Novel Transgenic Mice for Inducible Gene Overexpression in Pancreatic Cells Define Glucocorticoid Receptor-Mediated Regulations of Beta Cells

Conditional gene deletion in specific cell populations has helped the understanding of pancreas development. Using this approach, we have shown that deleting the glucocorticoid receptor (GR) gene in pancreatic precursor cells leads to a doubled beta-cell mass. Here, we provide genetic tools that permit a temporally and spatially controlled expression of target genes in pancreatic cells using the Tetracycline inducible system. To efficiently target the Tetracycline transactivator (tTA) in specific cell populations, we generated Bacterial Artificial Chromosomes (BAC) transgenic mice expressing the improved Tetracycline transactivator (itTA) either in pancreatic progenitor cells expressing the transcription factor Pdx1 (BAC-Pdx1-itTA), or in beta cells expressing the insulin1 gene (BAC-Ins1-itTA). In the two transgenic models, itTA-mediated activation of reporter genes was efficient and subject to regulation by Doxycycline (Dox). The analysis of a tetracycline-regulated LacZ reporter gene shows that in BAC-Pdx1-itTA mice, itTA is expressed from embryonic (E) day 11.5 in all pancreatic precursor cells. In the adult pancreas, itTA is active in mature beta, delta cells and in few acinar cells. In BAC-Ins1-itTA mice tTA is active from E13.5 and is restricted to beta cells in fetal and adult pancreas. In both lines, tTA activity was suppressed by Dox treatment and re-induced after Dox removal. Using these transgenic lines, we overexpressed the GR in selective pancreatic cell populations and found that overexpression in precursor cells altered adult beta-cell fraction but not glucose tolerance. In contrast, GR overexpression in mature beta cells did not alter beta-cell fraction but impaired glucose tolerance with insufficient insulin secretion. In conclusion, these new itTA mouse models will allow fine-tuning of gene expression to investigate gene function in pancreatic biology and help us understand how glucocorticoid signaling affects on the long-term distinct aspects of beta-cell biology

Glucocorticoids Inhibit Basal and Hormone-Induced Serotonin Synthesis in Pancreatic Beta Cells

International audienceDiabetes is a major complication of chronic Glucocorticoids (GCs) treatment. GCs induce insulin resistance and also inhibit insulin secretion from pancreatic beta cells. Yet, a full understanding of this negative regulation remains to be deciphered. In the present study, we investigated whether GCs could inhibit serotonin synthesis in beta cell since this neurotransmitter has been shown to be involved in the regulation of insulin secretion. To this aim, serotonin synthesis was evaluated in vitro after treatment with GCs of either islets from CD1 mice or MIN6 cells, a beta-cell line. We also explored the effect of GCs on the stimulation of serotonin synthesis by several hormones such as prolactin and GLP 1. We finally studied this regulation in islet in two in vivo models: mice treated with GCs and with liraglutide, a GLP1 analog, and mice deleted for the glucocorticoid receptor in the pancreas. We showed in isolated islets and MIN6 cells that GCs decreased expression and activity of the two key enzymes of serotonin synthesis, Tryptophan Hydroxylase 1 (Tph1) and 2 (Tph2), leading to reduced serotonin contents. GCs also blocked the induction of serotonin synthesis by prolactin or by a previously unknown serotonin activator, the GLP-1 analog exendin-4. In vivo, activation of the Glucagon-like-Peptide-1 receptor with liraglutide during 4 weeks increased islet serotonin contents and GCs treatment prevented this increase. Finally, islets from mice deleted for the GR in the pancreas displayed an increased expression of Tph1 and Tph2 and a strong increased serotonin content per islet. In conclusion, our results demonstrate an original inhibition of serotonin synthesis by GCs, both in basal condition and after stimulation by prolactin or activators of the GLP-1 receptor. This regulation may contribute to the deleterious effects of GCs on beta cells

Correction: Glucocorticoids Inhibit Basal and Hormone-Induced Serotonin Synthesis in Pancreatic Beta Cells.

[This corrects the article DOI: 10.1371/journal.pone.0149343.]

Fetal PGC-1 alpha Overexpression Programs Adult Pancreatic beta-Cell Dysfunction

International audienceAdult beta-cell dysfunction, a hallmark of type 2 diabetes, can be programmed by adverse fetal environment. We have shown that fetal glucocorticoids (GCs) participate in this programming through inhibition of beta-cell development. Here we have investigated the molecular mechanisms underlying this regulation. We showed that GCs stimulate the expression of peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1 alpha), a coregulator of the GCs receptor (GR), and that the overexpression of PGC-1 alpha represses genes important for beta-cell development and function. More precisely, PGC-1 alpha inhibited the expression of the key beta-cell transcription factor pancreatic duodenal homeobox 1 (Pdx1). This repression required the GR and was mediated through binding of a GR/PGC-1 alpha complex to the Pdx1 promoter. To explore PGC-1 alpha function, we generated mice with inducible beta-cell PGC-1 alpha overexpression. Mice overexpressing PGC-1 alpha exhibited at adult age impaired glucose tolerance associated with reduced insulin secretion, decreased beta-cell mass, and beta-cell hypotrophy. Interestingly, PGC-1 alpha expression in fetal life only was sufficient to impair adult beta-cell function whereas beta-cell PGC-1 alpha overexpression from adult age had no consequence on beta-cell function. Altogether, our results demonstrate that the GR and PGC-1 alpha participate in the fetal programming of adult beta-cell function through inhibition of Pdx1 expression. Diabetes 62:1206-1216, 201

The Pdx1-itTA activates lacZ expression in beta, delta and in acinar cells.

<p>(A) (E) (I) (M) and (Q) LacZ expression revealed by Xgal staining (blue) on adult pancreatic sections from a Pdx1-itTA/LacZtetOhGR mouse. (B) Immunofluorescence for insulin (green), (C) merge of A and B and (D) magnified view of inset in C. (F) Immunofluorescence for glucagon (green), (G) merge of E and F and (H) magnified view of inset in G. (J) Immunofluorescence for somatostatin (green), (K) merge of I and J and (L) magnified view of inset in K. (N) Immunofluorescence for PP (green), (O) merge of M and N and (P) magnified view of inset in O. (R) Immunofluorescence for amylase (green), (S) merge of Q and R and (T) magnified view of inset in S. Scale bar = 50 µm except for D, H, L, P and T where scale bar = 10 µm.</p

The Ins1-itTA is active in cells expressing insulin during fetal life.

<p>(A) LacZ expression in pancreatic buds at E13.5 in the dorsal (B) and ventral (C) part of the pancreas from Ins1-itTA/LacZtetOhGR fetuses. (D) LacZ expression is found at E15.5 in scattered cells. (E) A view of the dissected pancreas at E15.5.</p

Generation and characterization of mice expressing the itTA under the control of Insulin1 regulatory elements.

<p>(A) Bacterial Artificial Chromosomes (BAC) containing around 200 kb of the genomic region of Insulin1 was obtained from BAC libraries developed by the CHORI, Oakland USA. The coding region of the gene was removed by homologous recombination in bacteria and replaced by the itTA cDNA. The construct was then injected in the pronuclei of fertilized eggs. itTA = humanized tetracycline transactivator; AMP = ampicillin resistance gene; PA = polyadenylation site; FRT = Flippase recognition target. (B) Lac Z expression revealed by Xgal staining (blue) in islets in Ins1-itTA/LacZtetOhGR mice. (C) Absence of blue staining in control mice carrying only the LacZtetOhGR transgene. Two islets are outlined. Scale bar = 50 µm.</p

Generation and characterization of mice expressing the itTA under the control of <i>Pdx1</i> regulatory elements.

<p>(A) Bacterial Artificial Chromosomes (BAC) containing 202 kb of the genomic region of Pdx1 was obtained from a BAC library developed by the CHORI, Oakland, USA. The coding region of the gene was removed by homologous recombination in bacteria and replaced by the itTA cDNA. The construct was then injected in the pronuclei of fertilized eggs. itTA = improved tetracycline transactivator; AMP = ampicillin resistance gene; PA = polyadenylation site; FRT = Flippase recognition target. Below, a scheme representing the LacZtetOhGR construct (B) Lac Z expression revealed by Xgal staining (blue) in islets and in exocrine tissue in Pdx-itTA/LacZtetOhGR mice. (C) Magnified view of the boxed area of B with arrows pointing at blue cells in the exocrine tissue. (D) Absence of blue staining in control mice carrying only the LacZtetOhGR transgene. Two islets are outlined. Scale bar = 50 µm.</p

The Ins1-itTA transgene activates lacZ expression only in beta cells.

<p>(A), (E), (I), (M) and (Q) LacZ expression revealed by Xgal staining (blue) on adult pancreatic sections from a Ins1-itTA/LacZtetOhGR mouse. (B) Immunofluorescence for insulin (green), (C) merge of A and B and (D) magnified view of inset in C. (F) Immunofluorescence for glucagon (green), (G) merge of E and F and (H) magnified view of inset in G. (J) Immunofluorescence for somatostatin (green), (K) merge of I and J and (L) magnified view of inset in K. (N) Immunofluorescence for PP (green), (O) merge of M and N and P magnified view of inset in O. (R) Immunofluorescence for amylase (green), (S) merge of Q and R and (T) magnified view of inset in S. Scale bar = 50 µm except for D, H, L, P and T where scale bar = 10 µm.</p