Introduction
The mature pancreas contains exocrine acinar cells that secrete digestive enzymes into the intestine, and endocrine islets that synthesise hormones such as insulin (beta cells), glucagon (alpha cells), somatostatin (delta cells) and pancreatic polypeptide (PP cells). The pancreas originates from the dorsal and ventral regions of the foregut endoderm directly posterior to the stomach. The first indication of pancreatic morphogenesis occurs in mice at embryonic day (E) 8.5 (E9.5 in rat), when the endoderm evaginates to form buds. Subsequently, the mesenchyme condenses around the underlying endoderm and the epithelial buds grow in size, while the exocrine and endocrine cells differentiate [
1].
During development, the endodermal region committed to form the pancreas initially produces the transcription factor pancreatic and duodenal homeobox 1 (Pdx-1), a marker of pancreatic progenitors also produced in mature beta cells [
2]. The basic helix–loop–helix factor neurogenin 3 (Neurog3) is then transiently produced in epithelial pancreatic progenitor cells prior to endocrine differentiation [
3]. Neurog3 controls the expression of NeuroD, which is another member of the basic helix–loop–helix transcription factor family [
4].
Pancreatic development is controlled by signals derived from tissues in contact with the endodermal region that gives rise to the pancreas [
5]. Pancreatic development also depends on environmental signals such as oxygen tension [
6,
7] and nutrients [
8]. We previously showed that glucose controls beta cell development [
9]. Specifically, we found that glucose favours pancreatic endocrine cell development by regulating the transition between Neurog3 and NeuroD [
9].
Typically, glucose is transported inside the cell and is phosphorylated into glucose 6-phosphate. It next enters three pathways: the glycolysis pathway to provide energy, the hexosamine biosynthetic pathway (HBP) and the pentose phosphate pathway [
10]. We previously demonstrated that the positive effect of glucose on beta cell development required its metabolism through the HBP [
11]. This pathway produces UDP-
N-acetyl-glucosamine (UDP-GlcNAc), a substrate for
N- and
O-glycosylation, as well as for
O-GlcNAcylation, which consists of the addition of a single GlcNAc moiety to serine and threonine residues (for a review, see Bouche et al [
12]). This last modification is highly dynamic and often compared with phosphorylation.
Interestingly, the pentose phosphate pathway is an alternative pathway for glucose metabolism that generates NADPH and synthesises pentose sugars [
13]. Xylulose 5-phosphate, an intermediate of the pentose phosphate pathway, activates type 2A protein phosphatase (PP2A), which in turn dephosphorylates and activates transcription factor carbohydrate-responsive element binding protein (ChREBP) [
14,
15]. ChREBP is a transcription factor that belongs to the basic helix–loop–helix leucine zipper family, which transactivates glucose-responsive genes such as those encoding acetyl-CoA carboxylase (ACCase) and liver-type pyruvate kinase (L-PK) by binding the carbohydrate-responsive element [
16]. To the best of our knowledge, little information is available concerning the role of ChREBP in pancreatic cell development.
Here, we tested the possible involvement of ChREBP in pancreatic development, and the role of ChREBP as a mediator of glucose effect on beta cell differentiation. For this purpose, we modulated ChREBP activity in an in vitro model of pancreatic development and analysed beta cell differentiation.
Results
Treatment with okadaic acid, a PP2A inhibitor, reversed xylitol-induced activation of
ACCase and
L-PK expression, an effect that was stronger at 2 nmol/l than at 0.02 nmol/l (Fig.
2). Such an effect was also observed with calyculin A, another PP2A inhibitor (data not shown). Interestingly, okadaic acid treatment also reversed glucose-induced activation of
ACCase and
L-PK expression, but the effect was observed only with 2 nmol/l okadaic acid (Fig.
2).
Discussion
Pancreatic beta cell development is a multistep process. Information is now available on the signals that regulate early steps of the process, i.e. the development of PDX1-positive pancreatic progenitors from definitive endoderm [
30‐
32]. On the other hand, less is known about signals controlling beta cell differentiation from PDX1-positive pancreatic progenitors. This step in development can be further divided into three consecutive steps: (1) the proliferation of PDX1-positive pancreatic progenitors; (2) their differentiation into acinar tissue or into NEUROG3-producing endocrine progenitors; and (3) the differentiation of NEUROG3-producing endocrine progenitors into insulin-producing beta cells [
33]. Here, by using activators and inhibitors of the pentose phosphate shunt, we demonstrated that this pathway modulated neither the proliferation of PDX1-positive pancreatic progenitors, nor acinar cell differentiation, nor the formation of NEUROG3-producing endocrine progenitors. On the other hand, we demonstrated that the positive effect of glucose and xylitol on pancreatic beta cell differentiation depended on the transcription factor ChREBP and occurred in NEUROG3-producing endocrine progenitors.
In this study, we used an in vitro bioassay that recapitulates the major steps occurring during beta cell development from PDX1-positive fetal pancreatic progenitor cells. In this model, PDX1-positive pancreatic progenitors first proliferate and next differentiate into endocrine or acinar tissue. We previously validated and used this model to define factors and conditions that regulate specific steps in beta cell development. Specifically, with this assay, we previously demonstrated that fibroblast growth factor 10 was an activator of the proliferation of PDX1-positive pancreatic progenitors [
18]. Here, we found that xylitol did not regulate cell proliferation during pancreatic development, as previously shown for glucose [
9]. We also previously used this model of in vitro pancreatic development to define signals and conditions that modulate the differentiation of PDX1-positive pancreatic progenitors into NEUROG3-positive endocrine progenitors. We demonstrated that small molecules such as histone deacetylase inhibitors [
23], branched amino acids [
34] and oxygen tension [
7] regulate the development of NEUROG3-positive endocrine progenitors from PDX1-positive pancreatic progenitors. Recently, using the same assay, we demonstrated that glucose regulates the next step in beta cell development, i.e. the differentiation of beta cells from NEUROG3-positive endocrine progenitors, and that glucose metabolism was implicated in this regulation at least in part through activation of the HBP [
9,
11].
In the present study, we demonstrated that the transcription factor ChREBP regulates beta cell differentiation. ChREBP regulates gene transcription in response to glucose (for a review, see [
24]). Specifically, the pentose phosphate pathway, and particularly xylulose 5-phosphate, selectively activates PP2A, which in turn dephosphorylates the transcription factor ChREBP [
14]. This allows ChREBP translocation into the nucleus and activation of ChREBP targets, such as the glycolytic gene
L-PK [
35] or the lipogenic genes
ACCase and
Fas (which encodes fatty acid synthase) [
36]. ChREBP production is most abundant in liver, and is small in kidney and white and brown adipose tissue [
16]. In adult islets, ChREBP controls the expression of a number of genes such as
L-PK [
37],
ACCase [
38],
Fas [
39], thioredoxin-interacting protein [
40] and aryl hydrocarbon receptor nuclear translocator [
41]. Interestingly, recent data suggest that ChREBP can also be activated by glucose 6-phosphate [
42] and by the HBP [
43,
44], placing ChREBP at the crossroads of all glucose metabolic pathways.
Little information has been available on the role of ChREBP during pancreatic development. Different arguments derived from the present study indicate that ChREBP is important for proper beta cell differentiation. First, as described in this study, ChREBP is expressed in the pancreas during development [
45]. Second, during prenatal life, ChREBP is highly enriched in the endocrine pathway, as demonstrated by its sharp decrease in pancreases from
Neurog3-deficient mice that lack endocrine cells. ChREBP can thus be added to the list of genes whose expression is enriched in pancreatic endocrine cells [
46]. Third, in the embryonic pancreas, xylitol, a precursor of xylulose 5-phosphate and an intermediary molecule of the pentose phosphate pathway, activates the expression of
ACCase and
L-PK, two direct targets of ChREBP, demonstrating that this pathway is functional during pancreatic development. Fourth, xylitol activates beta cell differentiation without affecting acinar cell development. This fits well with the fact that ChREBP is highly enriched in cells of the endocrine pathway. In addition, okadaic acid and calyculin A, two inhibitors of the serine/threonine PP2A, which inhibit ChREBP activity [
47,
48], suppress the effects of xylitol on beta cell development. Finally, the production of dnChREBP suppresses the effects of both glucose and GlcNAc, a substrate of the HBP, on beta cell development. Taken together, such data demonstrate that ChREBP tightly regulates beta cell development.
We have recently demonstrated that the HBP mediates, at least in part, the effect of glucose on beta cell differentiation [
11]. Specifically, we had proposed that the HBP controlled an
O-GlcNAcylated factor necessary for proper beta cell differentiation [
11]. It is now established that ChREBP is
O-GlcNAcylated [
43,
44]. We propose that glucose controls beta cell differentiation, by promoting ChREBP activity, first by allowing its dephosphorylation, and second through the HBP, which would regulate ChREBP
O-GlcNAcylation. These two steps, controlled by glucose, would permit efficient beta cell differentiation. One element supporting this hypothesis is the fact that, in pancreases infected with an Ad-dnChREBP, the positive effect of GlcNAc on beta cell differentiation is lost (Fig.
6). Therefore, the effect of glucose on beta cell development, via its metabolism through the HBP, requires an active ChREBP. ChREBP is thus at the crossroads of the different glucose metabolic pathways regulating beta cell differentiation from pancreatic progenitors.
The phenotype of mice deficient in ChREBP was recently analysed [
16]. Such mice were glucose-intolerant, but their pancreatic phenotype was not described. In light of our data, it would be interesting to study pancreatic beta cell development in such mice. It will also be interesting to determine whether glucose, through ChREBP activation, controls human beta cell differentiation. Such information would be important in protocols aiming at generating human beta cells from stem cells.
Acknowledgements
We would like to thank H.C. Towle (Department of Biochemistry, Molecular Biology and Biophysics, Minneapolis, Minnesota) for the Ad-dnChREBP and S. Fabrega (core facility: Viral Vectors and Gene Transfer, University Paris Descartes, Institut Fédératif de Recherche Necker Enfants Malades IFR94, Paris, France) for the production of GFP adenovirus and for the virus amplification.