GLP1R agonists such as Exendin-4 (E) together with DYRK1A inhibitors such as Harmine (H) significantly increase human β-cell replication in vitro and in vivo. Importantly, 3-month treatment with H+E combination markedly increases human beta cell mass (∼7-fold) in islets transplanted in immunosuppressed mice, beyond the increase induced by proliferation. Therefore, we addressed whether H+E also enhances β-cell survival and that contributes to the increased human β-cell mass expansion in vivo. Treatment with H+E significantly decreased β-cell apoptosis in dispersed primary human islet cells treated in vitro with either thapsigargin (ER stress) , cytokines (inflammation) and H2O2 (ROS) , while the drugs alone or vehicle (V) did not induce such effect. Importantly, H+E treatment for 7 days significantly reduced β-cell apoptosis in human islet grafts transplanted in immunosuppressed mice assessed by insulin and TUNEL staining. Human β-cell mass analysis of these grafts by iDISCO+ revealed a significant ∼50% increase in H+E-treated mice compared with mice treated with drugs alone or V. Human α-cell mass was unchanged. To address the signaling pathways modulated by H+E after 7-day treatment, we performed RNA-seq analysis of these human islet grafts. We identified 29 differentially expressed human genes (> 2-fold, p<0.05) in H+E-treated human islet grafts. GSEA analysis revealed cell adhesion, survival, vascularization and secretion as the main pathways induced by H+E. Among the upregulated genes, VGF (VGF Nerve Growth Factor Inducible) is known to regulate insulin secretion and β-cell survival. We found that H+E treatment of human islets in vitro increases 2-to-3-fold VGF mRNA and secretion. We are now determining VGF involvement in H+E-induced human β-cell survival. In conclusion, we have uncovered a novel prosurvival function of H+E in human β-cells with the potential implication of VGF in these effects. These studies can lead to the discovery of future β-cell protection and regeneration therapies for diabetes. Disclosure C.Rosselot: None. A.F.Stewart: None. A.Garcia-ocana: Consultant; Sun Pharmaceutical Industries Ltd. Y.Li: None. D.Guevara: None. K.A.Beliard: None. R.Kang: None. P.Wang: None. K.Thakkar: None. G.Lu: None. R.J.Devita: None. Funding DYRK Inhibitors for Human Beta Cell Expansion RDK105015
Glucose enhances mitochondrial function, insulin secretion and Myc expression in β-cells. β-cell-specific Myc knockout mice show glucose intolerance, hypoinsulinemia and lack of adaptive β-cell mass expansion following high-fat diet feeding. However, whether Myc regulates GSIS and mitochondrial function in β-cells is unknown. Here, we tested the effects of the Myc activity inhibitor 10058-F4 (1RH) in GSIS, mitochondrial function and metabolism in islets using islet perifusion, Seahorse and metabolomics/transcriptomics approaches. Mouse and human islets incubated 6h with 40µM 1RH displayed impaired phase 1 and phase 2 insulin secretion induced by 11mM glucose. Insulin secretion was not affected in the presence of 25mM KCl suggesting that Myc is required for glucose- but not membrane depolarization-induced insulin secretion. Since adequate mitochondrial function is essential for GSIS, we measured oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) to analyze mitochondrial respiration and glycolytic flux in these islets. 1RH significantly reduced OCR, ECAR and glucose-induced ATP production. Conversely, the Myc inducer harmine (10µM), increased basal OCR, ATP production, and ECAR and partially reversed 1RH effects on mitochondrial function. RNAseq of mouse islets treated with 1RH in 11mM glucose revealed reduced expression of both oxidative phosphorylation and β-cell signature genes but enhanced gene expression of glycolysis, ROS, fatty acid metabolism and autophagy/mitophagy pathways. Metabolomics analysis of mouse islets treated with 1RH in 11mM glucose confirmed the reduction in ATP and highlighted the accumulation of L-palmitoylcarnitine and palmitic acid suggesting inefficient β-oxidation. In conclusion, Myc is required for GSIS and mitochondrial function in β-cells. Impaired Myc action can lead to unbalanced metabolism and autophagy/mytophagy leading to β-cell dysfunction. Disclosure G.Lu: None. R.Kang: None. P.Diaz-pozo: None. J.Lee: None. M.Li: None. V.M.Victor: None. D.Scott: None. A.Garcia-ocana: Consultant; Sun Pharmaceutical Industries Ltd. Funding National Institutes of Health (R01DK126450)
Loss of β cells leads to diabetes. α cells are refractory to spontaneous conversion to β cells, but a small percentage are reprogrammed to β cells with acute β cell loss. β cell heterogeneity and identification of β cell subpopulations and their transcriptomic profiles have been reported. However, α cell subpopulations in the human islet have not been clearly defined yet. Moreover, whether specific human α cell subpopulations can potentially evolve into β cells is unknown. Here, we integrated scRNA- and snRNA-seq data of human islets from 3 adult healthy donors to determine α cell subpopulations and their transcriptomic profiles. We extracted the α cell cluster (7,535 cells) and using Louvain resolution 0.8, we identified 5 subclusters: 1) α cells (GCGhigh/TTRhigh/PTPRT high); 2) αβ-Transition-1 (αβ-Tr1) cells; 3) αβ-Tr2 cells; 4) αβ1 cells (GCGhigh/INSlow/IAPPlow); and 5) αβ2 cells (GCGlow/INShigh/IAPPhigh). To investigate the transcriptomics connectivity among clusters, we performed RNA velocity and Partition-based Graph Abstraction (PAGA) analyses. PAGA showed that αβ-Tr1 cells (15.3% of cells) are the root subcluster from where α cells and αβ-Tr2 cells are formed, suggesting a precursor nature. αβ-Tr2 cells are the origin of αβ1 and αβ2 cells. We next projected this finding into the pseudotime algorithm and analyzed gene expression gradients in the trajectories. Interestingly, the αβ-Tr1/αβ-Tr2/αβ2 cells trajectory showed a gradual increase in the expression of β cell genes (INS, ZNF385D, CASR, MAFA) while decreasing expression of ribosomal genes, GCG and MALAT1. Single cell pathway analysis with escape package showed that αβ-Tr1 cells are enriched in ribosome and translation gene pathways and that αβ-Tr2 cells are enriched in transdifferentiation genes INSM1, PDX1 and SMAD3. In summary, these studies identified 5 α cell subpopulations with different transcriptomic profiles and highlight the potential plasticity of αβ-Tr cells to generate αβ cells. Disclosure R.Kang: None. J.Lee: None. A.Garcia-ocana: Consultant; Sun Pharmaceutical Industries Ltd. G.Lu: None.
Type 1 diabetes (T1D) results from loss of both immune tolerance and functional β-cells. Administration of harmine (H) plus exendin-4 (E) markedly induces human β-cell expansion. Anti-CD3 antibody treatment reduces C-peptide loss in T1D patients. Here, we tested whether combination therapy with anti-CD3 antibody and H+E enhances T1D remission in non-obese diabetic (NOD) mice. First, we tested whether H+E protects human β-cells against inflammation and ER stress. We found that H+E, but not the drugs alone, significantly reduced both thapsigargin- and cytokine-induced human β-cell apoptosis. Single-cell RNAseq of human islets treated with cytokines and H+E showed reduced IL1β and IFNγ signaling in β-cells. Apoptosis genes such as CYLD and RIPK1 were downregulated and prosurvival genes such as HIF1A and VEGFA were upregulated in β-cells of H+E-treated islets. Next, treatment of early-onset diabetic NOD mice with H+E (daily ip) for 8 weeks improved glucose homeostasis but failed to induce long-lasting immune tolerance and T1D remission. Therefore, we next treated early-onset diabetic NOD mice with low-dose anti-CD3 antibody daily for 3 days (40µg/day) followed by 8 weeks treatment with H+E or vehicle (V) (Alzet pumps) . Treatment with anti-CD3 and H+E eliminated hyperglycemia in 100% of the mice vs. only 20% remission in anti-CD3 and V-treated mice. CD3+ T cell numbers were significantly and similarly reduced (∼50%) in mice treated with H+E or V. Preliminary results indicate that activated T cells in pancreatic lymph nodes (PLNs) and islet insulitis were significantly reduced, and T regulatory cells in spleen and PLNs were increased in H+E-treated mice. TUNEL+ β-cells were decreased and Ki67+ β-cells were increased in the pancreas of H+E-treated mice. Collectively, these results indicate that combination therapy with low-dose anti-CD3 antibody and H+E enhances T1D remission in diabetic NOD mice by increasing β-cell viability and favoring immune tolerance. Disclosure G.Lu: None. R.Kang: None. Y.Li: None. P.Wang: None. C.Rosselot: None. R.J.Devita: None. A.F.Stewart: None. A.Garcia-ocana: Consultant; Sun Pharmaceutical Industries Ltd. Funding NIH DK105015
Single-cell RNA sequencing (scRNA-seq) of human islets requires cell dissociation and does not provide information on the transcriptional status of islet cells. On the other hand, single-nucleus RNA sequencing (snRNA-seq) does not require cell dissociation and provides abundant information on intronic sequences that can be used to identify actively transcribed genes. Based on this, we seek to compare scRNA-seq and snRNA-seq approaches in human islets to determine: 1) whether similar cell clusters could be detected; 2) whether new gene markers could be discovered in human islet endocrine cells using intronic reads; 3) whether human beta cell subpopulations could be identified based on INS expression dynamics; and 4) whether snRNA-seq could be used in human islet grafts transplanted in immunosuppressed mice. We performed scRNA-seq and snRNA-seq on three pairs of human islets obtained from three healthy adult human donors using exon only or exon plus intron reads, respectively. Analysis of integrated data revealed similar human islet cell clusters. In the snRNA-seq data, however, top differentially expressed genes were identified as new markers of human endocrine cells such as ZNF385D (β-cells) , PTPRT (α-cells) , LRFN5 (δ-cells) and CACNA2D3 (PP cells) . These markers also accurately define endocrine cell populations in human islet grafts. Additionally, we distinguished several beta cell sub-clusters- INS rich cluster, HNRNPA2B1 (vesicle) rich cluster and active INS transcribing cluster. In conclusion, snRNA-seq analysis of human islet cells is a previously unrecognized tool for the identification of human islet cell types in samples where nuclear RNA processing is required. By comparing INS expression between scRNA-seq and snRNA-seq data sets, we can detect different beta cell sub-populations with distinct gene expression patterns representing different biological dynamic states. Disclosure R.Kang: None. Y.Li: None. C.Rosselot: None. D.Scott: None. A.Garcia-ocana: Consultant; Sun Pharmaceutical Industries Ltd. G.Lu: None. Funding NIH DK105015
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