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)

Inflammatory bowel disease (IBD) affects almost 7 million people worldwide and is increasing in incidence. While the precise pathogenesis of IBD remains unknown, the production of inflammatory cytokines and chemokines play a central role. We have previously found that N,N-dimethylacetamide (DMA), a widely used non-toxic drug excipient, suppresses cytokine and chemokine secretion in vitro and prevents inflammation-induced preterm birth in vivo. Using sandwich enzyme-linked immunosorbent assays (ELISAs), we tested whether DMA attenuates cytokine and chemokine secretion from LPS- or TNFa-stimulated human intestinal epithelial cells and human monocytes and HMGB1 release from RAW 264.7 cells. To test our hypothesis that the mechanism of DMA’s effects in in-vitro and in-vivo models of IBD is inhibition of the NF-kB pathway, we used western blotting to track levels of the nuclear factor kappa B (NF-kB) inhibitory molecule I kappa B alpha (IkBa) in THP-1 human monocytes in the absence or presence of DMA. Finally, we induced colitis in C57Bl/6 mice with dextran sodium sulfate (DSS) and then tested whether daily i.p injections of DMA at 2.1 g/kg/day attenuates clinical and histopathologic signs of colitis. DMA attenuated cytokine and chemokine release from human intestinal epithelial cells and human monocytes and HMGB1 release from RAW 264.7 cells. Importantly, DMA prevented degradation of IkBa in THP-1 cells, thereby suggesting one mechanism for DMA’s effects. Finally, we show here, for the first time, that DMA attenuates clinical and histologic features of DSS-induced colitis. Based on these data, DMA should be further explored in preclinical and clinical trials for its potential as novel drug therapy for IBD.

Type 1 diabetes (T1D) results from loss of immune tolerance and β-cells. Currently, research on T1D treatments focus on regeneration and preservation of β-cells and restoration of immune tolerance. The sulfated synthetic polysaccharide dextran sulfate (DS), a mild anticoagulant and blood volume expander used in clinic, inhibits dendritic cell maturation and activation. DS administration increases circulating hepatocyte growth factor (HGF) in rodents and humans. In this study, we tested the effects of DS on: T1D development in NOD mice; β-cell survival, function and mitochondrial activity in cytokine-treated islets; and, the profile of immune activation and tolerance markers. DS treatment reduced spontaneous T1D onset in female NOD mice from 70% to 20%. T1D prevention in the wake of DS treatment was associated with preserved plasma insulin and β-cell mass, and reduced β-cell death. DS synergized with HGF in protecting mitochondrial function of cytokine-treated islets in vitro, maintaining maximal respiration, ATP production and glucose-stimulated insulin secretion. Pancreas histology showed decreased immune cell infiltration in islets of DS-treated mice. Splenocytes and pancreatic lymph nodes of DS-treated mice displayed decreased IFNγ+ CD8+ and CD4+, and increased IL4+ CD4+ and FoxP3+ CD4+ cells. In vitro, DS+HGF increased both the inhibitory phenotype of bone marrow derived macrophages (BMDM) downregulating IL1β, TNFα, and iNOS and increasing IL10, and the number of FoxP3+ CD4+ treated with αCD3 and αCD28 in the absence of TGF β. DS+HGF enhanced mitochondrial mass, potential, oxidative phosphorylation and expression of OXPHOS complexes, metabolic features of inhibitory BMDMs and regulatory T cells. Collectively, these results indicate that DS and HGF ameliorate T1D in NOD mice by alleviating the metabolic impairment induced by inflammation in islets and by boosting mitochondrial function to preserve β-cell function and upregulate M2 macrophages and regulatory T cells. DS and HGF can be of great value for treating T1D. Disclosure G. Lu: None. T. Zhang: None. D. Homann: None. A. Garcia-Ocaña: None. Funding National Institutes of Health (DK113079, DK020541, W81XWH-17-1-0363)

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