RESUMO
OBJECTIVE: ß-cell dedifferentiation has been revealed as a pathological mechanism underlying pancreatic dysfunction in diabetes. We previously showed that increased miR-7 levels trigger ß-cell dedifferentiation and diabetes. We used ß-cell-specific miR-7 overexpressing mice (Tg7) to test the hypothesis that loss of ß-cell identity triggered by miR-7 overexpression alters islet gene expression and islet microenvironment in diabetes. METHODS: We performed bulk and single-cell RNA sequencing (RNA-seq) in islets obtained from ß-cell-specific miR-7 overexpressing mice (Tg7). We carried out loss- and gain-of-function experiments in MIN6 and EndoC-bH1 cell lines. We analysed previously published mouse and human T2D data sets. RESULTS: Bulk RNA-seq revealed that ß-cell dedifferentiation is associated with the induction of genes associated with epithelial-to-mesenchymal transition (EMT) in prediabetic (2-week-old) and diabetic (12-week-old) Tg7 mice. Single-cell RNA-seq (scRNA-seq) indicated that this EMT signature is enriched specifically in ß-cells. These molecular changes are associated with a weakening of ß-cell: ß-cell contacts, increased extracellular matrix (ECM) deposition, and TGFß-dependent islet fibrosis. We found that the mesenchymal reprogramming of ß-cells is explained in part by the downregulation of Pdx1 and its inability to regulate a myriad of epithelial-specific genes expressed in ß-cells. Notable among genes transactivated by Pdx1 is Ovol2, which encodes a transcriptional repressor of the EMT transcription factor Zeb2. Following compromised ß-cell identity, the reduction in Pdx1 gene expression causes a decrease in Ovol2 protein, triggering mesenchymal reprogramming of ß-cells through the induction of Zeb2. We provided evidence that EMT signalling associated with the upregulation of Zeb2 expression is a molecular feature of islets in T2D subjects. CONCLUSIONS: Our study indicates that miR-7-mediated ß-cell dedifferentiation induces EMT signalling and a chronic response to tissue injury, which alters the islet microenvironment and predisposes to fibrosis. This research suggests that regulators of EMT signalling may represent novel therapeutic targets for treating ß-cell dysfunction and fibrosis in T2D.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Proteínas de Homeodomínio/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo , Animais , Células Cultivadas , Diabetes Mellitus Tipo 2/genética , Regulação para Baixo , Transição Epitelial-Mesenquimal/genética , Feminino , Células HEK293 , Proteínas de Homeodomínio/genética , Humanos , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Análise de Sequência de RNA , Transativadores/genética , Fatores de Transcrição/genética , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genéticaRESUMO
Allosteric regulation promises to open up new therapeutic avenues by increasing drug specificity at G-protein-coupled receptors (GPCRs). However, drug discovery efforts are at present hampered by an inability to precisely control the allosteric site. Herein, we describe the design, synthesis, and testing of PhotoETP, a light-activated positive allosteric modulator of the glucagon-like peptide-1 receptor (GLP-1R), a classâ B GPCR involved in the maintenance of glucose homeostasis in humans. PhotoETP potentiates Ca(2+) , cAMP, and insulin responses to glucagon-like peptide-1 and its metabolites following illumination of cells with blue light. PhotoETP thus provides a blueprint for the production of small-molecule classâ B GPCR allosteric photoswitches, and may represent a useful tool for understanding positive cooperativity at the GLP-1R.
Assuntos
Compostos de Anilina/farmacologia , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Regulação Alostérica/efeitos da radiação , Compostos de Anilina/síntese química , Compostos de Anilina/química , Animais , Compostos Azo/química , Células CHO , Cálcio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Humanos , Insulina/metabolismo , Isomerismo , Luz , Pirimidinas/química , Pirimidinas/farmacologia , Raios UltravioletaRESUMO
Incretin mimetics are set to become a mainstay of typeâ 2 diabetes treatment. By acting on the pancreas and brain, they potentiate insulin secretion and induce weight loss to preserve normoglycemia. Despite this, incretin therapy has been associated with off-target effects, including nausea and gastrointestinal disturbance. A novel photoswitchable incretin mimetic based upon the specific glucagon-like peptide-1 receptor (GLP-1R) agonist liraglutide was designed, synthesized, and tested. This peptidic compound, termed LirAzo, possesses an azobenzene photoresponsive element, affording isomer-biased GLP-1R signaling as a result of differential activation of second messenger pathways in response to light. While the trans isomer primarily engages calcium influx, the cis isomer favors cAMP generation. LirAzo thus allows optical control of insulin secretion and cell survival.
