The regenerative process in the pancreas is of particular interest because diabetes results from an inadequate number of insulinproducing beta cells and pancreatic cancer may arise from the uncontrolled growth of progenitor/stem cells. Continued and substantial growth of islet tissue occurs after birth in rodents and humans, with additional compensatory growth in response to increased demand. In rodents there is clear evidence of pancreatic regeneration after some types of injury, with proliferation of preexisting differentiated cell types accounting for some replacement. Additionally, neogenesis or the budding of new islet cells from pancreatic ducts has been reported, but the existence and identity of a progenitor cell have been debated. We hypothesized that the progenitor cells are duct epithelial cells that after replication undergo a regression to a less differentiated state and then can form new endocrine and exocrine pancreas. To directly test whether ductal cells serve as pancreatic progenitors after birth and give rise to new islets, we generated transgenic mice expressing human carbonic anhydrase II (CAII) promoter: Cre recombinase (Cre) or inducible CreER TM to cross with ROSA26 loxP-Stop-loxP LacZ reporter mice. We show that CAII-expressing cells within the pancreas act as progenitors that give rise to both new islets and acini normally after birth and after injury (ductal ligation). This identification of a differentiated pancreatic cell type as an in vivo progenitor of all differentiated pancreatic cell types has implications for a potential expandable source for new islets for replenishment therapy for diabetes.diabetes ͉ islets of Langerhans ͉ lineage tracing

SUMMARY Integrative organ crosstalk regulates key aspects of energy homeostasis, and its dysregulation may underlie metabolic disorders such as obesity and diabetes. To test the hypothesis that crosstalk between the liver and pancreatic islets modulates β cell growth in response to insulin resistance, we used the liver-specific insulin receptor knockout (LIRKO) mouse, a unique model that exhibits dramatic islet hyperplasia. Using complementary in vivo parabiosis and transplantation assays, as well as in vitro islet culture approaches, we demonstrate that humoral, nonneural, non-cell-autonomous factor(s) induces β cell proliferation in LIRKO mice. Furthermore, we report that a hepatocyte-derived factor(s) stimulates mouse and human β cell proliferation in ex vivo assays, independent of ambient glucose and insulin levels. These data implicate the liver as a critical source of β cell growth factor(s) in insulin-resistant states.

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Type 2 diabetes (T2D) is characterized by reduction of β-cell mass and dysfunctional insulin secretion. Understanding β-cell phenotype changes as T2D progresses should help explain these abnormalities. The normal phenotype should differ from the state of overwork when β-cells compensate for insulin resistance to keep glucose levels normal. When only mild hyperglycaemia develops, β-cells are subjected to glucotoxicity. As hyperglycaemia becomes more severe, so does glucotoxicity. β-Cells in all four of these situations should have separate phenotypes. When assessing phenotype with gene expression, isolated islets have artefacts resulting from the trauma of isolation and hypoxia of islet cores. An advantage comes from laser capture microdissection (LCM), which obtains β-cell-rich tissue from pancreatic frozen sections. Valuable data can be obtained from animal models, but the real goal is human β-cells. Our experience with LCM and gene arrays on frozen pancreatic sections from cadaver donors with T2D and controls is described. Although valuable data was obtained, we predict that the approach of taking fresh samples at the time of surgery is an even greater opportunity to markedly advance our understanding of how β-cell phenotype evolves as T2D develops and progresses.

BackgroundRecently, natural mutation of Tyrosine kinase 2 (Tyk2) gene has been shown to determine susceptibility to murine virus-induced diabetes. In addition, a previous human genome-wide study suggested the type 1 diabetes (T1D) susceptibility region to be 19p13, where the human TYK2 gene is located (19p13.2).MethodsPolymorphisms of TYK2 gene at the promoter region and exons were studied among 331 healthy controls, and 302 patients with T1D and 314 with type 2 diabetes (T2D) in the Japanese.FindingsA TYK2 promoter haplotype with multiple genetic polymorphisms, which are in complete linkage disequilibrium, named TYK2 promoter variant, presenting decreased promoter activity, is associated with an increased risk of not only T1D (odds ratio (OR), 2.4; 95% confidence interval (CI), 1.2 to 4.6; P = 0.01), but also T2D (OR, 2.1; 95% CI, 1.1 to 4.1; P = 0.03). The risk is high in patients with T1D associated with flu-like syndrome at diabetes onset and also those without anti-glutamic acid decarboxylase autoantibody.InterpretationThe TYK2 promoter variant is associated with an overall risk for diabetes, serving a good candidate as a virus-induced diabetes susceptibility gene in humans.FundingMinistry of Education, Culture, Sports, Science and Technology and of Health, Labor and Welfare of Japan.

Aims/hypothesisIt is widely accepted that production of insulin, glucagon, somatostatin and pancreatic polypeptide in islet cells is specific to beta, alpha, delta and pancreatic polypeptide cells, respectively. We examined whether beta cells express other genes encoding islet hormones.MethodsNested RT-PCR was performed on single beta cells of transgenic mice with green fluorescent protein (GFP) driven by mouse insulin I promoter (MIP-GFP).ResultsOnly 55% of adult beta cells expressed the insulin gene alone, while others expressed two or more islet hormone genes; 4% expressed all four hormone genes. In embryonic and neonatal cells, 60% to 80% of GFP+ cells co-expressed pancreatic polypeptide and insulin genes in contrast to 29% in adult. To clarify cell fate, we conducted lineage tracing using rat insulin II promoter-cre mice crossed with reporter mice Gt(ROSA)26Sor-loxP-flanked STOP-cassette-GFP. All GFP+ cells expressed insulin I and II genes, and showed similar heterogeneity of co-expression to that seen in MIP-GFP mice. Although we report expression of other hormone genes in a significant proportion of beta cells, our lineage tracing results demonstrate that after inducing InsII (also known as Ins2) expression, beta cell progenitors do not redifferentiate to non-beta cells.Conclusions/interpretationThis study shows co-expression of multiple hormone genes in beta cells of adult mice as well as in embryos and neonates. This finding could: (1) represent residual expression from beta cell precursors; (2) result from alternative developmental pathways for beta cells; or (3) denote the differentiation potential of these cells. It may be linked to functional heterogeneity. This heterogeneity in gene expression may provide a means to characterise the functional, cellular and developmental heterogeneity seen in beta cells.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-009-1570-x) contains supplementary material, which is available to authorised users.

There is growing information about the heterogeneity of pancreatic ␤-cells and how it relates to insulin secretion. This study used the approach of flow cytometry to sort and analyze ␤-cells from transgenic mice expressing green fluorescent protein (GFP) under the control of the mouse insulin I gene promoter. Three populations of ␤-cells with differing GFP brightness could be identified, which were classified as GFP-low, GFP-medium, and GFP-bright. The GFP-medium population comprised about 70% of the total. The GFP-low population had less insulin secretion as determined by the reverse hemolytic plaque assay and reduced insulin gene expression. Additionally, all three subpopulations of ␤-cells were found in mice of varying ages (embryonic d 15.5 and postnatal wk 1-9). The three populations from the youngest had larger cells (forward scatter) and less granularity (side scatter) than those from the adults. This approach opens up new ways to advance knowledge about ␤-cell heterogeneity. (Endocrinology 153: 5180 -5187, 2012) S tudies have described the heterogeneity of ␤-cells on a functional level with differences in insulin secretion and biosynthesis as well as morphology and various biological markers (1-10). Moreover, we and others have found heterogeneity in sorted single ␤-cells with regard to very low expression of mRNA of the hormone products of other islet cell types (11, 12). Our study used sorted ␤-cells from transgenic mice expressing green fluorescent protein (GFP) under the control of the mouse insulin I gene promoter (MIP) (13). The introduction of this transgene does not appear to be deleterious, because these mice have normal pancreatic development, glucose tolerance, islet histology, and ␤-cell secretory function (13). Our previous analysis of single GFP-positive cells from these mice with nested PCR revealed that only 55% of the ␤-cells expressed the insulin gene alone and 4% of the cells expressed genes of four islet hormones (insulin, glucagon, somatostatin, and pancreatic polypeptide) (11,12). Another example of heterogeneity in adult ␤-cells was shown with a dual-reporter construct (Pdx1-monomeric red fluorescent protein/insulin-enhanced GFP dual-reporter lentivirus) (14). Most mouse and human ␤-cells were clearly positive for both markers, but 10 -25% Pdx (ϩ) and insulin (low) cells are presumably true ␤-cells but show lower insulin expression. Yet another example of heterogeneity was recently demonstrated by the recent finding that 20 -25% of islets in rats appear to have reduced functional activity as suggested by low oxygen tension and reduced blood flow (15). However, little is understood about how the ␤-cells in these islets are related to the heterogeneity found in other studies.In our previous work with MIP-GFP mice, we noticed variability of GFP expression in ␤-cells and hypothesized that the GFP intensity regulated by the MIP might be a marker of ␤-cell heterogeneity that was reflected by differences in insulin promoter activity. Therefore, single ␤-cells were sorted on the basis of G...

Accumulating evidence suggests that viruses play an important role in the development of diabetes. Although the diabetogenic encephalomyocarditis strain D virus induces diabetes in restricted lines of inbred mice, the susceptibility genes to virus-induced diabetes have not been identified. We report here that novel Tyrosine kinase 2 (Tyk2) gene mutations are present in virus-induced diabetes-sensitive SJL and SWR mice. Mice carrying the mutant Tyk2 gene on the virus-resistant C57BL/6 background are highly sensitive to virus-induced diabetes. Tyk2 gene expression is strongly reduced in Tyk2-mutant mice, associated with low Tyk2 promoter activity, and leads to decreased expression of interferon-inducible genes, resulting in significantly compromised antiviral response. Tyk2-mutant pancreatic β-cells are unresponsive even to high dose of Type I interferon. Reversal of virus-induced diabetes could be achieved by β-cell-specific Tyk2 gene expression. Thus, reduced Tyk2 gene expression in pancreatic β-cells due to natural mutation is responsible for susceptibility to virus-induced diabetes.