in vivo and in vitro, it remains unclear as to what extent the autophagic status in each ß cell is different from its neighboring cells. To address this question, GFP-LC3 reporter mice, which can visualize the autophagic status of each ß cell as green-fluorescent puncta, were crossed with obese diabetic db/db mice. Imaging of green-fluorescent puncta in the islets of GFP-LC3 mice revealed that ß cells are a heterogeneous population, as the density of GFP-LC3 puncta in each cell was variable. Furthermore, the variability was greater in GFP-LC3; db/db mice than in non-diabetic GFP-LC3; db/+ mice. Furthermore, when GFP-LC3 mice were treated with a low dose of S961, which antagonizes insulin signaling without inducing overt hyperglycemia, the number of ß cells with a high density of GFP puncta was increased, suggesting that insulin resistance affects autophagic status independently of glucose profiles. These results suggest that pancreatic ß cells under metabolic stress are heterogeneous regarding their autophagic status, which provides insights into the cellular dynamics of each ß cell rather than the whole ß-cell population.

Activation of GLP-1 and gastrin signalling induces in vivo reprogramming of pancreatic exocrine cells into beta cells in mice.

AIMS/HYPOTHESIS: Lineage conversion of non-beta cells into insulin-producing cells has been proposed as a therapy for the cure of diabetes. Glucagon-like peptide-1 (GLP-1) and its derivatives can induce beta cell neogenesis in vitro and beta cell mass expansion in vivo, but GLP-1 signalling has not been shown to regulate cell fate decisions in vivo. We therefore tested the impact of GLP-1 receptor (GLP1R) expression on beta cell differentiation in vivo. METHODS: Mice overexpressing GLP1R in pancreatic exocrine cells were generated by Cre-mediated recombination in sex-determining region Y-box 9 (SOX9)-expressing cells and then treated with exendin-4 and/or gastrin. Histological analysis was performed to detect cellular reprogramming from the exocrine lineage into insulin-producing cells. RESULTS: Whereas no newly generated beta cells were detected in the mice treated with exendin-4 alone, treatment with gastrin only induced the conversion of exocrine cells into insulin-producing cells. Furthermore, the overexpression of GLP1R, together with gastrin and exendin-4, synergistically promoted beta cell neogenesis accompanied by the formation of islet-like clusters. These newly generated beta cells expressed beta cell specific transcription factors, such as pancreatic and duodenal homeobox 1 (PDX1), NK6 homeobox 1 (NKX6.1) and musculoaponeurotic fibrosarcoma oncogene family A (MafA). These mice showed no histological evidence of pancreatitis or pancreatic dysplasia in their acini and had normal plasma amylase levels. CONCLUSIONS/INTERPRETATION: Activation of GLP-1 and gastrin signalling induces beta cell neogenesis in the exocrine lineage without any deleterious pancreatic changes, which may lead to a potential therapy to cure diabetes by generating surrogate beta cells.

Expression mechanism of tryptophan hydroxylase 1 in mouse islets during pregnancy.

Serotonin signaling plays key roles in augmentation of pancreatic ß-cell function during pregnancy. Increased expression of tryptophan hydroxylase 1 (Tph1), a rate-limiting enzyme for serotonin synthesis by lactogenic hormones, is involved in this phenomenon. To investigate its mechanisms, we here performed 5'-RACE and identified ß-cell-specific transcription initiation sites for Tph1. Prolactin enhanced the expression of mRNA containing these exons; however, reporter gene plasmids containing the proximal 5'-flanking region of these exons did not show prolactin responsiveness in MIN6 cells. Prolactin-induced Tph1 expression was inhibited by a Jak2 inhibitor and was partially inhibited by an MEK1/2 or PI3K inhibitor. Therefore, we analyzed interferon γ-activated sequences (GAS) and found GAS-A about 9-kbp upstream of the transcription start site. The reporter gene plasmid containing the GAS-A region linked to a heterologous promoter showed increased promoter activity by prolactin, which was inhibited by the forced expression of a dominant-negative mutant form of Stat5A and a Jak2 inhibitor. Chromatin immunoprecipitation analysis showed that prolactin treatment augmented Stat5 binding to the GAS-A region in MIN6 cells, as well as in isolated mouse islets, and that Stat5 recognized the GAS-A region in pregnant mouse islets. In addition, the transactivation activity of Stat5 was enhanced by prolactin through the Erk and PI3K pathways in MIN6 cells. Finally, serotonin expression was attenuated in islets of ß-cell-specific Stat5-deficient mice compared with that of control littermates during pregnancy. Our findings suggest that prolactin-induced Tph1 expression is mediated by the activation of Jak2/Stat5, Erk, and PI3K pathways in ß cells.

SET7/9 Enzyme Regulates Cytokine-induced Expression of Inducible Nitric-oxide Synthase through Methylation of Lysine 4 at Histone 3 in the Islet ß Cell.

SET7/9 is an enzyme that methylates histone 3 at lysine 4 (H3K4) to maintain euchromatin architecture. Although SET7/9 is enriched in islets and contributes to the transactivation of ß cell-specific genes, including Ins1 and Slc2a, SET7/9 has also been reported to bind the p65 subunit of nuclear factor κB in non-ß cells and modify its transcriptional activity. Given that inflammation is a central component of ß cell dysfunction in Type 1 and Type 2 diabetes, the aim of this study was to elucidate the role of SET7/9 in proinflammatory cytokine signaling in ß cells. To induce inflammation, ßTC3 insulinoma cells were treated with IL-1ß, TNF-α, and IFN-γ. Cytokine treatment led to increased expression of inducible nitric-oxide synthase, which was attenuated by the diminution of SET7/9 using RNA interference. Consistent with previous reports, SET7/9 was co-immunoprecipitated with p65 and underwent cytosolic to nuclear translocation in response to cytokines. ChIP analysis demonstrated augmented H3K4 mono- and dimethylation of the proximal Nos2 promoter with cytokine exposure. SET7/9 was found to occupy this same region, whereas SET7/9 knockdown attenuated cytokine-induced histone methylation of the Nos2 gene. To test this relationship further, islets were isolated from SET7/9-deficient and wild-type mice and treated with IL-1ß, TNF-α, and IFN-γ. Cytokine-induced Nos2 expression was reduced in the islets from SET7/9 knock-out mice. Together, our findings suggest that SET7/9 contributes to Nos2 transcription and proinflammatory cytokine signaling in the pancreatic ß cell through activating histone modifications.

Human IAPP-induced pancreatic ß cell toxicity and its regulation by autophagy.

Pancreatic islets in patients with type 2 diabetes mellitus (T2DM) are characterized by loss of ß cells and formation of amyloid deposits derived from islet amyloid polypeptide (IAPP). Here we demonstrated that treatment of INS-1 cells with human IAPP (hIAPP) enhances cell death, inhibits cytoproliferation, and increases autophagosome formation. Furthermore, inhibition of autophagy increased the vulnerability of ß cells to the cytotoxic effects of hIAPP. Based on these in vitro findings, we examined the pathogenic role of hIAPP and its relation to autophagy in hIAPP-knockin mice. In animals fed a standard diet, hIAPP had no toxic effects on ß cell function; however, hIAPP-knockin mice did not exhibit a high-fat-diet-induced compensatory increase in ß cell mass, which was due to limited ß cell proliferation and enhanced ß cell apoptosis. Importantly, expression of hIAPP in mice with a ß cell-specific autophagy defect resulted in substantial deterioration of glucose tolerance and dispersed cytoplasmic expression of p62-associated toxic oligomers, which were otherwise sequestrated within p62-positive inclusions. Together, our results indicate that increased insulin resistance in combination with reduced autophagy may enhance the toxic potential of hIAPP and enhance ß cell dysfunction and progression of T2DM.

Repetitive hypoglycemia increases circulating adrenaline level with resultant worsening of intimal thickening after vascular injury in male Goto-Kakizaki rat carotid artery.

Hypoglycemia associated with diabetes management is a potential risk for cardiovascular diseases. However, the effect of hypoglycemic episodes including a surge of sympathetic activity on the progression of neointima formation after vascular injury remains largely unknown. In this study, insulin was injected intraperitoneally into nonobese diabetic Goto-Kakizaki (GK) rats, once every 3 days for 4 weeks after balloon injury of carotid artery to induce hypoglycemia. Then, we evaluated balloon injury-induced neointima formation. Insulin treatment enhanced neointima formation and increased the number of proliferating cell nuclear antigen (PCNA)-positive cells in the carotid artery. Injection of glucose with insulin prevented hypoglycemia and abrogated intimal thickening. Also, bunazosin, an α1 adrenergic receptor antagonist, prevented intimal thickening and accumulation of PCNA-positive cells induced by insulin treatment despite the presence of concomitant hypoglycemia and high adrenaline levels. Incubation of cultured smooth muscle cells with adrenaline resulted in a significant increase in their proliferation and G0/G1 to S phase progression, which was associated with activation of extracellular signal-regulated kinase, enhanced expression of cell cycle regulatory molecules such as cyclin D1, and cyclin E, and phosphorylation of retinoblastoma protein. These adrenaline-induced effects were abrogated by bunazosin. Our data indicated that increased adrenaline induced by repetitive hypoglycemia promotes intimal thickening and smooth muscle cell proliferation after endothelial denudation in GK rats.

Exendin-4 improves ß-cell function in autophagy-deficient ß-cells.

Autophagy is cellular machinery for maintenance of ß-cell function and mass. The implication of autophagy failure in ß-cells on the pathophysiology of type 2 diabetes and its relation to the effect of treatment of diabetes remains elusive. Here, we found increased expression of p62 in islets of db/db mice and patients with type 2 diabetes mellitus. Treatment with exendin-4, a glucagon like peptide-1 receptor agonist, improved glucose tolerance in db/db mice without significant changes in p62 expression in ß-cells. Also in ß-cell-specific Atg7-deficient mice, exendin-4 efficiently improved blood glucose level and glucose tolerance mainly by enhanced insulin secretion. In addition, we found that exendin-4 reduced apoptotic cell death and increased proliferating cells in the Atg7-deficient islets, and that exendin-4 counteracted thapsigargin-induced cell death of isolated islets augmented by autophagy deficiency. Our results suggest the potential involvement of reduced autophagy in ß-cell dysfunction in type 2 diabetes. Without altering the autophagic state in ß-cells, exendin-4 improves glucose tolerance associated with autophagy deficiency in ß-cells. This is mainly achieved through augmentation of insulin secretion. In addition, exendin-4 prevents apoptosis and increases the proliferation of ß-cells associated with autophagy deficiency, also without altering the autophagic machinery in ß-cells.