Project description:A hallmark of type 2 diabetes (T2D) is endocrine islet beta-cell failure, which can occur via cell dysfunction, loss-of-identity, and/or death. How each is induced remains largely unknown. Here, we use mouse beta-cells that are deficient for Myelin transcription factors (Myt TFs, including Myt1, 2, and 3) to address this question. We have reported that inactivating all three Myt genes in pancreatic progenitor cells (MytPancD) causes beta-cell failure and late onset diabetes in mice. Their lower expression in human beta-cells is correlated with beta-cell dysfunction and SNPs in Myt2 and Myt3 are associated in higher risk of T2D. We now show that these Myt TF-deficient beta-cells also de-differentiate by reactivating several progenitor markers, assayed by both immuno-assays and RNAseq. Intriguingly, mosaic Myt TF inactivation in only a portion of islet beta-cells does not results in overt diabetes, but this creates a condition where Myt TF-deficient beta-cells stay alive while dedifferentiating and trans-differentiating into Ppy-expressing cells. By transplanting MytPancD islets into the anterior eye chambers of immune-compromised mice, we directly show that glycemic and obesity-related conditions influence cell fate. These findings suggest that the observed beta-cell defects in T2D depend on not only their inherent genetic/epigenetic defects, but also the metabolic load.
Project description:Pancreatic islet endocrine cell and endothelial cell (EC) interactions mediated by vascular endothelial growth factor-A (VEGF-A) signaling are important for islet endocrine cell differentiation and the formation of highly vascularized islets. To dissect how VEGF-A signaling modulates intra-islet vasculature and innervation, islet microenvironment, and beta cell mass, we transiently increased VEGF-A production by beta cells. VEGF-A induction dramatically increased the number of intra-islet ECs but led to beta cell loss. After withdrawal of the VEGF-A stimulus, beta cell mass, function, and islet structure normalized as a result of a robust, but transient, burst in proliferation of pre-existing beta cells. Bone marrow-derived macrophages recruited to the site of beta cell injury were crucial for the beta cell proliferation, which was independent of pancreatic location and circulating factors such as glucose. Identification of the signals responsible for the proliferation of adult, terminally differentiated beta cells will improve strategies aimed at beta cell regeneration and expansion.
Project description:We measured changes in the human islet proteome following 72-hr exposure to 3 mM R-beta-hydroxybutyrate. Islets from 12 metabolically healthy human islet donors were obtained from the Alberta Diabetes Institute Islet Core
Project description:Endocrine islet beta cells comprise heterogenous cell subsets. Yet the origin, stability, and physiological significance of these subsets remain largely unknown. Using combinatorial cell lineage tracing, scRNA-seq, and DNA methylation analysis, we show here that embryonic islet progenitors with differential gene expression and DNA methylation produce stable beta-cell subtypes of different function and viability in adult mice. Differentially expressed genes, including the Myt transcription factors, voltage-gated channels, and Ca2+-sensor synaptotagmins, contribute to the functional differences of these subtypes. Maternal overnutrition, a major diabetes risk factor, reduces the proportion of endocrine progenitors of the better-functionality beta-cell subtype. Intriguingly, the gene signature that defines mouse beta-cell subtypes can reliably divide human cells into two populations, with the proportion of better-functionality beta cells reduced in diabetic donors. These results establish that some beta-cell subtypes are determined via DNA methylation in embryonic islet progenitors, which is regulated by diabetes-causing maternal factors. The implication is that modulating DNA methylation in islet progenitors can be explored to improve beta-cell function in the prevention and therapy of diabetes.
Project description:Islet-enriched transcription factors (TFs) exert broad control over cellular processes in pancreatic α and β cells and changes in their expression are associated with developmental state and diabetes. However, the implications of heterogeneity in TF expression across islet cell populations are not well understood. To define this TF heterogeneity and its consequences for cellular function, we profiled >40,000 cells from normal human islets by scRNA-seq and stratified α and β cells based on combinatorial TF expression. Subpopulations of islet cells co-expressing ARX/MAFB (α cells) and MAFA/MAFB (β cells) exhibited greater expression of key genes related to glucose sensing and hormone secretion relative to subpopulations expressing only one or neither TF. Moreover, all subpopulations were identified in native pancreatic tissue from multiple donors. By Patch-seq, MAFA/MAFB co-expressing β cells showed enhanced electrophysiological activity. Thus, these results indicate combinatorial TF expression in islet α and β cells predicts highly functional, mature subpopulations.
Project description:Fresh frozen sections of islets obtained by surgery were laser capture microdissected using autofluorescence to guide selection of beta cell areas of the islet. RNA was extracted and amplified with 2 rounds of T7 linear amplification. Two technical replicates were hybridized to Affymetrix U95Av2 arrays.
Project description:Endocrine enriched genes in adult islet beta cells were identified and compared with that of induced beta cells (with M3 transcription factors) in adult. The control sample is non-beta pancreatic cells. Gene expression profile comparison of 3 samples, 3 independent repeats for each sample indicate a high degree of similarity between endogenous and induced beta cells in adult mouse.
Project description:We knocked out the critical autophagy enzyme, ATG7, in the β-cells of mice (ATG7Δβ-cell) then monitored blood glucose to assess the phenotype induced by this KO model. We found that all ATG7Δβ-cell mice developed diabetes between 11-15 weeks of age. We isolated islets from ATG7Δβ-cell and littermate control mice several weeks prior to diabetes development (7-10 weeks of age) and performed bulk islet proteomics. The purpose of this experiment was to understand the islet biological process pathways altered by dysfunctional β-cell autophagy in the ATG7Δβ-cell model.
Project description:Endocrine islet beta cells comprise heterogenous cell subsets. Yet the origin, stability, and physiological significance of these subsets remain largely unknown. Using combinatorial cell lineage tracing, scRNA-seq, and DNA methylation analysis, we show here that embryonic islet progenitors with differential gene expression and DNA methylation produce stable beta-cell subtypes of different function and viability in adult mice. Differentially expressed genes, including the Myt transcription factors, voltage-gated channels, and Ca2+-sensor synaptotagmins, contribute to the functional differences of these subtypes. Maternal overnutrition, a major diabetes risk factor, reduces the proportion of endocrine progenitors of the better-functionality beta-cell subtype. Intriguingly, the gene signature that defines mouse beta-cell subtypes can reliably divide human cells into two populations, with the proportion of better-functionality beta cells reduced in diabetic donors. These results establish that some beta-cell subtypes are determined via DNA methylation in embryonic islet progenitors, which is regulated by diabetes-causing maternal factors. The implication is that modulating DNA methylation in islet progenitors can be explored to improve beta-cell function in the prevention and therapy of diabetes.
Project description:Pancreatic islet endocrine cell and endothelial cell (EC) interactions mediated by vascular endothelial growth factor-A (VEGF-A) signaling are important for islet endocrine cell differentiation and the formation of highly vascularized islets. To dissect how VEGF-A signaling modulates intra-islet vasculature and innervation, islet microenvironment, and β cell mass, we transiently increased VEGF-A production by β cells. VEGF-A induction dramatically increased the number of intra-islet ECs but led to β cell loss. After withdrawal of the VEGF-A stimulus, β cell mass, function, and islet structure normalized as a result of a robust, but transient, burst in proliferation of pre-existing β cells. Bone marrow-derived macrophages (MΦs) recruited to the site of β cell injury were crucial for the β cell proliferation, which was independent of pancreatic location and circulating factors such as glucose. Identification of the signals responsible for the proliferation of adult, terminally differentiated β cells will improve strategies aimed at β cell regeneration and expansion. Examination of RNA profiles from isolated whole islets from RIP-rtTA; TetO-VEGF-A mice with no doxycycline (Dox) treatment (3 samples) and after 1 week of Dox (3 sample); and islet-derived macrophages (3 samples) and endothelial cells (3 samples) isolated from dispersed purified islets from RIP-rtTA; TetO-VEGF-A mice after 1 week Dox treatment by fluorescence-activated cell sorting using antibodies against CD11b and CD31, respectively.