[Show abstract][Hide abstract] ABSTRACT: Endoplasmic reticulum (ER) stress-mediated apoptosis may play a crucial role in loss of pancreatic beta cell mass, contributing to the development of diabetes. Here we show that induction of 4E-BP1, the suppressor of the mRNA 5' cap-binding protein eukaryotic initiation factor 4E (eIF4E), is involved in beta cell survival under ER stress. 4E-BP1 expression was increased in islets under ER stress in several mouse models of diabetes. The Eif4ebp1 gene encoding 4E-BP1 was revealed to be a direct target of the transcription factor ATF4. Deletion of the Eif4ebp1 gene increased susceptibility to ER stress-mediated apoptosis in MIN6 beta cells and mouse islets, which was accompanied by deregulated translational control. Furthermore, Eif4ebp1 deletion accelerated beta cell loss and exacerbated hyperglycemia in mouse models of diabetes. Thus, 4E-BP1 induction contributes to the maintenance of beta cell homeostasis during ER stress and is a potential therapeutic target for diabetes.
[Show abstract][Hide abstract] ABSTRACT: Gene transfer with adenovirus vectors has been used extensively for pancreatic islet research. However, infection efficiency varies among reports. We reevaluated the infection efficiency, defined here as the percentage of islet cells expressing transgenes, in mouse islets. When the isolated islets were infected with adenoviruses, the infection efficiency was found to be 30-40% and the transduced cells were distributed in the islet periphery. Collagenase treatment of isolated islets before infection increased the infection efficiency to 70%, but with suppression of glucose-stimulated insulin secretion. To explore more efficient strategies, we employed arterial delivery of virus particles to islets in situ. Delivery of adenovirus (approximately 10(8) particles per pancreas) through the celiac and superior mesenteric arteries is highly efficient, resulting in more than 90% transduction without impairing glucose-stimulated insulin secretion. Arterial delivery of an adenovirus harboring glycerol kinase cDNA allowed us to observe glycerol-stimulated insulin secretion from mouse islets, which was not observed when we employed the conventional method. Furthermore, the arterial delivery method combined with a tetracycline-inducible adenovirus system induced efficient and controlled transgene expression. Our data provide new insights into gene transduction methods using recombinant adenoviruses in mouse islets, and are therefore anticipated to contribute to future basic and clinical islet research applications.
[Show abstract][Hide abstract] ABSTRACT: The WFS1 gene, encoding an endoplasmic reticulum (ER) membrane glycoprotein, is mutated in Wolfram syndrome characterized by diabetes mellitus and optic atrophy. Herein, Ca(2+) dynamics were examined in WFS1-knockdown and -overexpressing HEK293 cells. Studies using ER-targeted Ca(2+)-sensitive photoprotein aequorin demonstrated WFS1 protein to positively modulate ER Ca(2+) levels by increasing the rate of Ca(2+) uptake. Furthermore, Ca(2+) imaging with Fura-2 showed the magnitude of the store-operated Ca(2+) entry to parallel WFS1 expression levels. These data indicate that WFS1 protein participates in the regulation of cellular Ca(2+) homeostasis, at least partly, by modulating the filling state of the ER Ca(2+) store.
[Show abstract][Hide abstract] ABSTRACT: Wolfram syndrome, an autosomal recessive disorder associated with diabetes mellitus and optic atrophy, is caused by mutations in the WFS1 gene encoding an endoplasmic reticulum (ER) membrane protein. Herein, we report that pancreatic islets of wfs1-deficient mice exhibit increases in phosphorylation of RNA-dependent protein kinase-like ER kinase, chaperone gene expressions and active XBP1 protein levels, indicating an enhanced ER stress response. We established wfs1-deficient MIN6 clonal beta-cells by crossing wfs1-deficient mice with mice expressing simian virus 40 large T antigen in beta-cells. These cells show essentially the same alterations in ER stress responses as wfs1-deficient islets, which were reversed by re-expression of WFS1 protein or overexpression of GRP78, a master regulator of the ER stress response. In contrast, these changes are not observed in heart, skeletal muscle or brown adipose tissues with WFS1-deficiency. The increased ER stress response was accompanied by reduced BrdU incorporation and increased caspase-3 cleavage, indicating impaired cell cycle progression and accelerated apoptotic processes in the mutant islets. These changes are associated with increased expression of the cell cycle regulator p21(CIP1) in wfs1-deficient islets and clonal beta-cells. Treatment of islets with thapsigargin, an ER stress inducer, caused upregulation of p21(CIP1). In addition, forced expression of p21(CIP1) resulted in reduced MIN6 beta-cell numbers, suggesting the ER stress-induced increase in p21(CIP1) expression to be involved in beta-cell loss in the mutant islets. These data indicate that WFS1-deficiency activates the ER stress response specifically in beta-cells, causing beta-cell loss through impaired cell cycle progression and increased apoptosis.
Human Molecular Genetics 06/2006; 15(10):1600-9. DOI:10.1093/hmg/ddl081 · 6.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abnormal glucagon secretion is often associated with diabetes mellitus. However, the mechanisms by which nutrients modulate glucagon secretion remain poorly understood. Paracrine modulation by beta- or delta-cells is among the postulated mechanisms. Herein we present further evidence of the paracrine mechanism. First, to activate cellular metabolism and thus hormone secretion in response to specific secretagogues, we engineered insulinoma INS-1E cells using an adenovirus-mediated expression system. Expression of the Na+-dependent dicarboxylate transporter (NaDC)-1 resulted in 2.5- to 4.6-fold (P < 0.01) increases in insulin secretion in response to various tricarboxylic acid cycle intermediates. Similarly, expression of glycerol kinase (GlyK) increased insulin secretion 3.8- or 4.2-fold (P < 0.01) in response to glycerol or dihydroxyacetone, respectively. This cell engineering method was then modified, using the Cre-loxP switching system, to activate beta-cells and non-beta-cells separately in rat islets. NaDC-1 expression only in non-beta-cells, among which alpha-cells are predominant, caused an increase (by 1.8-fold, P < 0.05) in glucagon secretion in response to malate or succinate. However, the increase in glucagon release was prevented when NaDC-1 was expressed in whole islets, i.e., both beta-cells and non-beta-cells. Similarly, an increase in glucagon release with glycerol was observed when GlyK was expressed only in non-beta-cells but not when it was expressed in whole islets. Furthermore, dicarboxylates suppressed basal glucagon secretion by 30% (P < 0.05) when NaDC-1 was expressed only in beta-cells. These data demonstrate that glucagon secretion from rat alpha-cells depends on beta-cell activation and provide insights into the coordinated mechanisms underlying hormone secretion from pancreatic islets.
[Show abstract][Hide abstract] ABSTRACT: Mutations of the WFS1 gene are responsible for two hereditary diseases, Wolfram syndrome and low frequency sensorineural hearing loss. The WFS1 protein is a glycoprotein located in the endoplasmic reticulum (ER) membrane but its function is poorly understood. Herein we show WFS1 mRNA and protein levels in pancreatic islets to be increased with ER-stress inducers, thapsigargin and dithiothreitol. Another ER-stress inducer, the N-glycosylation inhibitor tunicamycin, also raised WFS1 mRNA but not protein levels. Site-directed mutagenesis showed both Asn-663 and Asn-748 to be N-glycosylated in mouse WFS1 protein. The glycosylation-defective WFS1 protein, in which Asn-663 and Asn-748 had been substituted with aspartate, exhibited an increased protein turnover rate. Consistent with this, the WFS1 protein was more rapidly degraded in the presence of tunicamycin. These data indicate that ER-stress and N-glycosylation play important roles in WFS1 expression and stability, and also suggest regulatory roles for this protein in ER-stress induced cell death.
Biochemical and Biophysical Research Communications 01/2005; 325(1):250-6. DOI:10.1016/j.bbrc.2004.10.017 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Wolfram syndrome, an autosomal recessive disorder characterized by juvenile-onset diabetes mellitus and optic atrophy, is caused by mutations in the WFS1 gene. In order to gain insight into the pathophysiology of this disease, we disrupted the wfs1 gene in mice. The mutant mice developed glucose intolerance or overt diabetes due to insufficient insulin secretion in vivo. Islets isolated from mutant mice exhibited a decrease in insulin secretion in response to glucose. The defective insulin secretion was accompanied by reduced cellular calcium responses to the secretagogue. Immunohistochemical analyses with morphometry and measurement of whole-pancreas insulin content demonstrated progressive beta-cell loss in mutant mice, while the alpha-cell, which barely expresses WFS1 protein, was preserved. Furthermore, isolated islets from mutant mice exhibited increased apoptosis, as assessed by DNA fragment formation, at high concentration of glucose or with exposure to endoplasmic reticulum-stress inducers. These results strongly suggest that WFS1 protein plays an important role in both stimulus-secretion coupling for insulin exocytosis and maintenance of beta-cell mass, deterioration of which leads to impaired glucose homeostasis. These WFS1 mutant mice provide a valuable tool for understanding better the pathophysiology of Wolfram syndrome as well as WFS1 function.
Human Molecular Genetics 07/2004; 13(11):1159-70. DOI:10.1093/hmg/ddh125 · 6.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To elucidate the role of intraepithelial lymphocytes (IEL) and enterocytes in the defense mechanism of the small intestine, we designed experiments to stimulate the IEL by anti-CD3epsilon, anti-TCRalphabeta, or anti-TCRgammadelta monoclonal antibodies (mAbs), and to examine the subsequent changes to the enterocytes. The enterocytes of the duodenum and jejunum, but not of the ileum, showed massive DNA fragmentation 30 min after intraperitoneal injection of anti-CD3 mAb. These responses were also induced by anti-TCRgammadelta mAb, but not by anti-TCRalphabeta mAb, and were completely inhibited by cyclosporin A. Nearly half of the enterocytes of the villi in the duodenum and jejunum were exfoliated into the lumen 4 h after the injection of the mAb. Administration of anti-CD3 mAb also induced DNA fragmentation in Fas-deficient MRL/lpr mice, indicating that the Fas-Fas ligand system was not involved in these events. The anti-CD3 mAb treatment also induced massive DNA fragmentation in the intestinal epithelium of the duodenum and jejunum in TNF-receptor-1-deficient mice, whereas TNF-alpha induced only the detachment of intestinal epithelium of wild-type mice, implying the dissociation of two independent factors and/or mechanisms for DNA fragmentation and the subsequent epithelial cell detachment in the murine duodenum and jejunum. The mAb failed to exfoliate the epithelium in TNF-R1-deficient mice. Thus, TCRgammadelta(+) IEL, when treated with anti-CD3 or anti-TCRgammadelta mAbs, induced rapid DNA fragmentation and subsequent detachment of the duodenal and jejunal epithelia, but not in the ileum ("the silent ileum"), partly because of the paucity of TCRgammadelta(+) IELs in the ileum.
Cell and Tissue Research 02/2004; 315(1):71-84. DOI:10.1007/s00441-003-0795-0 · 3.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The intestine, which is exposed to nutrition and to food-derived antigens and microbes including viruses and bacteria, might be an important site for the immune response. Crucial structural and functional differences exist between the small and large intestine, regional differences even having been demonstrated within the small intestine. Accordingly, intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) might be heterogeneous among the different intestinal regions. The aim of this study has been to describe, as accurately as possible, the numbers and T-cell receptor (TCR) phenotypes of IELs and LPLs present in distinct regions of the murine small intestine under physiological conditions. Using an immunohistological technique to differentiate IELs from LPLs, the differential enumeration of IELs and LPLs in distinct regions of the murine small intestine, based upon their definition originally determined by their location, has been performed for the first time and has demonstrated that (1) there are more IELs than LPLs in the duodenum and jejunum, but more LPLs than IELs in the ileum, (2) in the duodenum and jejunum, TCRgammadelta IELs account for 70%-75% of the total CD3(+) IELs, a much greater percentage than previously reported, (3) the ratio of TCRgammadelta to TCRalphabeta IELs is inverted in the ileum, with more than 75% IELs being TCRalphabeta-positive, (4) the lamina propria forms one functional unit throughout the small intestine in terms of the TCR subset components (TCRalphabeta:TCRgammadelta=3:1), and (5) the ileum is entirely different from other regions of the small intestine. To deepen our understanding of the functional significance of the small intestine as an immunologically competent organ, the precise distributions of IELs and LPLs, the ratio of their various subsets, and the strict distinction of IELs and LPLs, as described in this study, is indispensable.
Cell and Tissue Research 08/2003; 313(1):47-53. DOI:10.1007/s00441-003-0706-4 · 3.57 Impact Factor