Two cell lines have been established from insulinomas obtained by targeted expression of the simian virus 40 T antigen gene in transgenic mice. These cell lines, designated MIN6 and MIN7, produce insulin and T antigen and have morphological characteristics of pancreatic beta cells. MIN6 cells exhibit glucose-inducible insulin secretion comparable with cultured normal mouse islet cells, whereas MIN7 cells do not. Both cell lines produce liver-type glucose transporter (GT) mRNA at high level. Brain-type GT mRNA is also present at considerable level in MIN7 cells, but is barely detectable in MIN6 cells, suggesting that exclusive expression of the liver-type GT is related to glucose-inducible insulin secretion. MIN6 cells do not express either major histocompatibility (MHC) class I or class II antigens on the cell surface. However, treatment with interferon-gamma induces high levels of MHC class I antigens, and a combination of interferon-gamma and tumor necrosis factor-alpha induces a MHC class II antigen on the cell surface. These results emphasize that the MIN6 cell line retains physiological characteristics of normal beta cells. The MIN6 cell line will be especially useful to analyze the molecular mechanisms by which beta cells regulate insulin secretion in response to extracellular glucose concentrations. We discuss a possible role of GT isoforms in glucose sensing by beta cells.

Extraembryonic endoderm (ExE) is differentiated from the inner cell mass of the late blastocyst-stage embryo to form visceral and parietal endoderm, both of which have an important role in early embryogenesis. The essential roles of Gata-6 and Gata-4 on differentiation of visceral endoderm have been identified by analyses of knockout mice. Here we report that forced expression of either Gata-6 or Gata-4 in embryonic stem (ES) cells is sufficient to induce the proper differentiation program towards ExE. We believe that this is the first report of a physiological differentiation event induced by the ectopic expression of a transcription factor in ES cells.

Embryonic stem (ES) cells can differentiate into many cell types. Recent reports have shown that ES cells can differentiate into insulin-producing cells. However, the differentiation is not efficient enough to produce insulin-secreting cells for future therapeutic use. Pdx-1, a homeodomain-containing transcription factor, is a crucial regulator for pancreatic development. We established an ES cell line in which exogenous pdx-1 expression was precisely regulated by the Tet-off system integrated into the ROSA26 locus. Using this cell line, we examined the effect of pdx-1 expression during in vitro differentiation via embryoid body formation. The results showed that pdx-1 expression clearly enhanced the expression of the insulin 2, somatostatin, Kir6.2, glucokinase, neurogenin3, p48, Pax6, PC2, and HNF6 genes in the resulting differentiated cells. Immunohistochemical examination also revealed that insulin was highly produced in most of the differentiated ES cells. Thus, exogenous expression of pdx-1 should provide a promising approach for efficiently producing insulin-secreting cells from human ES cells for future therapeutic use in diabetic patients. Diabetes

The differentiation programs of spermatogenesis and oogenesis are largely independent. In the early stages, however, the mechanisms partly overlap. Here we demonstrated that a germ-cell-specific basic helix-loop-helix (bHLH) transcription factor gene, Sohlh2, is required for early spermatogenesis and oogenesis. SOHLH2 was expressed in mouse spermatogonia from the undifferentiated stage through differentiation and in primordial-to-primary oocytes. Sohlh2-null mice, produced by gene targeting, showed both male and female sterility, owing to the disrupted differentiation of mature (KIT(+)) spermatogonia and oocytes. The Sohlh2-null mice also showed the downregulation of genes involved in spermatogenesis and oogenesis, including the Sohlh1 gene, which is essential for these processes. Furthermore, we showed that SOHLH2 and SOHLH1 could form heterodimers. These observations suggested that SOHLH2 might coordinate with SOHLH1 to control spermatogonial and oocyte genes, including Sohlh1, to promote the differentiation of KIT(+) germ cells in vivo. This study lays the foundation for further dissection of the bHLH network that regulates early spermatogenesis and oogenesis.

Embryonic stem (ES) cells can differentiate into many cell types and are expected to be useful for tissue engineering. Recent reports have shown that ES cells can differentiate into insulin-producing cells in response to the transient expression of the pdx-1 gene, after the removal of feeder cells. To investigate the lineage of insulin-producing cells and their in vitro differentiation, we introduced the ␤geo gene, encoding a ␤-galactosidase-neomycin phosphotransferase fusion protein under the control of the mouse insulin 2 promoter, into ES cells that had been adapted to feederfree culture, and analyzed insulin gene expression during their in vitro differentiation. We also examined the expression of transcription factors that are related to the differentiation of the pancreas. X-gal staining analysis revealed ␤-galactosidase-positive cells on the surface and in the center of the embryoid body that proliferated during differentiation. Glucose-responsive insulin-producing cells, derived from our feeder-free ES cells, expressed insulin 2, pdx-1, Pax4, and Isl1 and also the glucagon, somatostatin, and PP genes. Moreover, the genes encoding p48, amylase, and carboxypeptidase A were also expressed. These results suggest that ES cells can differentiate not only into endocrine cells but also into exocrine cells of the pancreas, without the initiation of pdx-1 expression. Diabetes 52:1163-1168, 2003 M ouse embryonic stem (ES) cells are continuous cell lines derived directly from the fetal founder tissue of the preimplantation embryo. They can be expanded in culture while retaining the functional attributes of pluripotent early embryo cells. Their capacity for multilineage differentiation is reproduced in culture, as ES cells can differentiate into a wide range of well-defined cell types. Cell transplantation to restore tissue function after disease or injury is in theory applicable to a huge variety of human diseases. Thus, the use of lineage-restricted differentiation techniques developed for ES cells will promote future cell therapy. Moreover, an in vitro ES cell differentiation system will be valuable for research into early embryonic cell lineage. We and other groups have shown that ES cells can differentiate into the endoderm cell lineage (1-5). Recently, several groups have reported that ES cells can be induced to differentiate into insulin-producing cells These reports indicate that ES cells can differentiate into insulin-producing cells, but the cell lineage of these insulin-producing cells is still unknown. Indeed, insulin gene expression has been observed not only in the pancreas but also in the yolk sac and embryonic brain in vivo (6,7). To assess the cell lineage of insulin-producing cells during their differentiation from ES cells, we have developed ES cell lines carrying a lacZ-neomycin phosphotransferase fusion gene (␤geo) under the mouse insulin 2 gene promoter and analyzed the lacZ expression pattern of clusters of differentiating ES cells. Moreover, we have examined the expression of a series of transc...

SummaryThe NSY (Nagoya-Shibata-Yasuda) mouse was established as an inbred strain of mouse with spontaneous development of diabetes mellitus, by selective breeding for glucose intolerance from outbred Jcl:ICR mice. NSY mice spontaneously develop diabetes mellitus in an age-dependent manner. The cumulative incidence of diabetes is 98 % in males and 31% in females at 48 weeks of age. Neither severe obesity nor extreme hyperinsulinaemia is observed at any age in these mice. Glucose-stimulated insulin secretion was markedly impaired in NSY mice after 24 weeks of age. In contrast, fasting plasma insulin level was higher in male NSY mice than that in male C3H/He mice (545 +73 vs 350+ 40 pmol/1, p < 0.05, at 36 weeks of age). Pancreatic insulin content was higher in male NSY mice than that in male C3H/He mice (76 + 8 vs 52 _+ 5 ng/mg wet weight, p < 0.05, at 36 weeks of age). Morphologically, no abnormal findings, such as hypertrophy or inflammatory changes in the pancreatic islets, were observed in NSY mice at any age. These data suggest that functional changes of insulin secretion in response to glucose from pancreatic beta cells may contribute to the development of non-insulin-dependent diabetes mellitus (NIDDM) in the NSY mouse. Although insulin sensitivity was not measured, fasting hyperinsulinaemia in NSY mice suggests that insulin resistance may also contribute to the pathogenesis of NIDDM. Since these findings are similar to the pathophysiologic features of human NIDDM patients, the NSY mouse is considered to be useful for investigating the pathogenesis and genetic predisposition to NIDDM. [Diabetologia (1995) 38: 503-508] Key words NSY mouse, non-insulin-dependent diabetes mellitus, animal model, insulin secretion, isolated islets.Non-insulin-dependent diabetes mellitus (NIDDM) is a heterogeneous disorder, caused by an interaction of genetic and environmental factors [1][2][3]. This heterogeneity in human NIDDM makes it difficult to clarify the genetics or pathogenesis of the disease. Animal models are invaluable for the analysis of heterogeneous disorders such as diabetes. This is eviReceived: 22 June 1994 and in revised form: 11 October 1994 Corresponding author: Dr. H. Ikegami, Department of Geriatric Medicine, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan Abbreviations: NIDDM, Non-insulin-dependent diabetes mellitus; IDDM, insulin-dependent diabetes mellitus; NSY mouse, Nagoya-Shibata-Yasuda mouse.denced by the recent progress in the understanding of the genetics and pathogenesis of insulin-dependent diabetes mellitus by use of excellent animal models, such as the nonobese diabetic (NOD) mouse and the Bio-Breeding (BB) rat [4]. Several animal models for NIDDM have been described. Although recent studies have revealed impaired insulin secretion in GK rats [5][6][7], most of the animal models for NIDDM are characterized by obesity, hyperinsulinaemia and islet hypertrophy [8].The NSY (Nagoya-Shibata-Yasuda) mouse is a spontaneous model of NIDDM with moderate obesity that was establ...

The number and exact locations of the major histocompatibility complex (MHC)-linked diabetogenic genes

A possible pathogenic mutation in the beta 3-adrenergic-receptor gene (Trp64Arg) has been reported to be associated with an earlier age of onset of non-insulin-dependent diabetes mellitus (NIDDM) and clinical features of the insulin resistance syndrome in Pima Indian, Finnish and French subjects. Since marked heterogeneity has been reported in the association of mutations of candidate genes with NIDDM between Japanese and other ethnic groups, we investigated the association of Trp64Arg with NIDDM in Japanese subjects. The allele frequency of the mutation (Arg) was slightly, but not significantly, higher in NIDDM than in control subjects (70 out of 342 alleles [20.5%] vs 40 out of 248 [16.1%], respectively, p > 0.2). When our data were combined with those of Pima Indian and Finnish subjects, however, the Arg/Arg genotype was significantly associated with NIDDM as compared with the other two genotypes (p < 0.005, relative risk [RR] 2.13, 95% confidence interval [CI] 1.28-3.55). The Arg allele was also associated with NIDDM (p < 0.05, RR 1.27, 95% CI 1.06-1.52). Japanese subjects homozygous for the mutation had a significantly higher body mass index (mean +/- SD: 25.5 +/- 3.9 kg/m2) than heterozygotes (22.6 +/- 4.1, p < 0.05) and normal homozygotes (22.8 +/- 3.8, p < 0.05). NIDDM patients homozygous for the mutation tended to have an earlier age of onset of NIDDM than those with other genotypes. These data suggest that the Trp64Arg mutation not only contributes to weight gain and age-at-onset of NIDDM but is also associated with susceptibility to NIDDM.