Regulation of Pancreatic Juxtaductal Endocrine Cell Formation by FoxO1

Abstract

An understanding of the mechanisms that govern pancreatic endocrine cell ontogeny may offer strategies for their somatic replacement in diabetic patients. During embryogenesis, transcription factor FoxO1 is expressed in pancreatic progenitor cells. Subsequently, it becomes restricted to ␤ cells and to a rare population of insulin-negative juxtaductal cells (FoxO1 Diabetes is characterized by complete or relative deficiency of insulin-producing ␤ cells (1). The growing societal and public health toll of the disease provides impetus to isolate or generate ␤ cells for cellular replacement purposes. Moreover, given that most of the newly found diabetes susceptibility genes appear to affect ␤-cell function rather than insulin action (12,41,43), that the two newest classes of antidiabetic medications are ␤-tropic (2), and that the main therapeutic failures in diabetes are seen in response to ␤-tropic agents (19, 48), studies of ␤-cell biology have wide-ranging implications beyond the replacement issue.Two approaches to ␤-cell generation have been championed: one endeavors to define culture conditions conducive to embryonic stem cell differentiation into ␤ cells (6), while the other is based on the hypothesis that endocrine cell progenitors, often identified with duct epithelial cells, exist in the adult pancreas and can yield functional ␤ cells (4, 42).Lineage-tracing studies indicate that pancreatic endocrine cells arise from a neurogenin 3 (Neurog3)-expressing progenitor pool set aside early in embryonic development (13) and that postnatal ␤-cell turnover is a result of limited ␤-cell replication and apoptosis (8,34,46). These data point to a limited role of pancreatic duct cells in the maintenance of ␤-cell mass through neogenesis from non-␤-cell precursors. Nonetheless, ductal endocrine cell neogenesis can occur following pancreatic duct ligation (17, 49), raising the possibility of generating endocrine cells by commandeering developmental pathways at the genetic level. Along these lines, we made the intriguing observation of a rare population of juxtaductal FoxO1 ϩ cells that do not express insulin. This finding, coupled with the role of FoxOs in governing developmental processes in diverse lineages and in the long-term stability of various tissues (24,32,35), prompted us to examine whether these cells are progenitors of duct-associated endocrine cells. In this study, we used a combination of developmental, genetic, and cell biology analyses to identify, isolate, and functionally characterize these cells. MATERIALS AND METHODS Antibodies and immunohistochemistry.We used the following antibodies: antipancytokeratin (Sigma), antivimentin (Santa Cruz), anti-Nkx2.2 (homeodomain protein) (Hybridoma Bank, University of Iowa), anti-FoxO1 (21), antipancreas and duodenum homeobox protein 1 (anti-Pdx1) (25), antiglucagon (Sigma), anti-insulin (Dako), antisomatostatin (Chemicon), anti-pancreatic polypeptide (anti-Pp) (Linco), antiamylase (Abcam), anti-green fluorescent protein (anti-GFP) (Santa Cruz), and anti-forkhead box-...

Effect of forkhead box O1 (FOXO1) on beta cell development in the human fetal pancreas

“…Moreover, nFOXO1 was highly co-localised with PDX-1 + and NGN3 + cell populations during this developmental period. These findings are consistent with observations in the mouse, where FOXO1 is widely produced between e9.5 and 14.5 [10][11][12][13][14], a stage corresponding to the human developmental window examined in the present study. However, following this period in the mouse, a global decline in the distribution of FOXO1 has been observed, whereby this transcription factor becomes restricted to endocrine progenitor cells by e17.5 and eventually becomes limited to beta cells postnatally [12,13].…”

“…Knockdown led to notable increases in the beta cell population as well as insulin mRNA levels, indicating that FOXO1 may be involved in beta cell differentiation during human fetal pancreatic development. These findings are in accord with a recent study reporting a selective increase in juxta-ductal beta cells following a conditional Foxo1 knockdown in pancreatic progenitors in Pdx-1-cre mice [13].…”

“…Co-localisation of FOXO1 and NGN3 has been observed in both human and mouse models [13]. Downregulation of FOXO1 mRNA in human fetal islets resulted in a significant upregulation of NGN3 mRNA levels and the number of NGN3 + cells, suggesting that NGN3 may be a potential target for FOXO1.…”

“…During mouse pancreatic organogenesis, FOXO1 and PDX-1 share similar production patterns; both are widely produced in the pancreatic epithelium between embryonic day (e) 9.5 and 14.5 and become exclusively produced postnatally in beta cells [10][11][12][13][14]. More recently, FOXO1 has been reported in murine myoblasts to interact with Notch signalling to regulate hairy and enhancer of split 1 (Hes1) gene expression; HES1 is a well-known repressor of neurogenin 3 (Ngn3 [also known as Neurog3]) [15,16].…”

Increased Atherosclerosis and Endothelial Dysfunction in Mice Bearing Constitutively Deacetylated Alleles of Foxo1 Gene

“…We used cells at passages 2-4 after serum starvation for 16 h in DMEM supplemented with 5 mM glucose and 0.25% (v/v) bovine serum albumin (BSA). FoxO1 immunocytochemistry was performed as described previously (26).…”