Ghrelin cells replace insulin-producing beta cells in two mouse models of pancreas development

Abstract

The pancreatic islet is necessary for maintaining glucose homeostasis. Within the pancreatic islet, the homeodomain protein Nkx2.2 is essential for the differentiation of all insulin-producing beta cells and a subset of glucagon-producing alpha cells (1). Mice lacking Nkx2.2 have relatively normal sized islets, but a large number of cells within the mutant islet fail to produce any of the four major islet hormones. In this study we demonstrate that Nkx2.2 mutant endocrine cells have been replaced by cells that produce ghrelin, an appetite-promoting peptide predominantly found in the stomach. Intriguingly, normal mouse pancreas also contains a small population of ghrelin-producing cells, defining a new islet "epsilon" cell population. The expansion of ghrelin-producing cells at the expense of beta cells may be a general phenomenon, because we demonstrate that Pax4 mutant mice display a similar phenotype. We propose that insulin and ghrelin cells share a common progenitor and that Nkx2.2 and Pax4 are required to specify or maintain differentiation of the beta cell fate. This finding also suggests that there is a genetic component underlying the balance between insulin and ghrelin in regulating glucose metabolism.

Fig. 1.

Ghrelin mRNA and protein are up-regulated in the islets of Nkx2.2 mutant mice. Immunohistochemical analysis of sectioned wild-type pancreas (a–e) or mutant pancreas (f–j) is shown. All data are from neonatal pancreata except c and h, which show pancreata from e16.5 embryos. Dashed lines delineate the pancreas at e16.6 (c and h) and an islet in neonates (d, f, and g). (a and f) Expression of amylase (green) and insulin (red). (b and g) Expression of amylase (green) and glucagon (red). (c and h) RNA in situ analysis of ghrelin mRNA in e16.5 pancreas. Incubation time for wild-type and mutant samples was identical. (d and i) Expression of ghrelin protein (red) and amylase (green). (e and j) Expression of ghrelin (red) and neuroendocrine marker chromogranin A (green).

Fig. 2.

Ghrelin-expressing cells define a unique subset of islet cells in the mouse that do not express the four islet hormones; in the absence of Nkx2.2, ghrelin-expressing cells replace insulin- and glucagon-expressing cell populations. Shown is immunofluorescence analysis of sectioned wild-type (a–e) and mutant (f–i) neonatal pancreas. Dashed lines delineate the islets. Red, ghrelin; green, islet hormones. (a and f) Insulin. (b, c, and g) Glucagon. (d and h) Somatostatin. (e and i) PP.

Fig. 3.

Transcription factor profile of ghrelin-positive cells in wild-type islets. Immunofluorescence analysis of sectioned embryonic pancreas shows ghrelin (green), transcription factors, and glucagon (red). (a and b) Ghrelin expression in wild-type pancreas at e10.5. (a) Ghrelin is not coexpressed with Pdx1. (b) Ghrelin-positive, glucagon-positive cell (white arrowhead) and ghrelin-positive, glucagon-negative cell (white arrow). (c and d) e12.5: ghrelin is not expressed with Pdx1 (c) or Nkx6.1 (d). (e and f) e11.5: Pax6 is expressed in some, but not all, ghrelin cells (e). Nkx2.2 is expressed at low levels or not at all in ghrelin-positive cells (arrowhead) (f). Nkx2.2-high-expressing cells do not coexpress ghrelin (white arrowhead).

Fig. 4.

Pax4 mutant islets also have increased levels of ghrelin-expressing ε cells. Shown is nonradioactive in situ analysis of ghrelin mRNA on sections of e14.5 pancreas. (a) A small number of ghrelin-expressing cells are normally expressed in the wild-type pancreas at e14.5. (b) The number of ghrelin-expressing cells is greatly increased in Pax4 mutant islets, where there is a concomitant 90% reduction of insulin-producing cells (ref. and data not shown).

Fig. 5.

Schematic of the transcription factor pathway specifying insulin-producing β cells. In the absence of Nkx2.2 or Pax4, the progenitor cells are switched to a ghrelin-producing ε cell fate.