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

OBJECTIVE: Insulin release from pancreatic ß-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 ß-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 ß-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 ß-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, ß-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 ß-cells results in progressive mitochondrial dysfunction. Consequently, insulin secretion in response to metabolic stimuli is impaired and ß-cell mass reduced. Our findings indicate that TFB2M plays an important functional role in pancreatic ß-cells. Perturbations of its actions may lead to loss of functional ß-cell mass, a hallmark of T2D.

Discriminative Prediction of A-To-I RNA Editing Events from DNA Sequence.

RNA editing is a post-transcriptional alteration of RNA sequences that, via insertions, deletions or base substitutions, can affect protein structure as well as RNA and protein expression. Recently, it has been suggested that RNA editing may be more frequent than previously thought. A great impediment, however, to a deeper understanding of this process is the paramount sequencing effort that needs to be undertaken to identify RNA editing events. Here, we describe an in silico approach, based on machine learning, that ameliorates this problem. Using 41 nucleotide long DNA sequences, we show that novel A-to-I RNA editing events can be predicted from known A-to-I RNA editing events intra- and interspecies. The validity of the proposed method was verified in an independent experimental dataset. Using our approach, 203 202 putative A-to-I RNA editing events were predicted in the whole human genome. Out of these, 9% were previously reported. The remaining sites require further validation, e.g., by targeted deep sequencing. In conclusion, the approach described here is a useful tool to identify potential A-to-I RNA editing events without the requirement of extensive RNA sequencing.

Characterization of stimulus-secretion coupling in the human pancreatic EndoC-ßH1 beta cell line.

AIMS/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-ßH1, 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-ßH1 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-ßH1 cells, similar to those observed in human islets. Respiration in EndoC-ßH1 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-ßH1 cells and human islets. While both cell lines are suitable in vitro models, with the caveat of replicating key findings in isolated islets, EndoC-ßH1 cells have the advantage of carrying the human genome, allowing studies of human genetic variants, epigenetics and regulatory RNA molecules.

Fetal PGC-1α overexpression programs adult pancreatic ß-cell dysfunction.

Adult ß-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 ß-cell development. Here we have investigated the molecular mechanisms underlying this regulation. We showed that GCs stimulate the expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), a coregulator of the GCs receptor (GR), and that the overexpression of PGC-1α represses genes important for ß-cell development and function. More precisely, PGC-1α inhibited the expression of the key ß-cell transcription factor pancreatic duodenal homeobox 1 (Pdx1). This repression required the GR and was mediated through binding of a GR/PGC-1α complex to the Pdx1 promoter. To explore PGC-1α function, we generated mice with inducible ß-cell PGC-1α overexpression. Mice overexpressing PGC-1α exhibited at adult age impaired glucose tolerance associated with reduced insulin secretion, decreased ß-cell mass, and ß-cell hypotrophy. Interestingly, PGC-1α expression in fetal life only was sufficient to impair adult ß-cell function whereas ß-cell PGC-1α overexpression from adult age had no consequence on ß-cell function. Altogether, our results demonstrate that the GR and PGC-1α participate in the fetal programming of adult ß-cell function through inhibition of Pdx1 expression.

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.