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1.
Diabetes ; 64(12): 4112-22, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26283735

ABSTRACT

Cystic fibrosis (CF) is the result of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). CF-related diabetes affects 50% of adult CF patients. How CFTR deficiency predisposes to diabetes is unknown. Herein, we examined the impact of the most frequent cftr mutation in humans, deletion of phenylalanine at position 508 (ΔF508), on glucose homeostasis in mice. We compared ΔF508 mutant mice with wild-type (WT) littermates. Twelve-week-old male ΔF508 mutants had lower body weight, improved oral glucose tolerance, and a trend toward higher insulin tolerance. Glucose-induced insulin secretion was slightly diminished in ΔF508 mutant islets, due to reduced insulin content, but ΔF508 mutant islets were not more sensitive to proinflammatory cytokines than WT islets. Hyperglycemic clamps confirmed an increase in insulin sensitivity with normal ß-cell function in 12- and 18-week-old ΔF508 mutants. In contrast, 24-week-old ΔF508 mutants exhibited insulin resistance and reduced ß-cell function. ß-Cell mass was unaffected at 11 weeks of age but was significantly lower in ΔF508 mutants versus controls at 24 weeks. This was not associated with gross pancreatic pathology. We conclude that the ΔF508 CFTR mutation does not lead to an intrinsic ß-cell secretory defect but is associated with insulin resistance and a ß-cell mass deficit in aging mutants.


Subject(s)
Aging , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Insulin Resistance , Insulin-Secreting Cells/metabolism , Insulin/metabolism , Mutation , Animals , Crosses, Genetic , Cystic Fibrosis/genetics , Cystic Fibrosis/metabolism , Cystic Fibrosis/pathology , Cystic Fibrosis/physiopathology , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Diabetes Mellitus/etiology , Down-Regulation , Female , Humans , Immunohistochemistry , Insulin/blood , Insulin Secretion , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/pathology , Islets of Langerhans/cytology , Islets of Langerhans/metabolism , Islets of Langerhans/pathology , Male , Mice, Inbred Strains , Mice, Mutant Strains , Tissue Culture Techniques
2.
Diabetes ; 63(3): 982-93, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24194502

ABSTRACT

The cellular and molecular mechanisms underpinning the compensatory increase in ß-cell mass in response to insulin resistance are essentially unknown. We previously reported that a 72-h coinfusion of glucose and Intralipid (GLU+IL) induces insulin resistance and a marked increase in ß-cell proliferation in 6-month-old, but not in 2-month-old, Wistar rats. The aim of the current study was to identify the mechanisms underlying nutrient-induced ß-cell proliferation in this model. A transcriptomic analysis identified a central role for the forkhead transcription factor FOXM1 and its targets, and for heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), a ligand of the EGF receptor (EGFR), in nutrient-induced ß-cell proliferation. Phosphorylation of ribosomal S6 kinase, a mammalian target of rapamycin (mTOR) target, was increased in islets from GLU+IL-infused 6-month-old rats. HB-EGF induced proliferation of insulin-secreting MIN6 cells and isolated rat islets, and this effect was blocked in MIN6 cells by the EGFR inhibitor AG1478 or the mTOR inhibitor rapamycin. Coinfusion of either AG1478 or rapamycin blocked the increase in FOXM1 signaling, ß-cell proliferation, and ß-cell mass and size in response to GLU+IL infusion in 6-month-old rats. We conclude that chronic nutrient excess promotes ß-cell mass expansion via a pathway that involves EGFR signaling, mTOR activation, and FOXM1-mediated cell proliferation.


Subject(s)
Cell Proliferation , ErbB Receptors/physiology , Forkhead Transcription Factors/physiology , Insulin-Secreting Cells/physiology , Signal Transduction/physiology , TOR Serine-Threonine Kinases/physiology , Animals , Cell Cycle , Cells, Cultured , Forkhead Box Protein M1 , Gene Expression Profiling , Insulin Resistance , Insulin-Secreting Cells/cytology , Male , Quinazolines/pharmacology , Rats , Rats, Wistar , Tyrphostins/pharmacology
3.
J Mol Endocrinol ; 47(3): 273-83, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21821716

ABSTRACT

Pancreatic ß-cells have a well-developed endoplasmic reticulum due to their highly specialized secretory function to produce insulin in response to glucose and nutrients. It has been previously reported that overexpression of activating transcription factor 6 (ATF6) reduces insulin gene expression in part via upregulation of small heterodimer partner. In this study, we investigated whether ATF6 directly binds to the insulin gene promoter, and whether its direct binding represses insulin gene promoter activity. A bioinformatics analysis identified a putative ATF6 binding site in the A5/Core region of the rat insulin II gene promoter. Direct binding of ATF6 was confirmed using several approaches. Electrophoretic mobility shift assays in nuclear extracts from MCF7 cells, isolated rat islets and insulin-secreting HIT-T15 cells showed ATF6 binding to the native A5/Core of the rat insulin II gene promoter. Antibody-mediated supershift analyses revealed the presence of both ATF6 isoforms, ATF6α and ATF6ß, in the complex. Chromatin immunoprecipitation assays confirmed the binding of ATF6α and ATF6ß to a region encompassing the A5/Core of the rat insulin II gene promoter in isolated rat islets. Overexpression of the active (cleaved) fragment of ATF6α, but not ATF6ß, inhibited the activity of an insulin promoter-reporter by 50%. However, the inhibitory effect of ATF6α was insensitive to mutational inactivation or deletion of the A5/Core. Therefore, although ATF6 binds directly to the A5/Core of the rat insulin II gene promoter, this direct binding does not appear to contribute to its repressive activity.


Subject(s)
Activating Transcription Factor 6/metabolism , Insulin/genetics , Promoter Regions, Genetic , Transcription, Genetic , Activating Transcription Factor 6/genetics , Animals , Base Sequence , Binding Sites/genetics , Consensus Sequence/genetics , Gene Expression , Gene Expression Regulation/drug effects , Islets of Langerhans/drug effects , Islets of Langerhans/metabolism , Lactones/pharmacology , Male , Molecular Sequence Data , RNA Precursors/metabolism , RNA, Messenger/metabolism , Rats , Sequence Alignment , Sesquiterpenes/pharmacology
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