Insulin Secreting Cells

Diabetes Freedom

Diabetes Cure Diet

Get Instant Access

Type 1 diabetes occurs secondary to the autoimmune-mediated destruction of insulin-producing p-cells in pancreatic islets. In contrast, insulin resistance is important in the development of type 2 diabetes, although p-cell dysfunction characterized by an inability to secrete adequate amounts of insulin to overcome insulin resistance also contributes to the pathogenesis of type 2 diabetes. Thus the development of insulin-secreting cells would provide an effective therapy for type 1 and, possibly, type 2 diabetes.

3.1.1. Pancreas Development

The molecular mechanisms of pancreatic development provide insight into the transcription factors needed to initiate the hierarchical cascade of gene expression that results in differentiation along an islet cell lineage. This knowledge will facilitate developing strategies to generate insulin-secreting cells from stem cells. The molecular and cellular mechanisms important for pancreatic development have been the subject of several recent reviews (14,18-20). A brief overview is presented here.

Pancreatic islet development is a complex process dependent on multiple factors, including expression of a series of transcription factors important for cell differentiation and transmission of signals generated from surrounding mesenchyme and blood vessels. Differentiation of endoderm precursor cells into islets is controlled by a cascade of transcriptional events directed by a series of transcription factors that are expressed in a temporal and cell-specific pattern (Fig. 1). Expression of Pdx-1, a homeodomain protein, is important for early pancreatic development, because mice and humans homozygous for mutations in the Pdxl gene are apancreatic. Subsequently, neurogenin3 (ngn3) expression is important for the differentiation of pancreatic endocrine cell types. Null mutations of the ngn3 gene abrogate islet development in mice (21,22). Additional transcription factors, including NeuroD1/p 2 and Pax 6, also affect islet cell development, whereas Pax 4, Nkx2.2, and Nkx6.1 are important for p-cell development, although some of these factors also contribute to the differentiation of a, 5, or pancreatic polypeptide cells in islets. As indicated in Fig. 1, many of these transcription factors are expressed not only during development but also in differentiated adult islet cells.

3.1.2. Insulin-Secreting Cells From ES Cells

As described elsewhere (Chapter 8), protocols to induce the differentiation of ES cells into insulin-secreting cells have been developed (23-25). To date, the efficiency of generating insulin-secreting cells using these protocols has been low, and the cells have, in general, been relatively hypofunctional compared with native islets. One approach to enhance the differentiation process has been to express transcription factors important in islet development.

The impact of constitutively expressing either Pdx-1 or Pax4 in ES cells was recently described (26). Pdx-1 functions at multiple levels of pancreatic development. It is important not only for development of the exocrine and endocrine pancreas, but it is also important for maintaining the differentiated p-cell pheno-type, as it regulates the expression of several genes important for p-cell function, including the genes that encode insulin, the glucose transporter GLUT2, and glucokinase (14,18-20). Pax4 is a paired domain homeobox transcription factor that is important for committing endocrine precursor cells along the p- and 5-cell

Fig. 1. Model for the role of transcription factors during islet differentiation. The proposed role for different transcription factors in islet differentiation is shown. For simplicity, the association of a single transcription factor with different developmental events is based on the timing of their expression or the timing of their predominant role in differentiation. Any given factor likely functions at multiple steps during differentiation, and expression of multiple factors is probably required at each step of differentiation. Also shown are differentiated adult islet cells. Below each cell is the hormonal product of that cell type and the transcription factors that are expressed in the differentiated adult 5, p, a, and pancreatic polypeptide cells.

Fig. 1. Model for the role of transcription factors during islet differentiation. The proposed role for different transcription factors in islet differentiation is shown. For simplicity, the association of a single transcription factor with different developmental events is based on the timing of their expression or the timing of their predominant role in differentiation. Any given factor likely functions at multiple steps during differentiation, and expression of multiple factors is probably required at each step of differentiation. Also shown are differentiated adult islet cells. Below each cell is the hormonal product of that cell type and the transcription factors that are expressed in the differentiated adult 5, p, a, and pancreatic polypeptide cells.

lineage, because islets from mice with a null mutation of the Pax4 gene lack p and 5 cells (14,18-20). Three different approaches have been used to differentiate native ES cells and ES cells expressing either Pdx-1 or Pax4: (1) spontaneous differentiation in embryoid bodies followed by adherent culture in standard medium, (2) selection of nestin-positive cells and differentiation using a protocol similar to that described by Lumelsky et al. (24), and (3) use of nestin-positive cells in histotypic culture that promotes the generation of spheroids (26). In cells undergoing spontaneous differentiation, Pax4- and Pdx-1-expressing cells generally showed increased expression of genes encoding transcription factors and other proteins important for or characteristic of differentiated islet cell function. Moreover, the amount of insulin mRNA and percentage of cells expressing insulin was increased in the Pdx-1- and Pax4-expressing cells, although the impact of Pax4 was greater than that of Pdx-1. After the selection and differentiation of nestin-positive cells, approximately 80% of Pax4-expressing cells produced insulin. Growth of cells in histotypic culture resulted in spheroids containing cells with insulin-positive granules, albeit at a density lower than that present in adult p cells. When transplanted into diabetic mice, differentiated nestin-positive Pax4-expressing and wild-type ES cells were equally efficacious in restoring euglycemia. Thus expression of transcription factors important for p-cell development and differentiation augments the in vitro differentiation of ES cells into insulin-secreting cells, although the functional consequences in vivo remain unclear. One problem with the approach described previously is that transcription factor expression during development is dynamic. Indeed, Pax4 is important for p-cell differentiation during development, but it is essentially absent in adult murine p cells (27). Pdx-1 expression is relatively uniform early in development, but is later heterogeneous with high levels in p cells and lower levels in undifferentiated precursor cells (19). Thus constitutive expression fails to reproduce the dynamic regulation of transcription factor expression characteristic of cellular differentiation.

3.1.3. Insulin-Secreting Cells From Tissue Stem Cells

An alternative approach to using ES cells is to redirect the differentiation of adult stem cells along an islet lineage. One means of accomplishing this has been to use cells of endodermal origin. This has been attempted using IEC-6 cells, which are immature rat intestinal stem cells that exhibit an undifferentiated morphology and limited expression of intestinal-specific genes (28). Various approaches have been used to direct the differentiation of these cells into insulin-secreting cells. Stable and constitutive expression of Pdx-1 in IEC-6 cells caused them to assume an enteroendocrine cell phenotype capable of expressing serotonin, cholecystokinin, gastrin, and somatostatin (29). To direct these cells along an islet cell lineage, the Pdx-1-expressing cells were subsequently treated with betacellulin (30,31). Betacellulin is a member of the epidermal growth factor family of peptides that is expressed in adult and fetal pancreas, signals through the ErbB family of tyrosine kinase receptors, and stimulates the proliferation of multiple cell types, including p cells (32,33). Several lines of evidence suggest that betacellulin plays a key role in islet cell proliferation or differentiation. Betacellulin enhances pancreatic regeneration after a 90% pancreatectomy by increasing p-cell proliferation and mass (34). It also increases DNA synthe sis in human fetal pancreatic epithelial cells and enhances p-cell development in fetal murine pancreatic explant cultures (33,35). Treatment of PDX-1-express-ing IEC-6 cells with betacellulin resulted in insulin expression and the formation of secretory granules. However, insulin secretion was neither glucose-dependent nor stimulated by arginine (30,31). Among the transcription factors induced by betacellulin treatment was Isl-1. Isl-1 is an LIM homeodomain factor that is important early in pancreatic development and is expressed in pancreatic epithelium and mesenchyme surrounding the pancreas (36). It is also expressed later in development in postmitotic endocrine cells and is present in mature islet cells (36). Its role in islet function is unclear. Overexpression of Isl-1 in Pdx-1-express-ing cells also resulted in insulin expression (30,31). Transplantation of IEC-6 cells expressing both Pdx-1 and Isl-1 into diabetic rats transiently decreased the blood glucose level, although euglycemia was not restored (30). These studies suggest that expressing specific transcription factors in tissue stem cells can redirect their differentiation along an islet lineage, but that additional factors will be needed to fully differentiate the cells.

Liver is a second endoderm-derived tissue that has been used as a source of cells that can be directed to differentiate into islets. Like pancreas, liver is derived from ventral endoderm, and both tissues express members of the hepatocyte nuclear family and exhibit glucose responsiveness (37). Indeed, it has been suggested that there is an endodermal progenitor cell common to liver and pancreas (38). In vivo expression of transcription factors has been used to differentiate liver cells into insulin-secreting cells (37). Adenoviral-mediated expression of Pdx-1 has successfully generated insulin-producing cells in liver (39,40). After expression of Pdx-1, liver produced not only insulin, but also other islet genes, including those encoding glucagon, somatostatin, and islet amyloid polypeptide. Expression of these genes, as well as the Pdxl gene, was prolonged as Pdx-1, insulin, and somatostatin expression was present 6-8 months after the initial infection. Glucagon expression was extinguished after about 4 months. Prolonged expression of Pdx-1, and presumably other islet proteins, appeared to be due to auto-induction of the native Pdxl gene by Pdx-1 expressed from the adenoviral vector (40). After Pdx-1 expression, the insulin content of the liver was increased 10- to 30-fold, but this was still only 1.3-3% of the insulin content of pancreas (40). Insulin produced by the liver was functional in that it was able to treat and prevent diabetes induced by streptozotocin, a p-cell toxin (39,40). The cells producing insulin were distinct from those that produced glucagon and were localized in proximity to the central vein. Mature hepatocytes reside in this region of the liver, although, because only a small percentage of infected cells expressed insulin, only a small subpopulation of cells appears to be capable of transdifferentiation. The nature of these cells that undergo transdifferentiation is not clear.

A similar approach has been used to develop insulin-producing cells from epithelial progenitor cells derived from fetal liver (41). These cells express markers of hepatocytes, bile ducts, and oval cells and are capable of differentiating into mature hepatocytes in vivo (42). Oval cells are thought to represent hepatic stem cells (43). Transduction of these progenitor cells with a lentivirus that constitutively expresses mRNA encoding Pdx-1 results in partial differentiation along an islet lineage (41). Despite expression of Pdx-1, these cells continued to express hepatocyte markers, including glycogen, dipeptidyl peptidase IV, and Y-glutamyl transpeptidase. Autoinduction of the endogenous Pdxl gene was again evident, and some transcription factors present in adult p cells (e.g., NeuroD1, Nkx6.1) were also expressed, whereas others such as Nkx2.2 and Pax6 were absent (41). Interestingly, neurogenin3, which is present in developing but not mature islets, was also present. Finally, insulin and the prohormone convertases PC1/3 and PC2 as well as islet amyloid polypeptide, glucagon, pancreatic polypeptide, and elastase were expressed. Thus proteins present in both the endocrine and exocrine pancreas were produced. It has not been established whether these different hormones and enzymes are coexpressed by the same or different cells. Importantly, these cells exhibit glucose-stimulated insulin secretion, albeit with a curve that is shifted to the right compared with native islets. This may reflect a lack of expression of GLUT2 and glucokinase and expression of only the Kir6.2 subunit of the ATP-sensitive potassium channel that is important for insulin secretion. Importantly, these cells appeared to secrete mature processed insulin and were able to reverse streptozotocin-induced diabetes.

In studies using an adenoviral vector capable of higher and more prolonged expression, in vivo Pdx-1 expression in the liver had a different effect. In this circumstance, insulin-producing cells were present, but cells exhibiting characteristics of exocrine cells, including expression of trypsin, were also present (44,45). Interestingly, insulin and trypsin were coexpressed by the same cells, and the latter induced a severe hepatitis (44,45). In contrast, use of this same adenoviral vector to express the transcription factor NeuroD1/Beta2 and betacellulin resulted in the formation of islet clusters capable of reversing streptozotocin-induced diabetes (44,45). The islet-like clusters were, in general, localized immediately underneath the liver capsule. Thus the cells from which islet-like structures were generated appeared to be distinct from those in the proximity of the central vein that differentiated into insulin-secreting cells following Pdx-1 expression. After expression of NeuroD1 and betacellulin, gluca-gon, somatostatin, and pancreatic polypeptide were also present in the islet-like structures. Unlike native islets, individual cells in the islet-like structures produced multiple hormones. Other genes characteristic of mature islets were also expressed, including those encoding the prohormone convertases PC1/3 and

PC2 and the Kir6.2 and SUR1 subunits of the ATP-sensitive potassium channel (44,45). Insulin granules were also present in the cells.

Was this article helpful?

0 0
Delicious Diabetic Recipes

Delicious Diabetic Recipes

This brilliant guide will teach you how to cook all those delicious recipes for people who have diabetes.

Get My Free Ebook


Post a comment