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

Rôle des glucocorticoïdes et de PGC-1a dans le développement et la fonction des cellules b pancréatiques

Une altération de l environnement fœtal peut augmenter le risque de développer certaines pathologies chez l adulte, d où la notion de programmation fœtale de maladies adultes. Nous avons montré dans des modèles murins que la sous-nutrition maternelle, associée à une surexposition aux glucocorticoïdes (GC) induit une altération de la masse et de la fonction des cellules b pancréatiques aboutissant à une intolérance au glucose chez l adulte. Dans ce travail de thèse, nous avons voulu définir les acteurs moléculaires de ces régulations. Nous avons montré chez la souris que la surexposition fœtale aux GC module l expression des gènes contrôlant la différenciation pancréatique et que les effets délétères des GC sur les cellules b nécessitaient la présence du récepteur aux GC (GR). Dans des lignées de cellules b, les GC diminuent l expression de facteurs de transcription importants pour la fonction des cellules b tels que Pdx1 et augmentent celle du coactivateur transcriptionnel PGC-1a, un partenaire du GR. Nous avons ensuite montré que PGC-1a participe aux effets des GC sur les cellules b et notamment sur la répression de Pdx1. Finalement, nous avons généré des souris surexprimant PGC-1a dans les cellules b et montré qu elles présentent une altération de la tolérance glucidique, de la sécrétion d insuline, de la masse et de la taille individuelle des cellules b. Enfin, nous avons montré que la surexpression uniquement fœtale suffit à induire le phénotype observé. En conclusion, nos travaux ont permis d identifier PGC-1a en tant que régulateur clé de la masse et de la fonction des cellules b, suggérant son rôle potentiel dans l étiologie du diabète de type 2PARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

Characterization of Stimulus-Secretion Coupling in the Human Pancreatic EndoC-βH1 Beta Cell Line

International audienceAIMS/HYPOTHESIS:Studies on beta cell metabolism are often conducted in rodent beta cell lines due to the lack of stable human beta cell lines. Recently, a human cell line, EndoC-βH1, was generated. Here we investigate stimulus-secretion coupling in this cell line, and compare it with that in the rat beta cell line, INS-1 832/13, and human islets.METHODS:Cells were exposed to glucose and pyruvate. Insulin secretion and content (radioimmunoassay), gene expression (Gene Chip array), metabolite levels (GC/MS), respiration (Seahorse XF24 Extracellular Flux Analyzer), glucose utilization (radiometric), lactate release (enzymatic colorimetric), ATP levels (enzymatic bioluminescence) and plasma membrane potential and cytoplasmic Ca2+ responses (microfluorometry) were measured. Metabolite levels, respiration and insulin secretion were examined in human islets.RESULTS:Glucose increased insulin release, glucose utilization, raised ATP production and respiratory rates in both lines, and pyruvate increased insulin secretion and respiration. EndoC-βH1 cells exhibited higher insulin secretion, while plasma membrane depolarization was attenuated, and neither glucose nor pyruvate induced oscillations in intracellular calcium concentration or plasma membrane potential. Metabolite profiling revealed that glycolytic and TCA-cycle intermediate levels increased in response to glucose in both cell lines, but responses were weaker in EndoC-βH1 cells, similar to those observed in human islets. Respiration in EndoC-βH1 cells was more similar to that in human islets than in INS-1 832/13 cells.CONCLUSIONS/INTERPRETATION:Functions associated with early stimulus-secretion coupling, with the exception of plasma membrane potential and Ca2+ oscillations, were similar in the two cell lines; insulin secretion, respiration and metabolite responses were similar in EndoC-βH1 cells and human islets. While both cell lines are suitable in vitro models, with the caveat of replicating key findings in isolated islets, EndoC-βH1 cells have the advantage of carrying the human genome, allowing studies of human genetic variants, epigenetics and regulatory RNA molecules

Mitochondrial transcription factor B2 is essential for mitochondrial and cellular function in pancreatic β-cells

Objective: Insulin release from pancreatic β-cells is controlled by plasma glucose levels via mitochondrial fuel metabolism. Therefore, insulin secretion is critically dependent on mitochondrial DNA (mtDNA) and the genes it encodes. Mitochondrial transcription factor B2 (TFB2M) controls transcription of mitochondrial-encoded genes. However, its precise role in mitochondrial metabolism in pancreatic β-cells and, consequently, in insulin secretion remains unknown. Methods: To elucidate the role of TFB2M in mitochondrial function and insulin secretion in vitro and in vivo, mice with a β-cell specific homozygous or heterozygous knockout of Tfb2m and rat clonal insulin-producing cells in which the gene was silenced were examined with an array of metabolic and functional assays. Results: There was an effect of gene dosage on Tfb2m expression and function. Loss of Tfb2m led to diabetes due to disrupted transcription of mitochondrial DNA (mtDNA) and reduced mtDNA content. The ensuing mitochondrial dysfunction activated compensatory mechanisms aiming to limit cellular dysfunction and damage of β-cells. These processes included the mitochondrial unfolded protein response, mitophagy, and autophagy. Ultimately, however, these cell-protective systems were overridden, leading to mitochondrial dysfunction and activation of mitochondrial-dependent apoptotic pathways. In this way, β-cell function and mass were reduced. Together, these perturbations resulted in impaired insulin secretion, progressive hyperglycemia, and, ultimately, development of diabetes. Conclusions: Loss of Tfb2m in pancreatic β-cells results in progressive mitochondrial dysfunction. Consequently, insulin secretion in response to metabolic stimuli is impaired and β-cell mass reduced. Our findings indicate that TFB2M plays an important functional role in pancreatic β-cells. Perturbations of its actions may lead to loss of functional β-cell mass, a hallmark of T2D

Consequences of GR overexpression on beta-cell mass and glucose homeostasis in Pdx1-itTA/LacZtetOhGR or Ins1-itTA/LacZtetOhGR mice.

<p>(A) Immunohistochemistry for the glucocorticoid receptor (GR, brown nuclear staining) on pancreatic sections from a control LacZtetOhGR, (B) a Pdx1-itTA/LacZtetOhGR mouse and (C) a Ins1-itTA/LacZtetOhGR mouse. (D) Beta-cell fraction, (E) beta-cell mass, (F) blood glucose during an intra-peritoneal glucose tolerance test and (G) corresponding Area Under the Curve (AUC) in adult Pdx1-itTA/LacZtetOhGR mice. (H) Beta-cell fraction, (I) beta-cell mass, (J) blood glucose during an intra-peritoneal glucose tolerance test and (K) corresponding Area Under the Curve (AUC) in adult Ins1-itTA/LacZtetOhGR mice. Results are expressed as means ± SD for n = 3–5 animals per group. * p<0.05 when comparing double transgenic mice (Pdx1-itTA/LacZtetOhGR or Ins1-itTA/LacZtetOhGR) to control (LacZtetOhGR) mice using a Mann-Whitney non parametric test. Scale bar = 100 µm.</p

Similarity of editing-sequence contexts in fruit fly, mouse and human.

<p>Each row shows the number of A-to-I editing-sequence contexts in species similar with those in fruit fly, mouse and human. Values in brackets refer to training datasets in this study.</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 flowchart of the proposed method.

<p>First, we obtain flanking sequences surrounding the known editing sites and background sets from the reference genome. Second, we measure the similarity of sequence segments pairs (background sets are not used during the training process for one-class classification). Then the generated similarity matrix is mapped to string kernel space. For this kernel matrix, the training process tries to find a maximal margin hyperplane, which is usually achieved by quadratic programming. Based on a generated model from the training process, prediction can be made, using any given sequence or mapped short reads from next generation sequencing by extracting adenine centered segments. L₁: length used for calculating edit distance. L₂: length used for calculating Hamming distance.</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