Pancreatic β Cell–specific Expression of Thioredoxin, an Antioxidative and Antiapoptotic Protein, Prevents Autoimmune and Streptozotocin-induced Diabetes

The cytotoxicity of reactive oxygen intermediates (ROIs) has been implicated in the destruction of pancreatic β cells in insulin-dependent diabetes mellitus (IDDM). Thioredoxin (TRX), a redox (reduction/oxidation)-active protein, has recently been shown to protect cells from oxidative stress and apoptosis. To elucidate the roles of oxidative stress in the development of autoimmune diabetes in vivo, we produced nonobese diabetic transgenic mice that overexpress TRX in their pancreatic β cells. In these transgenic mice, the incidence of diabetes was markedly reduced, whereas the development of insulitis was not prevented. Moreover, induction of diabetes by streptozotocin, an ROI-generating agent, was also attenuated by TRX overexpression in β cells. This is the first direct demonstration that an antioxidative and antiapoptotic protein protects β cells in vivo against both autoimmune and drug-induced diabetes. Our results strongly suggest that oxidative stress plays an essential role in the destruction of β cells by infiltrating inflammatory cells in IDDM

Impaired Anaphylactic Responses with Intact Sensitivity to Endotoxin in Mice Lacking a Platelet-activating Factor Receptor

Platelet-activating factor (PAF) is a potent phospholipid mediator with diverse biological activities in addition to its well-known ability to stimulate platelet aggregation. Pharmacologic studies had suggested a role for PAF in pregnancy, neuronal cell migration, anaphylaxis, and endotoxic shock. Here we show that disruption of the PAF receptor gene in mice caused a marked reduction in systemic anaphylactic symptoms. Unexpectedly, however, the PAF receptor–deficient mice developed normally, were fertile, and remained sensitive to bacterial endotoxin. These mutant mice clearly show that PAF plays a dominant role in eliciting anaphylaxis, but that it is not essential for reproduction, brain development, or endotoxic shock

Residual laminin-binding activity and enhanced dystroglycan glycosylation by LARGE in novel model mice to dystroglycanopathy

Hypoglycosylation and reduced laminin-binding activity of α-dystroglycan are common characteristics of dystroglycanopathy, which is a group of congenital and limb-girdle muscular dystrophies. Fukuyama-type congenital muscular dystrophy (FCMD), caused by a mutation in the fukutin gene, is a severe form of dystroglycanopathy. A retrotransposal insertion in fukutin is seen in almost all cases of FCMD. To better understand the molecular pathogenesis of dystroglycanopathies and to explore therapeutic strategies, we generated knock-in mice carrying the retrotransposal insertion in the mouse fukutin ortholog. Knock-in mice exhibited hypoglycosylated α-dystroglycan; however, no signs of muscular dystrophy were observed. More sensitive methods detected minor levels of intact α-dystroglycan, and solid-phase assays determined laminin binding levels to be ∼50% of normal. In contrast, intact α-dystroglycan is undetectable in the dystrophic Largemyd mouse, and laminin-binding activity is markedly reduced. These data indicate that a small amount of intact α-dystroglycan is sufficient to maintain muscle cell integrity in knock-in mice, suggesting that the treatment of dystroglycanopathies might not require the full recovery of glycosylation. To examine whether glycosylation defects can be restored in vivo, we performed mouse gene transfer experiments. Transfer of fukutin into knock-in mice restored glycosylation of α-dystroglycan. In addition, transfer of LARGE produced laminin-binding forms of α-dystroglycan in both knock-in mice and the POMGnT1 mutant mouse, which is another model of dystroglycanopathy. Overall, these data suggest that even partial restoration of α-dystroglycan glycosylation and laminin-binding activity by replacing or augmenting glycosylation-related genes might effectively deter dystroglycanopathy progression and thus provide therapeutic benefits

Microarray Analysis of Novel Candidate Genes Responsible for Glucose-Stimulated Insulin Secretion in Mouse Pancreatic β Cell Line MIN6

<div><p>Elucidating the regulation of glucose-stimulated insulin secretion (GSIS) in pancreatic islet β cells is important for understanding and treating diabetes. MIN6 cells, a transformed β-cell line derived from a mouse insulinoma, retain GSIS and are a popular <i>in vitro</i> model for insulin secretion. However, in long-term culture, MIN6 cells' GSIS capacity is lost. We previously isolated a subclone, MIN6 clone 4, from the parental MIN6 cells, that shows well-regulated insulin secretion in response to glucose, glybenclamide, and KCl, even after prolonged culture. To investigate the molecular mechanisms responsible for preserving GSIS in this subclone, we compared four groups of MIN6 cells: Pr-LP (parental MIN6, low passage number), Pr-HP (parental MIN6, high passage number), C4-LP (MIN6 clone 4, low passage number), and C4-HP (MIN6 clone 4, high passage number). Based on their capacity for GSIS, we designated the Pr-LP, C4-LP, and C4-HP cells as “responder cells.” In a DNA microarray analysis, we identified a group of genes with high expression in responder cells (“responder genes”), but extremely low expression in the Pr-HP cells. Another group of genes (“non-responder genes”) was expressed at high levels in the Pr-HP cells, but at extremely low levels in the responder cells. Some of the responder genes were involved in secretory machinery or glucose metabolism, including <i>Chrebp</i>, <i>Scgn</i>, and <i>Syt7</i>. Among the non-responder genes were <i>Car2</i>, <i>Maf</i>, and <i>Gcg</i>, which are not normally expressed in islet β cells. Interestingly, we found a disproportionate number of known imprinted genes among the responder genes. Our findings suggest that the global expression profiling of GSIS-competent and GSIS-incompetent MIN6 cells will help delineate the gene regulatory networks for insulin secretion.</p> </div

Transgenic Expression of a Single Transcription Factor Pdx1 Induces Transdifferentiation of Pancreatic Acinar Cells to Endocrine Cells in Adult Mice

<div><p>A promising approach to new diabetes therapies is to generate β cells from other differentiated pancreatic cells <i>in vivo</i>. Because the acinar cells represent the most abundant cell type in the pancreas, an attractive possibility is to reprogram acinar cells into β cells. The transcription factor Pdx1 (Pancreas/duodenum homeobox protein 1) is essential for pancreatic development and cell lineage determination. Our objective is to examine whether exogenous expression of Pdx1 in acinar cells of adult mice might induce reprogramming of acinar cells into β cells. We established a transgenic mouse line in which Pdx1 and EGFP (enhanced green fluorescent protein) could be inducibly expressed in the acinar cells. After induction of Pdx1, we followed the acinar cells for their expression of exocrine and endocrine markers using cell-lineage tracing with EGFP. The acinar cell-specific expression of Pdx1 in adult mice reprogrammed the acinar cells as endocrine precursor cells, which migrated into the pancreatic islets and differentiated into insulin-, somatostatin-, or PP (pancreatic polypeptide)-producing endocrine cells, but not into glucagon-producing cells. When the mice undergoing such pancreatic reprogramming were treated with streptozotocin (STZ), the newly generated insulin-producing cells were able to ameliorate STZ-induced diabetes. This paradigm of <i>in vivo</i> reprogramming indicates that acinar cells hold promise as a source for new islet cells in regenerative therapies for diabetes.</p></div

Gtsf1l and Gtsf2 Are Specifically Expressed in Gonocytes and Spermatids but Are Not Essential for Spermatogenesis.

The unknown protein family 0224 (UPF0224) includes three members that are expressed in germ-line cells in mice: Gtsf1, Gtsf1l, and BC048502 (Gtsf2). These genes produce proteins with two repeats of the CHHC Zn-finger domain, a predicted RNA-binding motif, in the N terminus. We previously reported that Gtsf1 is essential for spermatogenesis and retrotransposon suppression. In this study, we investigated the expression patterns and functions of Gtsf1l and Gtsf2. Interestingly, Gtsf1l and Gtsf2 were found to be sequentially but not simultaneously expressed in gonocytes and spermatids. Pull-down experiments showed that both GTSF1L and GTSF2 can interact with PIWI-protein complexes. Nevertheless, knocking out Gtsf1, Gtsf2, or both did not cause defects in spermatogenesis or retrotransposon suppression in mice

Acinar-to-ductal metaplasia induced by adenovirus-mediated pancreatic expression of Isl1.

Tubular complexes (TCs) are aggregates of duct-like monolayered cells in the developing and regenerating pancreas. Recent studies showed that TCs have regenerative potential, including islet neogenesis. We previously delivered adenovirus vector (AdV) into exocrine cells of the pancreas by intra-common bile ductal (ICBD) injection, and found that AdV expressing Pdx1, a pancreas-specific transcription factor, causes TC formation and islet neogenesis. We also established RTF-Pdx1-EGFP mice, which ubiquitously express Pdx1 when tetracycline is removed from the drinking water. However, exogenous Pdx1 expression in adult RTF-Pdx1-EGFP mice did not cause any pathological changes in the pancreas during three weeks of observation after tetracycline withdrawal. To examine whether the host immune response induced by AdV was involved in TC formation, we delivered AdVs expressing pancreas-related transcription factors or an irrelevant protein into the pancreas of RTF-Pdx1-EGFP mice. Histological analyses showed that both AdV injection and Pdx1 expression are required for TC formation. We also analyzed the effects of these ICBD-injected AdVs. AdV expressing Isl1, a proendocrine transcription factor, effectively induced TC formation through acinar-to-ductal metaplasia, and exogenous Pdx1 expression facilitated this process. Considering the regenerative potential of TCs, a strategy that efficiently induces TC formation may lead to novel therapies for diabetes

Quantitative RT-PCR analysis of imprinted genes.

<p>Expression of imprinted genes, <i>Rian</i> (A), <i>Plagl1</i> (B), <i>Dlk1</i> (C), and <i>Kcnq1</i> (D) in Pr-LP, Pr-HP, C4-LP, and C4-HP MIN6 cells. <i>Plagl1</i>, <i>Dlk1</i>, and <i>Rian</i> were confirmed to be responder genes, whereas <i>Kcnq1</i> was a non-responder gene. Values are means ± SD and n = 4–5. *<i>P</i><0.05.</p

Insulin content and secretion of MIN6 cells.

<p>Insulin content of Pr-LP, Pr-HP, C4-LP, and C4-HP MIN6 cells (A). The insulin content of Pr-HP cells was lower than that of Pr-LP, C4-LP, or C4-HP cells. Values are means ± SD and n = 5–6. *<i>P</i><0.05. Insulin secretion/insulin content from Pr-LP, Pr-HP, C4-LP, and C4-HP MIN6 cells stimulated with 3 mM (3G), 25 mM (25G) glucose, 100 nM glybenclamide (SU), or 30 mM KCl (B, C). Values are means ± SD and n = 5–6. *<i>P</i><0.05 v.s. insulin secretion at 3 mM glucose.+<i>P</i><0.05 v.s. insulin secretion of Pr-LP, C4-LP, and C4-HP cells at 3 mM glucose by Student's <i>t</i>-test.</p