Beta Cell Identity Research Articles

Enzymatically stable analogue of the gut-derived peptide xenin on beta-cell transdifferentiation in high fat fed and insulin-deficient Ins1Cre/+ ;Rosa26-eYFP mice.

The antidiabetic effects of the gut hormone xenin include augmenting insulin secretion and positively affecting pancreatic islet architecture. The current study has further probed pancreatic effects through sub-chronic administration of the long-acting xenin analogue, xenin-25[Lys13 PAL], in both high fat fed (HFF) and streptozotocin (STZ)-induced insulin-deficient Ins1Cre/+ ;Rosa26-eYFP transgenic mice. Parallel effects on metabolic control and pancreatic islet morphology, including islet beta-cell lineage tracing were also assessed. Xenin-25[Lys13 PAL] treatment reversed body weight loss induced by STZ, increased plasma insulin and decreased blood glucose levels. There were less obvious effects on these parameters in HFF mice, but all xenin-25[Lys13 PAL] treated mice exhibited decreased pancreatic alpha-cell areas and circulating glucagon. Xenin-25[Lys13 PAL] treatment fully, or partially, returned overall islet and beta-cell areas in STZ- and HFF mice to those of lean control animals, respectively, and was consistently associated with decreased beta-cell apoptosis. Interestingly, xenin-25[Lys13 PAL] also increased beta-cell proliferation and decreased alpha-cell apoptosis in STZ mice, with reduced alpha-cell growth noted in HFF mice. Lineage tracing studies revealed that xenin-25[Lys13 PAL] reduced the number of insulin positive pancreatic islet cells that lost their beta-cell identity, in keeping with a decreased transition of insulin positive to glucagon positive cells. These beneficial effects on islet cell differentiation were linked to maintained expression of Pdx1 within beta-cells. Xenin-25[Lys13 PAL] treatment was also associated with increased numbers of smaller sized islets in both models. Benefits of xenin-25[Lys13 PAL] on diabetes includes positive modulation of islet cell differentiation, in addition to promoting beta-cell growth and survival.

258-OR: DNA Methylation Regulates the Identity of Pancreatic Beta Cells

Diabetes is associated with the loss of beta-cell identity and function. Thus, understanding the mechanisms that simultaneously regulate beta-cell function and identity is critical for developing beta-cell replacement therapies. In this study, we show that the de novo DNA methyltransferase, Dnmt3a, orchestrates beta-cell identity and function. Previously, we have shown that loss of Dnmt3a in pancreatic beta-cells prevents the developmental metabolic reprogramming, resulting in loss of glucose stimulated insulin secretion (GSIS). To gain insights into the role of new DNA methylation patterns established in early pancreas development, we deleted Dnmt3a in the embryonic pancreatic progenitors in mice. RNA sequencing of Dnmt3aKO and control islets in adult life revealed that loss of Dnmt3a in progenitors leads to dysregulation of key genes involved in the maintenance of beta-cell identity and function. Notably, loss of Dnmt3a led to the upregulation of Tyrosine Hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, along with dysregulation of several genes involved in Dopamine signaling. Specifically, a large proportion of beta-cells in KO was marked by TH expression, compared to controls. Bisulfite sequencing analysis revealed that TH expression is repressed in beta-cells due to DNA methylation during differentiation of endocrine progenitors. In addition, the TH positive beta-cells displayed G2/M cell cycle arrest, along with elevated ER-stress markers. This alteration of cell identity was accompanied by impaired glucose tolerance and a GSIS profile resembling functionally immature beta-cells in the KO mice. The Dnmt3aKO mice did not show any significant change in beta-cell mass, while delta- and alpha- cell masses were increased at 7 month. Taken together, our results show that de novo methylation during early pancreatic development is critical for the establishment of beta-cell identity and function. Disclosure N. Parveen: None. J.K. Wang: None. S. Dhawan: None. Funding National Institutes of Health (R01DK120523)

1683-P: Upregulation of Pancreatic Islet EGF Receptor Improves Beta-Cell Identity and In Vivo Vascularisation in a Directly Observed Transplant Model

Aims: Long-term outcomes for islet transplantation remain suboptimal. An important determinant of islet transplantation success is vascularisation of engrafted islets. The epidermal growth factor receptor (EGFR) is a key angiogenic and pro-survival factor. In an in vivo mouse model, we investigated if EGFR overexpression in pancreatic islets improves engraftment. Methods: Donor islets from C57BL/6 mice were infected with adenoviruses generated to encoding human EGFR alongside a GFP reporter. To assess the effects of EGFR over-expression on beta-cell identity and function, qPCR was run to determine expression of angiogenesis, beta-cell enriched and beta-cell disallowed genes compared with islets infected with control (empty vector) virus. To assess the effects of EGFR-overexpression on implantation in vivo, EGFR-overexpressing or control (empty vector) islets were transplanted into the anterior chambers of the eyes of syngeneic recipients. Using confocal microscopy, islet volume (GFP signal) and blood vessel density (following IV administration of dextran conjugated Texas Red) were assessed regularly and longitudinally over 30 days. Results: qPCR results showed that EGFR-overexpressing islets had greater gene expression of angiogenic factors. Consistent with preserved beta-cell identity, beta-cell enriched genes were upregulated in EGFR-overexpressing islets compared to control islets, whilst beta-cell disallowed genes were repressed. EGFR-overexpressing islets demonstrating greater density compared to control islets at all time points (n=5 animals per group, 1-5 islets per eye, p<0.05). Conclusion: We are the first to demonstrate that EGFR overexpression can promote the vascularisation of transplanted islets in vivo. The transcriptional profiling of these islets suggests an improved potential for beta-cell regenerative capacity and function. Disclosure V. Salem: None. U. Ali: None. K. Suba: None. S. Bitsi: None. T. Lopes: None. A. Martin Alonso: None. Y.S. Patel: None. I. Leclerc: Consultant; Spouse/Partner; Sun Pharmaceutical Industries Ltd. Research Support; Spouse/Partner; Servier. B. Owen: None. G.A. Rutter: Consultant; Self; Sun Pharmaceutical Industries Ltd. Research Support; Self; Servier, Sun Pharmaceutical Industries Ltd. S.M. Rothery: None. A. Tomas: None. Funding Diabetes UK (15/0005317)

Dapagliflozin exerts positive effects on beta cells, decreases glucagon and does not alter beta- to alpha-cell transdifferentiation in mouse models of diabetes and insulin resistance

Loss of beta cell identity and subsequent transdifferentiation of beta-to-alpha cells is implicated in the pathogenesis of diabetes. In addition, sodium-glucose transport protein 2 (SGLT2) inhibition has been linked to altered alpha-cell function. To investigate these phenomenon, lineage tracing of beta-cells was examined following 10–12 days dapagliflozin (1 or 5 mg/kg, once daily, as appropriate) treatment in multiple low-dose streptozotocin (STZ), high fat fed (HFF) or hydrocortisone (HC) transgenic Ins1Cre/+/Rosa26-eYFP mouse models of diabetes and insulin resistance. As anticipated, STZ, HFF and HC treated mice developed characteristic features of insulin deficiency or resistance. Dapagliflozin elicited differing beneficial effects depending on the aetiology of syndrome studied. The SGLT2 inhibitor efficiently promoted (P < 0.001) weight loss in HFF and STZ mice, whilst in HC mice it reduced (P < 0.001) energy intake, without an impact on body weight. Despite lacking significant effects on glycaemia, 1 mg/kg dapagliflozin consistently decreased both plasma and pancreatic glucagon. This was associated with increased pancreatic insulin in STZ and HFF mice. In STZ and HFF mice, beta cell proliferation and Pdx1 expression were enhanced by dapagliflozin, with a further increase in overall glucagon staining in HFF islets. Islet, beta- and alpha-cell areas were increased in dapagliflozin treated HC mice, which appeared to be linked to decreased alpha- and beta-cell apoptosis. Although the diabetes-like syndromes induced clear alterations in islet cell transdifferentiation, treatment with dapagliflozin (1 mg/kg) had no significant impact on this process, with 5 mg/kg marginally decreasing loss of beta-cells identity in STZ mice. These data suggest that SGLT2 inhibitors have positive effects on beta cells and decrease plasma and pancreatic glucagon, independent of changes in ambient glucose levels. Our combined data indicate that SGLT2 inhibitors do not directly induce hyperglucagonaemia.

Beta cell identity changes with mild hyperglycemia: Implications for function, growth, and vulnerability

ObjectiveAs diabetes develops, marked reductions of insulin secretion are associated with very modest elevations of glucose. We wondered if these glucose changes disrupt beta cell differentiation enough to account for the altered function. MethodsRats were subjected to 90% partial pancreatectomies and those with only mild glucose elevations 4 weeks or 10 weeks after surgery had major alterations of gene expression in their islets as determined by RNAseq. ResultsChanges associated with glucose toxicity demonstrated that many of the critical genes responsible for insulin secretion were downregulated while the expression of normally suppressed genes increased. Also, there were marked changes in genes associated with replication, aging, senescence, stress, inflammation, and increased expression of genes controlling both class I and II MHC antigens. ConclusionsThese findings suggest that mild glucose elevations in the early stages of diabetes lead to phenotypic changes that adversely affect beta cell function, growth, and vulnerability.

Stearoyl CoA desaturase is a gatekeeper that protects human beta cells against lipotoxicity and maintains their identity

Aims/hypothesisDuring the onset of type 2 diabetes, excessive dietary intake of saturated NEFA and fructose lead to impaired insulin production and secretion by insulin-producing pancreatic beta cells. The majority of data on the deleterious effects of lipids on functional beta cell mass were obtained either in vivo in rodent models or in vitro using rodent islets and beta cell lines. Translating data from rodent to human beta cells remains challenging. Here, we used the human beta cell line EndoC-βH1 and analysed its sensitivity to a lipotoxic and glucolipotoxic (high palmitate with or without high glucose) insult, as a way to model human beta cells in a type 2 diabetes environment.MethodsEndoC-βH1 cells were exposed to palmitate after knockdown of genes related to saturated NEFA metabolism. We analysed whether and how palmitate induces apoptosis, stress and inflammation and modulates beta cell identity.ResultsEndoC-βH1 cells were insensitive to the deleterious effects of saturated NEFA (palmitate and stearate) unless stearoyl CoA desaturase (SCD) was silenced. SCD was abundantly expressed in EndoC-βH1 cells, as well as in human islets and human induced pluripotent stem cell-derived beta cells. SCD silencing induced markers of inflammation and endoplasmic reticulum stress and also IAPP mRNA. Treatment with the SCD products oleate or palmitoleate reversed inflammation and endoplasmic reticulum stress. Upon SCD knockdown, palmitate induced expression of dedifferentiation markers such as SOX9, MYC and HES1. Interestingly, SCD knockdown by itself disrupted beta cell identity with a decrease in mature beta cell markers INS, MAFA and SLC30A8 and decreased insulin content and glucose-stimulated insulin secretion.Conclusions/interpretationThe present study delineates an important role for SCD in the protection against lipotoxicity and in the maintenance of human beta cell identity.Data availabilityMicroarray data and all experimental details that support the findings of this study have been deposited in in the GEO database with the GSE130208 accession code.

MiR-132 controls pancreatic beta cell proliferation and survival through Pten/Akt/Foxo3 signaling

ObjectiveMicroRNAs (miRNAs) play an integral role in maintaining beta cell function and identity. Deciphering their targets and precise role, however, remains challenging. In this study, we aimed to identify miRNAs and their downstream targets involved in the regeneration of islet beta cells following partial pancreatectomy in mice. MethodsRNA from laser capture microdissected (LCM) islets of partially pancreatectomized and sham-operated mice were profiled with microarrays to identify putative miRNAs implicated in beta cell regeneration. Altered expression of the selected miRNAs, including miR-132, was verified by RT-PCR. Potential targets of miR-132 were selected through bioinformatic data mining. Predicted miR-132 targets were validated for their changed RNA, protein expression levels, and signaling upon miR-132 knockdown and/or overexpression in mouse MIN6 and human EndoC-βH1 insulinoma cells. The ability of miR-132 to foster beta cell proliferation in vivo was further assessed in pancreatectomized miR-132−/− and control mice. ResultsPartial pancreatectomy significantly increased the number of BrdU+/insulin+ islet cells. Microarray profiling revealed that 14 miRNAs, including miR-132 and -141, were significantly upregulated in the LCM islets of the partially pancreatectomized mice compared to the LCM islets of the control mice. In the same comparison, miR-760 was the only downregulated miRNA. The changed expression of these miRNAs in the islets of the partially pancreatectomized mice was confirmed by RT-PCR only in the case of miR-132 and -141. Based on previous knowledge of its function, we focused our attention on miR-132. Downregulation of miR-132 reduced the proliferation of MIN6 cells while enhancing the levels of pro-apoptotic cleaved caspase-9. The opposite was observed in miR-132 overexpressing MIN6 cells. Microarray profiling, RT-PCR, and immunoblotting of the latter cells demonstrated their downregulated expression of Pten with concomitant increased levels of pro-proliferative factors phospho-Akt and phospho-Creb and inactivation of pro-apoptotic Foxo3a via its phosphorylation. Downregulation of Pten was further confirmed in the LCM islets of pancreatectomized mice compared to the sham-operated mice. Moreover, overexpression of miR-132 correlated with increased proliferation of EndoC-βH1 cells. The regeneration of beta cells following partial pancreatectomy was lower in the miR-132/212−/− mice than the control littermates. ConclusionsThis study provides compelling evidence about the critical role of miR-132 for the regeneration of mouse islet beta cells through the downregulation of its target Pten. Hence, the miR-132/Pten/Akt/Foxo3 signaling pathway may represent a suitable target to enhance beta cell mass.

Structure-functional changes in eNAMPT at high concentrations mediate mouse and human beta cell dysfunction in type 2 diabetes

Aims/hypothesisProgressive decline in functional beta cell mass is central to the development of type 2 diabetes. Elevated serum levels of extracellular nicotinamide phosphoribosyltransferase (eNAMPT) are associated with beta cell failure in type 2 diabetes and eNAMPT immuno-neutralisation improves glucose tolerance in mouse models of diabetes. Despite this, the effects of eNAMPT on functional beta cell mass are poorly elucidated, with some studies having separately reported beta cell-protective effects of eNAMPT. eNAMPT exists in structurally and functionally distinct monomeric and dimeric forms. Dimerisation is essential for the NAD-biosynthetic capacity of NAMPT. Monomeric eNAMPT does not possess NAD-biosynthetic capacity and may exert distinct NAD-independent effects. This study aimed to fully characterise the structure-functional effects of eNAMPT on pancreatic beta cell functional mass and to relate these to beta cell failure in type 2 diabetes.MethodsCD-1 mice and serum from obese humans who were without diabetes, with impaired fasting glucose (IFG) or with type 2 diabetes (from the Body Fat, Surgery and Hormone [BodyFatS&H] study) or with or at risk of developing type 2 diabetes (from the VaSera trial) were used in this study. We generated recombinant wild-type and monomeric eNAMPT to explore the effects of eNAMPT on functional beta cell mass in isolated mouse and human islets. Beta cell function was determined by static and dynamic insulin secretion and intracellular calcium microfluorimetry. NAD-biosynthetic capacity of eNAMPT was assessed by colorimetric and fluorescent assays and by native mass spectrometry. Islet cell number was determined by immunohistochemical staining for insulin, glucagon and somatostatin, with islet apoptosis determined by caspase 3/7 activity. Markers of inflammation and beta cell identity were determined by quantitative reverse transcription PCR. Total, monomeric and dimeric eNAMPT and nicotinamide mononucleotide (NMN) were evaluated by ELISA, western blot and fluorometric assay using serum from non-diabetic, glucose intolerant and type 2 diabetic individuals.ResultseNAMPT exerts bimodal and concentration- and structure-functional-dependent effects on beta cell functional mass. At low physiological concentrations (~1 ng/ml), as seen in serum from humans without diabetes, eNAMPT enhances beta cell function through NAD-dependent mechanisms, consistent with eNAMPT being present as a dimer. However, as eNAMPT concentrations rise to ~5 ng/ml, as in type 2 diabetes, eNAMPT begins to adopt a monomeric form and mediates beta cell dysfunction, reduced beta cell identity and number, increased alpha cell number and increased apoptosis, through NAD-independent proinflammatory mechanisms.Conclusions/interpretationWe have characterised a novel mechanism of beta cell dysfunction in type 2 diabetes. At low physiological levels, eNAMPT exists in dimer form and maintains beta cell function and identity through NAD-dependent mechanisms. However, as eNAMPT levels rise, as in type 2 diabetes, structure-functional changes occur resulting in marked elevation of monomeric eNAMPT, which induces a diabetic phenotype in pancreatic islets. Strategies to selectively target monomeric eNAMPT could represent promising therapeutic strategies for the treatment of type 2 diabetes.

Adipsin preserves beta cells in diabetic mice and associates with protection from type 2 diabetes in humans.

Type 2 diabetes is characterized by insulin resistance and a gradual loss of pancreatic beta cell mass and function1,2. Currently, there are no therapies proven to prevent beta cell loss and some, namely insulin secretagogues, have been linked to accelerated beta cell failure, thereby limiting their use in type 2 diabetes3,4. The adipokine adipsin/complement factor D controls the alternative complement pathway and generation of complement component C3a, which acts to augment beta cell insulin secretion5. In contrast to other insulin secretagogues, we show that chronic replenishment of adipsin in diabetic db/db mice ameliorates hyperglycemia and increases insulin levels while preserving beta cells by blocking dedifferentiation and death. Mechanistically, we find that adipsin/C3a decreases the phosphatase Dusp26; forced expression of Dusp26 in beta cells decreases expression of core beta cell identity genes and sensitizes to cell death. In contrast, pharmacological inhibition of DUSP26 improves hyperglycemia in diabetic mice and protects human islet cells from cell death. Pertaining to human health, we show that higher concentrations of circulating adipsin are associated with a significantly lower risk of developing future diabetes among middle-aged adults after adjusting for body mass index (BMI). Collectively, these data suggest that adipsin/C3a and DUSP26-directed therapies may represent a novel approach to achieve beta cell health to treat and prevent type 2 diabetes.

2184-P: Foxo1-CoF Repressor (FCoR) Regulates Pancreatic Alpha- and Beta-Cell Identity by both DNA and Histone Methylation

We have previously identified a Foxo1 binding protein, FCoR, which suppresses Foxo1 activity by increasing acetylation. FCoR is expressed in pancreatic α- and β-cells. FCoR expression is decreased in high-fat fed mice and in Leprdb/db mice, and FcorKO mice exhibited decreased insulin secretion, increased α-cell mass and β to α-cell conversion, suggesting FCoR involvement in the pathophysiology of diabetes. In FcorKO islets, nucleus localization of Foxo1 was increased, and Foxo1 acetylation was significantly decreased compared to WT. These data suggest that FCoR regulates acetylation and activity of Foxo1 in islets. The qPCR in isolated islets from FcorKO showed increased Arx, Mafb and Gcg expression compared to WT, supporting the phenotype of increased α-cell mass. Interestingly, expression level of Mafa and Neurod1, which are the β-cell markers and target genes of Foxo1, were also significantly increased, confirming activation of Foxo1 in the absence of FCoR. FCoR suppresses the expression of Aristaless related homeobox (Arx), which is essential for α-cell differentiation, via increased methylation of the CG rich promoter region of Arx. One of the epigenetic modifications of histone, the tri-methylation at H3K4 (H3K4me3), is an important marker of Arx expression in α-cells. Chromatin immunoprecipitation assay revealed that H3K4me3 was significantly decreased in αTC cells transduced with Flag-FCoR compared to LacZ-transduced-cells, suggesting that FCoR suppresses Arx expression through epigenetic regulation involving both DNA and histone methylation. Our data show that FCoR regulates Foxo1 activity in islets, and that FCoR regulates pancreatic α- and β-cell identity by epigenetic regulation of DNA and histone methylation. Disclosure N. Kodani: Research Support; Self; Lilly Diabetes, Novartis Pharmaceuticals Corporation. M. Kobayashi: None. O. Kikuchi: None. T. Kitamura: None. H. Itoh: None. J. Nakae: None.

332-LB: Mir-132 Controls Pancreatic Beta-Cell Proliferation and Survival through the Pten/Akt/Foxo3 Signaling

Aim and Hypothesis: microRNAs (miRNAs) play an integral role in maintaining beta cell function and identity. We aimed to identify miRNAs and their downstream targets involved in regeneration of islet beta cells following partial pancreatectomy (pP) in mice. Methods: RNA from laser capture microdissected islets of pP and sham-operated mice were profiled with microarrays to identify miRNAs implicated in control of beta cell regeneration. Altered expression of selected miRNAs, including miR-132, was verified by RT-PCR. Potential targets of miR-132 were selected through bioinformatic data mining. Predicted miR-132 targets were validated for their changed RNA and protein expression levels and signaling upon miR-132 knockdown and overexpression in mouse MIN6 cells and human EndoC-βH1 insulinoma cells. The ability of miR-132 to foster beta cell proliferation in vivo was further assessed in pP miR-132-/-and control mice. Results: pP significantly increased the number of BrdU+/insulin+ positive islet cells. Microarray profiling revealed 14 miRNAs, including miR-132, to be significantly upregulated in LCM islets of partially pancreatectomized mice and confirmed by RT-PCR. Downregulation of miR-132 in MIN6 cells reduced proliferation while enhancing the levels of pro-apoptotic cleaved caspase-9. Microarray profiling, RT-PCR and immunoblotting revealed downregulated expression of Pten, with concomitant increased levels of pro-proliferative factors as well as inactivation of pro-apoptotic Foxo3. Overexpression of miR-132 in human insulinoma cells EndoC-βH1 validated the findings. In vivo regeneration of beta cells was reduced in pP miR-132-/- mice compared to control mice. Conclusions: Our study provides compelling evidence for miR-132 being critical for regeneration of mouse islet beta cells in vivo through downregulation of its target Pten. Hence, the miR-132/Pten/Akt/Foxo3 signaling pathway may represent a suitable target to enhance beta cell regeneration. Disclosure S. Kersting: None. H. Mziaut: None. Funding German Center for Diabetes Research (to S.K.)

105-OR: Lineage Determination of Endocrine Progenitors in Murine and Human Pancreatic Development

Generation of functional beta cells for regenerative medicine would benefit from an increased understanding of pancreatic endocrine cell differentiation. During pancreatic development, expression of Ngn3 defines the progenitor cells that will differentiate into one of five endocrine lineages, including the beta cell. Here, we identify and further characterize a distinct endocrine progenitor population defined by differential expression of a transcription factor named Fev in murine and human endocrine cell development. Using single-cell RNA-sequencing and genetic lineage tracing, we build a transcriptomic profile of murine Fev+ progenitors that is distinct from that of Ngn3+ progenitors and reconstruct lineage relationships among Ngn3+, Fev+, and differentiated, hormone+ cells. We further identify candidate transcriptional regulators along the differentiation trajectory of this Fev+ progenitor population toward the alpha or beta cell lineages. Translating our findings in murine pancreatic development to that of human, we identify an analogous FEV+ population and putative gene regulators of endocrine cell fate in human fetal pancreas. In a stepwise differentiation platform designed to generate beta cells from human embryonic stem cells (hESCs), we also uncover robust FEV expression in endocrine progenitors prior to their differentiation into insulin-producing beta cells. Single-cell RNA-sequencing and lineage reconstruction of hESC-derived cells at the differentiated beta cell stage reveal FEV+ cells that may represent endocrine progenitors that have failed to acquire a beta cell identity; this failure of FEV+ endocrine cell differentiation may facilitate in uncovering the required molecular signals and genetic circuitry that push endocrine progenitors toward a beta cell fate. This work establishes a new model of pancreatic endocrine cell differentiation and identifies a Fev+ endocrine progenitor as a new cell stage in endocrine lineage determination. Disclosure D.M. Wong: None. L. Byrnes: None. J.O. Bouza: None. G. Peixoto: None. R. McMullen: None. J.B. Sneddon: Advisory Panel; Spouse/Partner; Akouos. Advisory Panel; Self; Encellin. Advisory Panel; Spouse/Partner; Encellin. Consultant; Self; Semma Therapeutics, Inc. Funding Nora Eccles Treadwell Foundation; University of California, San Francisco

2141-P: Nrf2 Mediates Glucose-Stimulated Beta-Cell Proliferation

Glucose catabolism drives adaptive beta cell mass expansion by promoting proliferation under conditions of increased insulin demand. Previously, we showed that induction of the antioxidant regulator, Nrf2, is required for glucose-stimulated β-cell proliferation, and that overexpression of Nrf2 stimulates human beta cell proliferation. Here we manipulate the Nrf2 pathway using pharmacological and genetic approaches to explore the link between glucose metabolism, Nrf2 activation and beta cell proliferation. Treatment of isolated mouse islets with either a Nrf2 inhibitor (brusatol) or Cre-recombinase deleted Nrf2, resulted in complete inhibition of glucose-stimulated proliferation (both n=3, p &amp;lt; 0.01). Conversely, stimulation of Nrf2 by CDDO-me or genetic gain of function (Cre-mediated depletion of Nrf2 inhibitor, Keap1) increased beta cell proliferation of beta cells from isolated mouse islets cultured in 5 mM glucose (4.9 ± 0.2 fold and 6.9 ± 1.4 fold, respectively; n=3, p &amp;lt; 0.05). Inhibition or depletion of Nrf2 was associated with a remarkable decrease in insulin content, suggesting that beta cell identity requires basal levels of Nrf2 expression. Importantly, isolated human beta cells displayed similar responses to Nrf2 loss and gain of function. Activation and nuclear translocation of Nrf2 requires dissociation from its inhibitor, Keap1, and phosphorylation of Ser-40 on Nrf2. Five minutes of 20 mM glucose treatment profoundly increased Ser-40 phosphorylation and translocation of Nrf2, while the same effect by CDDO-Me treatment took 50 minutes in INS1 cells. The glucose-mediated activation and translocation of Nrf2 was blocked by the antioxidant N-acetylcysteine (NAC), which also attenuated glucose-stimulated proliferation of isolated mouse beta cells. In addition, the activation and phosphorylation of Nrf2 was prevented by the pan-PKC inhibitor, calphostin C. We conclude that manipulation of the Nrf2 pathway is an exciting novel target for regulation of beta cell proliferation. Disclosure S. Baumel-Alterzon: None. L.S. Katz: None. G. Brill: None. A. Garcia-Ocaña: None. D. Scott: None. Funding American Diabetes Association (1-17-IBS-116 to D.S.); National Institutes of Health

327-LB: Transgenic Overexpression of MicroRNA-30d Impairs Glucose Homeostasis by Regulating Beta-Cell Proliferation and Dedifferentiation

MicroRNAs (miRNAs) are small non-coding RNAs that bind to target mRNAs and inhibit the translation in mammals. Abnormal miRNA functions are closely associated with pancreatic beta-cell loss and dysfunction in type 2 diabetes. Downregulation of miR-30d has been observed in the islets of diabetic mice. To investigate the in vivo function of miR-30d in pancreatic beta cells, we generated the transgenic mice that overexpressed miR-30d specifically in the pancreatic beta cells. Compared to wild type mice, the transgenic mice that overexpressed miR-30d resulted in hyperglycemia and glucose intolerance with reduced beta-cell mass and insulin production following high fat diet treatment. Strikingly, loss of beta-cell mass was partially due to beta-cell dedifferentiation to glucagon-producing alpha-cells, resulted in increased plasma glucagon level. The transcriptomic analysis combined with bioinformatic analysis of islets isolated from transgenic verses control mice identified connective tissue growth factor (CTGF) and Cyclin E2 as miR-30d potential targets. Moreover, several β-cell disallowed genes (e.g., MCT4/SLC16A3, LDHA) were observed upregulated in transgenic mice, which have been reported as positive markers of dedifferentiated human β-cells from type 2 diabetic patients. Taken together, miR-30d plays an essential role in maintaining mature beta-cell identity. Disclosure X. Tang: None. Funding National Institutes of Health

SAT-161 Macrophage Alterations in Islets of Obese Mice Linked to Beta Cell Compensation or Beta Cell Dedifferentiation in Diabetes

Background: Pancreatic beta cell dedifferentiation may be an important contributing factor to beta cell failure in type 2 diabetes, but the triggering stimuli are poorly understood. Macrophage levels are elevated in the islets of humans and mice with type 2 diabetes, but their effect on beta cell identity is not clear. Our goal was to examine the gene expression changes in islet-associated macrophages in obesity models with opposing disposition to diabetes development and to assess their potential contribution to beta cell (mal)adaptation. Methods: Islets were isolated from lean control, obese diabetes-prone db/db and obese diabetes-resistant ob/ob mice. Macrophages were sorted using flow cytometry. Islets were treated ex vivo with clodronate-containing liposomes to deplete macrophages. Gene expression was assessed by real-time RT-PCR. Results: Macrophage levels were increased in islets from db/db mice but not in islets from ob/ob mice compared to controls. Macrophages from db/db and ob/ob islets displayed distinct changes in gene expression, suggesting differential shifts in functional state. Macrophages from db/db islets maintained pro-inflammatory gene expression, whereas macrophages from ob/ob islets shifted towards an anti-inflammatory gene expression pattern with elevated levels of transforming growth factor beta 1 (Tgfb1) and reduced interleukin 1 beta (IL1b). Clodronate-liposome treatment of islets depleted macrophages, as evidenced by reduced mRNA expression of CD11b and F4/80. The depletion of macrophages in db/db islets increased the expression of beta cell identity genes. The mRNA levels of islet-associated transcription factors (Mafa and Pdx1), glucose transporter (Glut2), ATP-sensitive potassium channel (kcnj11), incretin receptor (Gipr) and adaptive unfolded protein response genes (Xbp1, Hspa5, Pdia4 and Fkbp11) were increased in db/db islets after macrophage depletion, whereas the mRNA levels of the deleterious UPR effector, Ddit3, were reduced. Furthermore, macrophage depletion in db/db islets reduced the expression of genes involved in inflammatory (IL1b, Fas and Nfkbia) and oxidative stress [catalase (Cat) and heme oxygenase-1 (Hmox1)]. In contrast, depletion of macrophages in islets of ob/ob mice did not affect beta cell identity gene expression. Conclusion: These novel findings suggest that distinct alterations in islet macrophages of obese mice are critically important for the maintenance of beta cell compensation or the loss of beta cell identity in diabetes. Depleting islet macrophages could be beneficial for reducing beta cell stress and preserving beta cell identity in type 2 diabetes.

OR05-3 Mir-21 Contributes to Cytokine-Induced Beta Cell Dysfunction via Inhibition of mRNAs Regulating Beta Cell Identity

A hallmark of diabetes is the loss of physical or functional β cell mass. Alterations in β cell microRNA (miRNA) profiles have been described in diabetes. MiRNAs have also been shown to serve as important regulators of β cell development and function, implicating them in β cell dysfunction during diabetes development. Our lab has previously demonstrated that β cell microRNA 21 (miR-21) is increased in models of diabetes. However, a comprehensive analysis of the β cell effects of miR-21 remain poorly defined, and the effects of miR-21 on in vivo glucose homeostasis have never been explored. To this end, we performed a comprehensive in silico analysis of bioinformatics databases to identify potential β cell targets of miR-21, which yielded multiple targets in the Transforming Growth Factor Beta 2 (Tgfb2) and Fibroblast Growth Factor Receptor 3 (Fgfr3) pathways associated with regulation of differentiation. We hypothesize that β cell miR-21 plays a critical role in inhibiting β cell function and inducing loss of β cell identity. To validate targets in vitro, we developed a model whereby miR-21 is upregulated using a dose dependent lentiviral Tetracycline-on system in INS1 cells. Overexpression of miR-21 led to a reduction in expression levels of several members of the Tgfb2 and Fgfr3 pathways as well as multiple transcription factors associated with β cell function and identity, and an increase in aldehyde dehydrogenase transcripts, consistent with β cell dedifferentiation. To verify direct interactions between miR-21 and candidate target mRNAs, a biotin pulldown experiment was performed using a 3’ biotinylated mature miR-21 construct and a 3’ biotinylated cel-miR-67 control construct. Several mRNAs associated with β cell identity were enriched in the pulldown, indicating a direct interaction with miR-21. Lineage tracing was performed within an in vivo zebrafish model of β cell specific oxidative stress in which β cells expressed a nuclear GFP signal. Whole body knock down of miR-21 by morpholino microinjection showed a protective effect in stressed β cells and rescued against a dedifferentiated phenotype. To test the effect of miR-21 on glucose tolerance in vivo, inducible β cell specific knockout (βmiR-21KO) and overexpression (βmiR-21) mice were generated by crossing Ins1tm1(CreERT2)Thor mice with miR-21 floxed mice and miR-21-CAG-Z-EGFP mice, respectively. When compared to littermate controls, intraperitoneal glucose tolerance tests (IPGTT) exhibited hyperglycemia in βmiR-21 mice and euglycemia in βmiR-21KO mice. Metabolic studies, including glucose stimulated insulin secretion (GSIS) and insulin tolerance tests (ITT) are ongoing in our mouse models. Our results implicate miR-21 as a regulator of β cell dedifferentiation during diabetes development.

MiR‐21 Contributes to Cytokine‐Induced Beta Cell Dysfunction via Inhibition of mRNAs Regulating Beta Cell Identity

A hallmark of diabetes is the loss of physical or functional β cell mass. Alterations in β cell microRNA (miRNA) profiles have been described in diabetes. MiRNAs have also been shown to serve as important regulators of β cell development and function, implicating them in β cell dysfunction during diabetes development. Our lab has previously demonstrated that β cell microRNA 21 (miR‐21) is increased in models of diabetes. However, a comprehensive analysis of the β cell effects of miR‐21 remain poorly defined, and the effects of miR‐21 on in vivo glucose homeostasis have never been explored. To this end, we performed a comprehensive in silico analysis of bioinformatics databases to identify potential β cell targets of miR‐21, which yielded multiple targets in the Transforming Growth Factor Beta 2 (Tgfb2) and Fibroblast Growth Factor Receptor 3 (Fgfr3) pathways associated with regulation of differentiation. We hypothesize that β cell miR‐21 plays a critical role in inhibiting β cell function and inducing loss of β cell identity. To validate targets in vitro, we developed a model whereby miR‐21 is upregulated using a dose dependent lentiviral Tetracycline‐on system in INS1 cells. Overexpression of miR‐21 led to a reduction in expression levels of several members of the Tgfb2 and Fgfr3 pathways as well as multiple transcription factors associated with β cell function and identity, and an increase in aldehyde dehydrogenase transcripts, consistent with β cell dedifferentiation. To verify direct interactions between miR‐21 and candidate target mRNAs, a biotin pulldown experiment was performed using a 3′ biotinylated mature miR‐21 construct and a 3′ biotinylated cel‐miR‐67 control construct. Several mRNAs associated with β cell identity were enriched in the pulldown, indicating a direct interaction with miR‐21. Lineage tracing was performed within an in vivo zebrafish model of β cell specific oxidative stress in which β cells expressed a nuclear GFP signal. Whole body knock down of miR‐21 by morpholino microinjection showed a protective effect in stressed β cells and rescued against a dedifferentiated phenotype. To test the effect of miR‐21 on glucose tolerance in vivo, inducible β cell specific knockout (βmiR‐21KO) and overexpression (βmiR‐21) mice were generated by crossing Ins1tm1(CreERT2)Thor mice with miR‐21 floxed mice and miR‐21‐CAG‐Z‐EGFP mice, respectively. When compared to littermate controls, intraperitoneal glucose tolerance tests (IPGTT) exhibited hyperglycemia in βmiR‐21 mice and euglycemia in βmiR‐21KO mice. Metabolic studies, including glucose stimulated insulin secretion (GSIS) and insulin tolerance tests (ITT) are ongoing in our mouse models. Our results implicate miR‐21 as a regulator of β cell dedifferentiation during diabetes development.Support or Funding InformationDeVault Fellowship through the Indiana University School of MedicineThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Macrophage alterations in islets of obese mice linked to beta cell disruption in diabetes.

Mild islet inflammation has been suggested as a contributing factor to beta cell failure in type 2 diabetes. Macrophage levels are elevated in the islets of humans and mice with type 2 diabetes, but their effects on beta cells are not understood. Our goal was to examine the gene expression changes in islet-associated macrophages in obesity models with opposing disposition to diabetes development and to assess their potential contribution to beta cell (mal)adaptation. Islets were isolated from lean control mice, obese diabetes-prone db/db mice and obese diabetes-resistant ob/ob mice. Macrophages were sorted using flow cytometry. Islets were treated ex vivo with clodronate-containing liposomes to deplete macrophages. Gene expression was assessed by real-time RT-PCR. Macrophage levels were increased in islets from db/db mice but not in islets from ob/ob mice compared with lean control mice. Macrophages from db/db and ob/ob islets displayed distinct changes in gene expression compared with control islet macrophages, suggesting differential shifts in functional state. Macrophages from db/db islets displayed increased expression of interferon regulatory factor 5 (Irf5), IL-1 receptor antagonist (Il1rn) and mannose receptor C-type 1 (Mrc1), whereas macrophages from ob/ob islets showed elevated levels of transforming growth factor beta 1 (Tgfb1) and reduced IL-1β (Il1b). Clodronate-liposome treatment of islets depleted macrophages, as evidenced by reduced mRNA expression of Cd11b (also known as Itgam) and F4/80 (also known as Adgre1) compared with PBS-liposome-treated islets. The depletion of macrophages in db/db islets increased the expression of genes related to beta cell identity. The mRNA levels of islet-associated transcription factors (Mafa and Pdx1), glucose transporter (Glut2 [also known as Slc2a2]), ATP-sensitive K+ channel (Kcnj11), incretin receptor (Gipr) and adaptive unfolded protein response (UPR) genes (Xbp1, Hspa5, Pdia4 and Fkbp11) were increased in db/db islets after macrophage depletion, whereas the mRNA levels of the deleterious UPR effector, Ddit3, were reduced. In contrast, depletion of macrophages in islets of ob/ob mice did not affect beta cell identity gene expression. The findings of this study suggest that distinct alterations in islet macrophages of obese mice are critically important for the disruption of beta cell gene expression in diabetes.

Pancreatic Beta Cell Differentiation From Human Pluripotent Stem Cells.

Insulin-expressing beta cells are crucial for the maintenance of systemic glucose homeostasis. Elucidation of the molecular and cellular mechanisms of beta cell development, expansion, survival, and function are required for full understanding of the molecular pathogenesis of diabetes. However, access to human beta cells for such studies is limited by virtue of the logistics of acquisition, prior medical status of donor, and imperfect culture systems for maintaining beta cell identity and function after isolation from human pancreas. Here, a technique for generation of beta cells from human pluripotent stem cells (hPSCs) by modification of key signaling pathways during islet development is described. Up to 70% C-peptide-positive beta cells can be obtained from endodermal anlagen after 27 days of differentiation with specific growth factors and small molecules. Although 50% of them are monohormonal C-peptide-positive cells and have molecular and cellular characteristics consistent with human beta cells in the Islets of Langerhans, a sub-population co-expressing other endocrine markers are also generated, indicating the immaturity of these cells. © 2018 by John Wiley & Sons, Inc.

DNA Hydroxymethylation Regulates Beta-Cell Maturation and Expansion

Epigenetic mechanisms play a central role in governing functional beta-cell mass. In particular, the methylation of DNA to generate 5-methylcytosine (5mC), directed by DNA methyltransferases, has been shown to regulate the establishment and maintenance of beta-cell identity and function. While the contribution of DNA methylation to beta-cell homeostasis and diabetes pathogenesis has been studied in detail, not much is known about the role of DNA demethylation in beta-cells. Oxidative conversion of 5mC to 5-hydroxymethylcytosine (5hmC) by Ten Eleven Translocation (Tet) enzymes constitutes a key step towards active DNA demethylation. In the present study, we provide evidence that the 5hmC landscape is dynamic, and undergoes specific changes during embryonic and postnatal islet development, islet adaptation, and in diabetes. We report that the Pdx1-positive pancreatic progenitors are marked by very low 5hmC, and beta-cell differentiation and functional maturation is accompanied by a progressive increase in 5hmC. However, under conditions during adaptive beta-cell expansion as well as in diabetes 5hmC levels decline in part due to the oxidative stress. These changes in 5hmC pattern are conserved in mouse and human pancreas, and are accompanied by the changes in Tet2 levels. We show that deletion of Tet2 in the pancreatic lineage results in improved glucose tolerance and beta-cell function. Together, our data show the importance of Tet2-dependent 5hmC patterning in maintaining a differentiated beta-cell phenotype, and highlight the remodeling of beta-cell epigenetic landscape as a key component of diabetes pathogenesis. Disclosure T. Gurlo: None. S. Georgia: None. S. Dhawan: None.