It is important for our understanding of the pancreatic islets to study whether new islets are able to form in the intact pancreas. We developed a new method to determine the total number and the mean volume of the pancreatic islets, and we used this method to study the expansion of the islet mass in ob/ob mice (n ‫؍‬ 8), using ob/؉ mice (n ‫؍‬ 8) as controls. The total islet volume was increased by a factor of 3.6 in ob/ob mice compared with ob/؉ mice, whereas, importantly, the total number of islets did not differ among ob/ob mice and ob/؉ mice (3,193 ؎ 160 islets in ob/ob mice vs. 3,184 ؎ 142 islets in ob/؉ mice, P ‫؍‬ 0.97). The coefficient of variation in the volume distribution of islets was equal in the two groups, showing that in ob/ob mice, the existing islets expand their volume by the same proportion, without a net formation of new islets. We suggest that the pancreatic islets should be considered as anatomically such complex structures that islet neogenesis does not spontaneously occur in an intact pancreas. Cells within the existing islets are presumably the most important sources for islet cell hyperplasia during expansion of the total islet mass. Diabetes 52:1716 -1722, 2003 Afundamental and yet unanswered question in the understanding of the pathogenesis of type 2 diabetes is the limitation in the formation of enough additional ␤-cells to maintain normoglycemia during an increased demand for insulin; even individuals with significant insulin resistance do not develop diabetes if they have an appropriate number of functioning ␤-cells, emphasizing the pathophysiological importance of the total ␤-cell mass.The ob/ob mouse has been extensively studied as a model of type 2 diabetes (1). These mice are obese and develop insulin resistance and diabetes because of an inherited inability to produce leptin (2), despite a marked expansion of the total mass of the pancreatic islets. It has been stated in the literature that both islet hyperplasia and islet hypertrophia are responsible for the increase in the total islet mass in ob/ob mice (3,4), but among the few studies that actually addressed this question by applying methods that, in theory, could measure the total number of islets (5,6), the results are in fact diverging. Previously, we found a linear correlation between the total islet volume and the volume-weighted mean volume in rats (7), a result that suggested-but, because of the nature of the method, could not prove-that the expansion of the islet mass during physiological growth in rats is caused by the growth of existing islets without the formation of new islets. We therefore developed a method relying on recent stereological methods to determine the total number and mean volume of the pancreatic islets, and we used this method to investigate the expansion of the islet mass in ob/ob mice. An understanding of whether new islets are able to form in the intact pancreas is important for understanding the islets as an anatomical structure, and, in addition, it could be important for understanding whi...

Background Epidemiological as well as animal studies have shown that environmental factors such as nutrition contribute to the development of diabetes. In this study we investigated whether the early introduction of a gluten‐free diet can influence the onset and/or incidence of diabetes, as well as insulitis and the number of gut mucosal lymphocytes, in non‐obese diabetic (NOD) mice. Methods Gluten‐free and standard Altromin diets (with the same milk protein and vitamin content) were given to breeding pairs of NOD mice as well as to the first generation of NOD female mice, which were then observed for 320 days. Results A substantially lower diabetes incidence (χ2=15.8, p=0.00007) was observed in NOD mice on the gluten‐free diet (15%, n=27) compared to mice on the standard diet (64%, n=28). In addition, mice on the gluten‐free diet developed diabetes significantly later (244±24 days SEM) compared to those on the standard diet (197±8 days, p=0.03). No differences in the number of CD3+, TCR‐γδ+, IgA+, and IgM+ cells in the small intestine were observed. Conclusion We showed that gluten‐free diet both delayed and to a large extent prevented diabetes in NOD mice that have never been exposed to gluten. Copyright © 1999 John Wiley & Sons, Ltd.

The findings show that perinatal determinants may influence the risk of subsequent development of type 1 diabetes in a sex-specific manner.

Key parameters of the endocrine pancreas, such as islet number, islet mass, ␤-cell mass, and ␣-cell mass, were studied in different strains of inbred mice to investigate the impact of genetic background on the size and structure of the endocrine pancreas. Six mice from each of seven different strains of inbred mice were included in the study. For all parameters investigated, there was a pronounced interstrain variation. ANCOVA showed that only mouse strain was statistically significant as an explanatory parameter for the number of islets. Mouse strain, body weight, and pancreas mass reached statistical significance as explanatory parameters for the islet mass, with mouse strain as the most significant predictor. These data show that genetic background is the most important predictor of both the number of islets and total islet volume. We also conclude that inbred mice could be a valuable resource to identify the genes responsible for the size and structure of the endocrine pancreas. Diabetes 54:133-137, 2005

Background Epidemiological as well as animal studies have shown that environmental factors such as nutrition contribute to the development of diabetes. In this study we investigated whether the early introduction of a gluten-free diet can in¯uence the onset and/or incidence of diabetes, as well as insulitis and the number of gut mucosal lymphocytes, in non-obese diabetic (NOD) mice.Methods Gluten-free and standard Altromin diets (with the same milk protein and vitamin content) were given to breeding pairs of NOD mice as well as to the ®rst generation of NOD female mice, which were then observed for 320 days.Results A substantially lower diabetes incidence (s 2 =15.8, p=0.00007) was observed in NOD mice on the gluten-free diet (15%, n=27) compared to mice on the standard diet (64%, n=28). In addition, mice on the gluten-free diet developed diabetes signi®cantly later (244t24 days SEM) compared to those on the standard diet (197t8 days, p=0.03). No differences in the number of CD3 + , TCR-cd + , IgA + , and IgM + cells in the small intestine were observed.Conclusion We showed that gluten-free diet both delayed and to a large extent prevented diabetes in NOD mice that have never been exposed to gluten.

The homeodomain protein PDX-1 is critical for pancreas development and is a key regulator of insulin gene expression. PDX-1 nullizygosity and haploinsufficiency in mice and humans results in pancreatic agenesis and diabetes, respectively. At embryonic day (e) 10.5, PDX-1 is expressed in all pluripotential gut-derived epithelial cells destined to differentiate into the exocrine and endocrine pancreas. At e15, PDX-1 expression is downregulated in exocrine cells, but remains high in endocrine cells. The aim of this study was to determine whether targeted overexpression of PDX-1 to the exocrine compartment of the developing pancreas at e15 would allow for respecification of the exocrine cells. Transgenic (TG) mice were generated in which PDX-1 was expressed in the exocrine pancreas using the exocrine-specific elastase-1 promoter. These mice exhibited a marked dysmorphogenesis of the exocrine pancreas, manifested by increased rates of replication and apoptosis in acinar cells and a progressive fatty infiltration of the exocrine pancreas with age. Interestingly, the TG mice exhibited improved glucose tolerance, but absolute ␤-cell mass was not increased. These findings indicate that downregulation of PDX-1 is required for the proper maintenance of the exocrine cell phenotype and that upregulation of PDX-1 in acinar cells affects ␤-cell function. The mechanisms underlying these observations remain to be elucidated. Diabetes 50: 1553-1561, 2001 P DX-1 (also known as IDX-1/STF-1/IPF-1) is a homeodomain transcription factor evolutionarily derived from the Parahox amphilox three-gene cluster and is a critical determinant of pancreas development in higher vertebrates (1-4). The early embryonic expression of PDX-1 demarcates the dorsal and ventral regions of the primitive mouse gut from which the two pancreatic buds emerge. As development proceeds, PDX-1 is expressed in the pancreas, the posterior portion of the developing primitive stomach epithelium, the anterior region of the duodenum, and the yolk sac (5). In the fully developed adult pancreas, PDX-1 expression is restricted predominantly to the endocrine pancreas (islets of Langerhans) in the insulin-producing ␤-cells and in a fraction of the somatostatin-producing ␦-cells, as well as in a subpopulation of cells in the pancreatic ducts that are pluripotential progenitors for ␤-cells (6,7). PDX-1 is a transcriptional activator of the insulin, somatostatin, glucokinase, and GLUT2 genes (2-4), and is involved in regulating glucose responsiveness of the insulin promoter (8,9).The importance of PDX-1 in pancreatic development is demonstrated by the consequences of the loss of PDX-1 function. Targeted disruption of the pdx-1 gene in mice and an inactivating mutation of pdx-1 (ipf-1) in a human infant (10) manifest as agenesis of the pancreas (11,12). Conditional disruption of the pdx-1 gene selectively in ␤-cells of mice using the Cre/Lox approach results in a progressive loss of ␤-cells and the development of diabetes in mice by age 5-6 months (13). Mice (14) and humans (10)...

We describe a method for unbiased assumption‐free estimation of the total number of β‐cells and the mean β‐cell volume in mouse and rat pancreas based on light microscopy. Such a method, which takes advantage of one of the most recent developments in stereology, the fractionator, has not previously been described. It relies on repeated fractionation of the tissue using systematic uniform random sampling combined with an unbiased counting principle. The method was applied to eight BALB/cBom male mice (56 days) and six Lewis/MOL male rats (47 days). In mice, the total number of β‐cells was 1.06±0.07±106 (mean±SEM) per pancreas with a mean β‐cell volume of 1280±17 μm3, while in rats the total β‐cell number was 2.76±0.42±106 per pancreas with a mean β‐cell volume of 1170±65 μm3. Furthermore, the results showed that in both species the biological variability in the total β‐cell volume is due to differences in the number of β‐cells rather than variability of the mean β‐cell volume. The method can be used to give a precise description of number and volume of β‐cells at different ages, and will make it possible to estimate the contributions of hyper/hypotrophia and hyperlhypoplasia to a given induced or spontaneous change in the total β‐cell mass.

Aims/hypothesis. Pancreatic ducts are considered as potential sites for neogenesis of beta cells. In vitro studies have reported formation of islets from postnatal human and rodent duct tissue. We examined whether postnatal human duct-cell preparations can generate new beta cells after transplantation. Methods. Pancreatic duct cells were prepared from the non-endocrine fraction of human donor pancreases that were processed for islet-cell isolation. Grafts containing 0.5 million duct cells with 1% contaminating insulin-positive cells were implanted under the kidney capsule of normoglycaemic nude mice. At 0.5 and 10 weeks post-transplantation, implants were examined for their cellular composition and for the volumes of their composing cell populations, i.e. cytokeratin 19-positive duct cells, synaptophysin-, insulin-and glucagon-positive endocrine cells. Results. Between week 0.5 and 10, duct-cell volume decreased by at least 90% whereas the change in insulin-positive cell volume depended on donor age. Implants from donors over10 years had a threefold decrease in their insulin-positive cell volume, while those from donors under 10 years had a 2.5-fold increase. After 10 weeks, the implants from the younger donors consisted of 19% insulin-positive cells occurring as single units or small cell clusters. Three percent of these insulin-positive cells also expressed the ductal marker CK 19 and were consistently found in conjunction with ductal epithelia; up to 1% was positive for the proliferation marker BrdU and located in small endocrine cell clusters. Conclusions/interpretation. These data indicate that duct cell preparations from donors under 10 years can generate insulin-positive cells. This process might involve differentiation of CK 19-positive-insulin cells that are formed at the duct epithelia as well as proliferation of insulin-positive cells within endocrine cell aggregates. [Diabetologia (2003) 46:830-838]