Beta Cell Identity Research Articles

Intra-islet insulin synthesis defects are associated with endoplasmic reticulum stress and loss of beta cell identity in human diabetes

Aims/hypothesisEndoplasmic reticulum (ER) stress and beta cell dedifferentiation both play leading roles in impaired insulin secretion in overt type 2 diabetes. Whether and how these factors are related in the natural history of the disease remains, however, unclear.MethodsIn this study, we analysed pancreas biopsies from a cohort of metabolically characterised living donors to identify defects in in situ insulin synthesis and intra-islet expression of ER stress and beta cell phenotype markers.ResultsWe provide evidence that in situ altered insulin processing is closely connected to in vivo worsening of beta cell function. Further, activation of ER stress genes reflects the alteration of insulin processing in situ. Using a combination of 17 different markers, we characterised individual pancreatic islets from normal glucose tolerant, impaired glucose tolerant and type 2 diabetic participants and reconstructed disease progression.Conclusions/interpretationOur study suggests that increased beta cell workload is accompanied by a progressive increase in ER stress with defects in insulin synthesis and loss of beta cell identity.Graphical abstract

Aging compromises human islet beta cell function and identity by decreasing transcription factor activity and inducing ER stress.

Pancreatic islet beta cells are essential for maintaining glucose homeostasis. To understand the impact of aging on beta cells, we performed meta-analysis of single-cell RNA sequencing datasets, transcription factor (TF) regulon analysis, high-resolution confocal microscopy, and measured insulin secretion from nondiabetic donors spanning most of the human life span. This revealed the range of molecular and functional changes that occur during beta cell aging, including the transcriptional deregulation that associates with cellular immaturity and reorganization of beta cell TF networks, increased gene transcription rates, and reduced glucose-stimulated insulin release. These alterations associate with activation of endoplasmic reticulum (ER) stress and autophagy pathways. We propose that a chronic state of ER stress undermines old beta cell structure function to increase the risk of beta cell failure and type 2 diabetes onset as humans age.

Oxidative stress-mediated beta cell death and dysfunction as a target for diabetes management.

The biggest drawback of a current diabetes therapy is the treatment of the consequences not the cause of the disease. Regardless of the diabetes type, preservation and recovery of functional pancreatic beta cells stands as the biggest challenge in the treatment of diabetes. Free radicals and oxidative stress are among the major mediators of autoimmune destruction of beta cells in type 1 diabetes (T1D) or beta cell malfunction and death provoked by glucotoxicity and insulin resistance in type 2 diabetes (T2D). Additionally, oxidative stress reduces functionality of beta cells in T2D by stimulating their de-/trans-differentiation through the loss of transcription factors critical for beta cell development, maturity and regeneration. This review summarizes up to date clarified redox-related mechanisms involved in regulating beta cell identity and death, underlining similarities and differences between T1D and T2D. The protective effects of natural antioxidants on the oxidative stress-induced beta cell failure were also discussed. Considering that oxidative stress affects epigenetic regulatory mechanisms involved in the regulation of pancreatic beta cell survival and insulin secretion, this review highlighted huge potential of epigenetic therapy. Special attention was paid on application of the state-of-the-art CRISPR/Cas9 technology, based on targeted epigenome editing with the purpose of changing the differentiation state of different cell types, making them insulin-producing with ability to attenuate diabetes. Clarification of the above-mentioned mechanisms could provide better insight into diabetes etiology and pathogenesis, which would allow development of novel, potentially more efficient therapeutic strategies for the prevention or reversion of beta cell loss.

Aging Impairs Adaptive Unfolded Protein Response and Drives Beta Cell Dedifferentiation in Humans.

Diabetes is an age-related disease; however, the mechanism underlying senescent beta cell failure is still unknown. The present study was designed to investigate whether and how the differentiated state was altered in senescent human beta cells by excluding the effects of impaired glucose tolerance. We calculated the percentage of hormone-negative/chromogranin A-positive endocrine cells and evaluated the expressions of forkhead box O1 (FoxO1) and Urocortin 3 (UCN3) in islets from 31 nondiabetic individuals, divided into young (<40 years), middle-aged (40-60 years) and elderly (>60 years) groups. We also assessed adaptive unfolded protein response markers glucose-regulated protein 94 (GRP94), and spliced X-box binding protein 1 (XBP1s) in senescent beta cells and their possible contributions to maintaining beta cell identity and differentiation state. We found an almost 2-fold increase in the proportion of dedifferentiated cells in elderly and middle-aged groups compared with the young group (3.1 ± 1.0% and 3.0 ± 0.9% vs 1.7 ± 0.5%, P < .001). This was accompanied by inactivation of FoxO1 and loss of UCN3 expression in senescent human beta cells. In addition, we demonstrated that the expression levels of adaptive unfolded protein response (UPR) components GRP94 and XBP1s declined with age. In vitro data showed knockdown GRP94 in Min6-triggered cells to dedifferentiate and acquire progenitor features, while restored GRP94 levels in H2O2-induced senescent Min6 cells rescued beta cell identity. Our finding highlights that the failure to establish proper adaptive UPR in senescent human beta cells shifts their differentiated states, possibly representing a crucial step in the pathogenesis of age-related beta cell failure.

Regulation of Pdx1 by oxidative stress and Nrf2 in pancreatic beta-cells.

The beta-cell identity gene, pancreatic duodenal homeobox 1 (Pdx1), plays critical roles in many aspects of the life of beta-cells including differentiation, maturation, function, survival and proliferation. High levels of reactive oxygen species (ROS) are extremely toxic to cells and especially to beta-cells due to their relatively low expression of antioxidant enzymes. One of the major mechanisms for beta-cell dysfunction in type-2 diabetes results from oxidative stress-dependent inhibition of PDX1 levels and function. ROS inhibits Pdx1 by reducing Pdx1 mRNA and protein levels, inhibiting PDX1 nuclear localization, and suppressing PDX1 coactivator complexes. The nuclear factor erythroid 2-related factor (Nrf2) antioxidant pathway controls the redox balance and allows the maintenance of high Pdx1 levels. Therefore, pharmacological activation of the Nrf2 pathway may alleviate diabetes by preserving Pdx1 levels.

202-OR: Early De Novo DNA Methylation Patterning Regulates Adult Beta-Cell Function and Survival Programs

DNA methylation is an important epigenetic mechanism that controls transcriptional programs to direct cell phenotype. Dynamic, stage-specific changes in DNA methylation coordinate the gene-expression patterns associated with cell differentiation throughout development. Our prior work has shown that the establishment of new DNA methylation patterns is essential for functional maturation of beta cells in postnatal life, while maintenance of existing methylation patterns sustains beta cell identity. While maintenance of DNA methylation in pancreatic progenitors is essential for their survival during differentiation, the requirement for new DNA methylation patterns in pancreatic progenitors has not been investigated. To address this, we generated pancreatic progenitor specific ablation of the de novo DNA methyltransferase, Dnmt3a, an enzyme that establishes new DNA methylation patterns, using Pdx1-Cre (Dnmt3aPKO) . RNA-sequencing of the Dnmt3aPKO islets reveals that early loss of Dnmt3a alters the beta cell identity, function, and survival programs. In addition, we observe the upregulation of genes associated with dopamine signaling and disruption of imprinted gene expression in the KO islets. Several of these gene-expression changes are distinct from those observed upon beta cell specific ablation of Dnmt3a, suggesting the unique contribution of early patterning to adult beta cell phenotype. Genome-wide profiling of DNA methylation patterns using reduced representation bisulfite sequencing (RRBS) in the Dnmt3aPKO islets points to disruption of similar programs associated with cell identity and function. These data also reveal interesting differences in the DNA methylation patterns in male and female Dnmt3aPKO mice. Collectively, our data suggest that stage specific DNA methylation patterning during beta cell differentiation controls specific aspects of the mature beta cell phenotype. Disclosure N.Parveen: None. J.K.Wang: None. A.P.Ham: None. S.Dhawan: None. Funding National Institutes of Health (R01-DK120523)

XBP1 maintains beta cell identity, represses beta-to-alpha cell transdifferentiation and protects against diabetic beta cell failure during metabolic stress in mice

Aims/hypothesisPancreatic beta cell dedifferentiation, transdifferentiation into other islet cells and apoptosis have been implicated in beta cell failure in type 2 diabetes, although the mechanisms are poorly defined. The endoplasmic reticulum stress response factor X-box binding protein 1 (XBP1) is a major regulator of the unfolded protein response. XBP1 expression is reduced in islets of people with type 2 diabetes, but its role in adult differentiated beta cells is unclear. Here, we assessed the effects of Xbp1 deletion in adult beta cells and tested whether XBP1-mediated unfolded protein response makes a necessary contribution to beta cell compensation in insulin resistance states.MethodsMice with inducible beta cell-specific Xbp1 deletion were studied under normal (chow diet) or metabolic stress (high-fat diet or obesity) conditions. Glucose tolerance, insulin secretion, islet gene expression, alpha cell mass, beta cell mass and apoptosis were assessed. Lineage tracing was used to determine beta cell fate.ResultsDeletion of Xbp1 in adult mouse beta cells led to beta cell dedifferentiation, beta-to-alpha cell transdifferentiation and increased alpha cell mass. Cell lineage-specific analyses revealed that Xbp1 deletion deactivated beta cell identity genes (insulin, Pdx1, Nkx6.1, Beta2, Foxo1) and derepressed beta cell dedifferentiation (Aldh1a3) and alpha cell (glucagon, Arx, Irx2) genes. Xbp1 deletion in beta cells of obese ob/ob or high-fat diet-fed mice triggered diabetes and worsened glucose intolerance by disrupting insulin secretory capacity. Furthermore, Xbp1 deletion increased beta cell apoptosis under metabolic stress conditions by attenuating the antioxidant response.Conclusions/interpretationThese findings indicate that XBP1 maintains beta cell identity, represses beta-to-alpha cell transdifferentiation and is required for beta cell compensation and prevention of diabetes in insulin resistance states.Graphical abstract

Loss of Human Beta Cell Identity in a Reconstructed Omental Stromal Cell Environment.

In human type 2 diabetes, adipose tissue plays an important role in disturbing glucose homeostasis by secreting factors that affect the function of cells and tissues throughout the body, including insulin-producing pancreatic beta cells. We aimed here at studying the paracrine effect of stromal cells isolated from subcutaneous and omental adipose tissue on human beta cells. We developed an in vitro model wherein the functional human beta cell line EndoC-βH1 was treated with conditioned media from human adipose tissues. By using RNA-sequencing and western blotting, we determined that a conditioned medium derived from omental stromal cells stimulates several pathways, such as STAT, SMAD and RELA, in EndoC-βH1 cells. We also observed that upon treatment, the expression of beta cell markers decreased while dedifferentiation markers increased. Loss-of-function experiments that efficiently blocked specific signaling pathways did not reverse dedifferentiation, suggesting the implication of more than one pathway in this regulatory process. Taken together, we demonstrate that soluble factors derived from stromal cells isolated from human omental adipose tissue signal human beta cells and modulate their identity.

Ac3IV, a V1a and V1b receptor selective vasopressin analogue, protects against hydrocortisone-induced changes in pancreatic islet cell lineage

The Avpr1a (V1a) and Avpr1b (V1b) receptor selective, vasopressin (AVP) analogue, Ac3IV has been shown to improve metabolism and pancreatic islet structure in diabetes and insulin resistance. The present study further investigates these actions by assessing the ability of Ac3IV to protect against pancreatic islet architectural disturbances induced by hydrocortisone (HC) treatment in transgenic Ins1Cre/+;Rosa26-eYFP mice, that possess beta-cell lineage tracing capabilities. HC intervention increased (p < 0.001) energy intake but reduced (p < 0.01) body weight gain, with no impact of Ac3IV. All HC mice had reduced (p < 0.05) circulating glucose, but plasma insulin and glucagon concentrations remained unchanged. However, HC mice presented with increased (p < 0.001) pancreatic insulin content, which was further augmented by Ac3IV. In addition, Ac3IV treatment countered HC-induced increases in islet-, beta- and alpha-cell areas (p < 0.01), as well as promoting islet number towards control levels. This was accompanied by reduced (p < 0.05) beta-cell growth, but enhanced (p < 0.001) alpha-cell proliferation. There were no changes in islet cell apoptotic rates in any of the groups of HC mice, but co-expression of CK19 with insulin in pancreatic ductal cells was reduced by Ac3IV. Assessment of beta-cell lineage revealed that Ac3IV partially protected against HC-mediated de-differentiation of mature beta-cells, whilst also decreasing (p < 0.01) beta- to alpha-cell transdifferentiation. Our data indicate that sustained activation of V1a and V1b receptors exerts positive islet cell transition effects to help retain beta-cell identity in HC mice.

Beneficial impact of Ac3IV, an AVP analogue acting specifically at V1a and V1b receptors, on diabetes islet morphology and transdifferentiation of alpha- and beta-cells.

Ac3IV (Ac-CYIQNCPRG-NH2) is an enzymatically stable vasopressin analogue that selectively activates Avpr1a (V1a) and Avpr1b (V1b) receptors. In the current study we have employed streptozotocin (STZ) diabetic transgenic Ins1Cre/+;Rosa26-eYFP and GluCreERT2;Rosa26-eYFP mice, to evaluate the impact of sustained Ac3IV treatment on pancreatic islet cell morphology and transdifferentiation. Twice-daily administration of Ac3IV (25 nmol/kg bw) to STZ-diabetic Ins1Cre/+;Rosa26-eYFP mice for 12 days increased pancreatic insulin (p<0.01) and significantly reversed the detrimental effects of STZ on pancreatic islet morphology. Such benefits were coupled with increased (p<0.01) beta-cell proliferation and decreased (p<0.05) beta-cell apoptosis. In terms of islet cell lineage tracing, induction of diabetes increased (p<0.001) beta- to alpha-cell differentiation in Ins1Cre/+;Rosa26-eYFP mice, with Ac3IV partially reversing (p<0.05) such transition events. Comparable benefits of Ac3IV on pancreatic islet architecture were observed in STZ-diabetic GluCreERT2;ROSA26-eYFP transgenic mice. In this model, Ac3IV provoked improvements in islet morphology which were linked to increased (p<0.05-p<0.01) transition of alpha- to beta-cells. Ac3IV also increased (p<0.05-p<0.01) CK-19 co-expression with insulin in pancreatic ductal and islet cells. Blood glucose levels were unchanged by Ac3IV in both models, reflecting the severity of diabetes induced. Taken together these data indicate that activation of islet receptors for V1a and V1b positively modulates alpha- and beta-cell turnover and endocrine cell lineage transition events to preserve beta-cell identity and islet architecture.

The role of long non-coding RNAs in the regulation of pancreatic beta cell identity.

Type 2 diabetes (T2D) is a widespread disease affecting millions in every continental population. Pancreatic β-cells are central to the regulation of circulating glucose, but failure in the maintenance of their mass and/or functional identity leads to T2D. Long non-coding RNAs (lncRNAs) represent a relatively understudied class of transcripts which growing evidence implicates in diabetes pathogenesis. T2D-associated single nucleotide polymorphisms (SNPs) have been identified in lncRNA loci, although these appear to function primarily through regulating β-cell proliferation. In the last decade, over 1100 lncRNAs have been catalogued in islets and the roles of a few have been further investigated, definitively linking them to β-cell function. These studies show that lncRNAs can be developmentally regulated and show highly tissue-specific expression. lncRNAs regulate neighbouring β-cell-specific transcription factor expression, with knockdown or overexpression of lncRNAs impacting a network of other key genes and pathways. Finally, gene expression analysis in studies of diabetic models have uncovered a number of lncRNAs with roles in β-cell function. A deeper understanding of these lncRNA roles in maintaining β-cell identity, and its deterioration, is required to fully appreciate the β-cell molecular network and to advance novel diabetes treatments.

IAPP-induced beta cell stress recapitulates the islet transcriptome in type 2 diabetes

Aims/hypothesisType 2 diabetes is characterised by islet amyloid and toxic oligomers of islet amyloid polypeptide (IAPP). We posed the questions, (1) does IAPP toxicity induce an islet response comparable to that in humans with type 2 diabetes, and if so, (2) what are the key transcriptional drivers of this response?MethodsThe islet transcriptome was evaluated in five groups of mice: beta cell specific transgenic for (1) human IAPP, (2) rodent IAPP, (3) human calpastatin, (4) human calpastatin and human IAPP, and (5) wild-type mice. RNA sequencing data was analysed by differential expression analysis and gene co-expression network analysis to establish the islet response to adaptation to an increased beta cell workload of soluble rodent IAPP, the islet response to increased expression of oligomeric human IAPP, and the extent to which the latter was rescued by suppression of calpain hyperactivation by calpastatin. Rank-rank hypergeometric overlap analysis was used to compare the transcriptome of islets from human or rodent IAPP transgenic mice vs humans with prediabetes or type 2 diabetes.ResultsThe islet transcriptomes in humans with prediabetes and type 2 diabetes are remarkably similar. Beta cell overexpression of soluble rodent or oligomer-prone human IAPP induced changes in islet transcriptome present in prediabetes and type 2 diabetes, including decreased expression of genes that confer beta cell identity. Increased expression of human IAPP, but not rodent IAPP, induced islet inflammation present in prediabetes and type 2 diabetes in humans. Key mediators of the injury responses in islets transgenic for human IAPP or those from individuals with type 2 diabetes include STAT3, NF-κB, ESR1 and CTNNB1 by transcription factor analysis and COL3A1, NID1 and ZNF800 by gene regulatory network analysis.Conclusions/interpretationBeta cell injury mediated by IAPP is a plausible mechanism to contribute to islet inflammation and dedifferentiation in type 2 diabetes. Inhibition of IAPP toxicity is a potential therapeutic target in type 2 diabetes.Graphical abstract

Characterisation of Ppy-lineage cells clarifies the functional heterogeneity of pancreatic beta cells in mice

Aims/hypothesisPancreatic polypeptide (PP) cells, which secrete PP (encoded by the Ppy gene), are a minor population of pancreatic endocrine cells. Although it has been reported that the loss of beta cell identity might be associated with beta-to-PP cell-fate conversion, at present, little is known regarding the characteristics of Ppy-lineage cells.MethodsWe used Ppy-Cre driver mice and a PP-specific monoclonal antibody to investigate the association between Ppy-lineage cells and beta cells. The molecular profiles of endocrine cells were investigated by single-cell transcriptome analysis and the glucose responsiveness of beta cells was assessed by Ca2+ imaging. Diabetic conditions were experimentally induced in mice by either streptozotocin or diphtheria toxin.ResultsPpy-lineage cells were found to contribute to the four major types of endocrine cells, including beta cells. Ppy-lineage beta cells are a minor subpopulation, accounting for 12–15% of total beta cells, and are mostly (81.2%) localised at the islet periphery. Unbiased single-cell analysis with a Ppy-lineage tracer demonstrated that beta cells are composed of seven clusters, which are categorised into two groups (i.e. Ppy-lineage and non-Ppy-lineage beta cells). These subpopulations of beta cells demonstrated distinct characteristics regarding their functionality and gene expression profiles. Ppy-lineage beta cells had a reduced glucose-stimulated Ca2+ signalling response and were increased in number in experimental diabetes models.Conclusions/interpretationOur results indicate that an unexpected degree of beta cell heterogeneity is defined by Ppy gene activation, providing valuable insight into the homeostatic regulation of pancreatic islets and future therapeutic strategies against diabetes.Data availabilityThe single-cell RNA sequence (scRNA-seq) analysis datasets generated in this study have been deposited in the Gene Expression Omnibus (GEO) under the accession number GSE166164 (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE166164).Graphical abstract

An inhibitor-mediated beta-cell dedifferentiation model reveals distinct roles for FoxO1 in glucagon repression and insulin maturation

ObjectiveThe loss of forkhead box protein O1 (FoxO1) signaling in response to metabolic stress contributes to the etiology of type II diabetes, causing the dedifferentiation of pancreatic beta cells to a cell type reminiscent of endocrine progenitors. Lack of methods to easily model this process in vitro, however, have hindered progress into the identification of key downstream targets and potential inhibitors. We therefore aimed to establish such an in vitro cellular dedifferentiation model and apply it to identify novel agents involved in the maintenance of beta-cell identity. MethodsThe murine beta-cell line, Min6, was used for primary experiments and high-content screening. Screens encompassed a library of small-molecule drugs representing the chemical and target space of all FDA-approved small molecules with an automated immunofluorescence readout. Validation experiments were performed in a murine alpha-cell line as well as in primary murine and human diabetic islets. Developmental effects were studied in zebrafish and C. elegans models, while diabetic db/db mouse models were used to elucidate global glucose metabolism outcomes. ResultsWe show that short-term pharmacological FoxO1 inhibition can model beta-cell dedifferentiation by downregulating beta-cell-specific transcription factors, resulting in the aberrant expression of progenitor genes and the alpha-cell marker glucagon. From a high-content screen, we identified loperamide as a small molecule that can prevent FoxO inhibitor-induced glucagon expression and further stimulate insulin protein processing and secretion by altering calcium levels, intracellular pH, and FoxO1 localization. ConclusionsOur study provides novel models, molecular targets, and drug candidates for studying and preventing beta-cell dedifferentiation.

Changes to the identity of EndoC-\u03b2H1 beta cells may be mediated by stress-induced depletion of HNRNPD

BackgroundBeta cell identity changes occur in the islets of donors with diabetes, but the molecular basis of this remains unclear. Protecting residual functional beta cells from cell identity changes may be beneficial for patients with diabetes.ResultsA somatostatin-positive cell population was induced in stressed clonal human EndoC-βH1 beta cells and was isolated using FACS. A transcriptomic characterisation of somatostatin-positive cells was then carried out. Gain of somatostatin-positivity was associated with marked dysregulation of the non-coding genome. Very few coding genes were differentially expressed. Potential candidate effector genes were assessed by targeted gene knockdown. Targeted knockdown of the HNRNPD gene induced the emergence of a somatostatin-positive cell population in clonal EndoC-βH1 beta cells comparable with that we have previously reported in stressed cells.ConclusionsWe report here a role for the HNRNPD gene in determination of beta cell identity in response to cellular stress. These findings widen our understanding of the role of RNA binding proteins and RNA biology in determining cell identity and may be important for protecting remaining beta cell reserve in diabetes.

176-OR: STIM1 Deletion Leads to Loss of Beta-Cell Identity in High-Fat Diet-Fed Female Mice

STIM1 is an endoplasmic reticulum (ER) calcium (Ca2+) sensor that replenishes ER Ca2+ stores in a process known as store operated calcium entry (SOCE). Previously, we have shown that SOCE is impaired in the diabetic pancreatic β cell; however, the consequences of reduced β cell SOCE have not been well studied. To this end, mice with pancreatic β cell specific deletion of STIM1 (STIM1Δβ) were generated by crossing STIM1flox/flox with Ins-Cre mice and fed a high fat diet (HFD) beginning at 8 wk of age. Male STIM1Δβ mice did not show changes in glucose tolerance or body composition. In contrast, female HFD-fed STIM1Δβ mice were heavier (25.1 vs. 28.3 g, p&amp;lt;0.05), had increased fat mass (4.9 vs. 8.2g, P&amp;lt;0.01), and exhibited significant glucose intolerance (AUC IPGTT 22461 vs. 29245 mg/dL*min; p&amp;lt;0.0001) with impaired insulin secretion by in-vivo GSIS (p&amp;lt;0.05) without differences insulin tolerance, energy expenditure or food intake. Bulk RNA-sequencing of wild-type and STIM1Δβ female islets revealed reduced expression of MafA, UCN3, and Ins1 and Ins2 in STIM1Δβ islets, indicating potential loss of β cell identity. Pathway analysis showed modulation of pathways related to 17-beta estradiol (E2) signaling, with downregulation of a G-protein coupled estrogen receptor 1 (GPER), while differentially expressed genes were enriched for functions related to apoptosis, lipid metabolism, and epithelial cell differentiation. Consistently, STIM1Δβ female mice had lower β cell mass (2.984 mg vs. 1.349 mg, p&amp;lt;0.05) and higher alpha cell mass (0.1822 mg vs. 0.4463 mg, p&amp;lt;0.05). Proteomics analysis performed in STIM1-deficient INS-1 cells revealed significantly increased glucagon expression. Further, lower mobilization of intracellular cAMP levels was noted in response to treatment with E2 or G1, an agonist of GPER. These findings suggest a role for STIM1 in the maintenance of pancreatic β cell identity and indicate a potential interaction between SOCE and E2 signaling in female mice. Disclosure P. Sohn: None. P. Krishnan: None. C. Lee: None. T. Kono: None. C. Evans-molina: Advisory Panel; Self; Provention Bio, Inc., Consultant; Self; Dompe, Other Relationship; Self; Bristol-Myers Squibb Company, Nimbus Pharmaceuticals, Pfizer Inc. Funding National Institutes of Health (5T32DK064466-17, 5F30DK123996); U.S. Department of Veterans Affairs (2-I01BX001733); National Institute of Diabetes and Digestive and Kidney Diseases (1R01DK093954-06A1); Indiana University School of Medicine; Gates Millennium Scholars Program

DNA Methylation Patterning and the Regulation of Beta Cell Homeostasis.

Pancreatic beta cells play a central role in regulating glucose homeostasis by secreting the hormone insulin. Failure of beta cells due to reduced function and mass and the resulting insulin insufficiency can drive the dysregulation of glycemic control, causing diabetes. Epigenetic regulation by DNA methylation is central to shaping the gene expression patterns that define the fully functional beta cell phenotype and regulate beta cell growth. Establishment of stage-specific DNA methylation guides beta cell differentiation during fetal development, while faithful restoration of these signatures during DNA replication ensures the maintenance of beta cell identity and function in postnatal life. Lineage-specific transcription factor networks interact with methylated DNA at specific genomic regions to enhance the regulatory specificity and ensure the stability of gene expression patterns. Recent genome-wide DNA methylation profiling studies comparing islets from diabetic and non-diabetic human subjects demonstrate the perturbation of beta cell DNA methylation patterns, corresponding to the dysregulation of gene expression associated with mature beta cell state in diabetes. This article will discuss the molecular underpinnings of shaping the islet DNA methylation landscape, its mechanistic role in the specification and maintenance of the functional beta cell phenotype, and its dysregulation in diabetes. We will also review recent advances in utilizing beta cell specific DNA methylation patterns for the development of biomarkers for diabetes, and targeting DNA methylation to develop translational approaches for supplementing the functional beta cell mass deficit in diabetes.

Micro-RNA-mediated Maintenance of Beta-cell Identity Reveals Targets for Reversing Beta-cell Dedifferentiation.

Micro-RNA-mediated Maintenance of Beta-cell Identity Reveals Targets for Reversing Beta-cell Dedifferentiation.

The hepatokine fetuin-A disrupts functional maturation of pancreatic beta cells

Aims/hypothesisNeonatal beta cells carry out a programme of postnatal functional maturation to achieve full glucose responsiveness. A partial loss of the mature phenotype of adult beta cells may contribute to a reduction of functional beta cell mass and accelerate the onset of type 2 diabetes. We previously found that fetuin-A, a hepatokine increasingly secreted by the fatty liver and a determinant of type 2 diabetes, inhibits glucose-stimulated insulin secretion (GSIS) of human islets. Since fetuin-A is a ubiquitous fetal glycoprotein that declines peripartum, we examined here whether fetuin-A interferes with the functional maturity of beta cells.MethodsThe effects of fetuin-A were assessed during in vitro maturation of porcine neonatal islet cell clusters (NICCs) and in adult human islets. Expression alterations were examined via microarray, RNA sequencing and reverse transcription quantitative real-time PCR (qRT-PCR), proteins were analysed by western blotting and immunostaining, and insulin secretion was quantified in static incubations.ResultsNICC maturation was accompanied by the gain of glucose-responsive insulin secretion (twofold stimulation), backed up by mRNA upregulation of genes governing beta cell identity and function, such as NEUROD1, UCN3, ABCC8 and CASR (Log2 fold change [Log2FC] > 1.6). An active TGFβ receptor (TGFBR)–SMAD2/3 pathway facilitates NICC maturation, since the TGFBR inhibitor SB431542 counteracted the upregulation of aforementioned genes and de-repressed ALDOB, a gene disallowed in mature beta cells. In fetuin-A-treated NICCs, upregulation of beta cell markers and the onset of glucose responsiveness were suppressed. Concomitantly, SMAD2/3 phosphorylation was inhibited. Transcriptome analysis confirmed inhibitory effects of fetuin-A and SB431542 on TGFβ-1- and SMAD2/3-regulated transcription. However, contrary to SB431542 and regardless of cMYC upregulation, fetuin-A inhibited beta cell proliferation (0.27 ± 0.08% vs 1.0 ± 0.1% Ki67-positive cells in control NICCs). This effect was sustained by reduced expression (Log2FC ≤ −2.4) of FOXM1, CENPA, CDK1 or TOP2A. In agreement, the number of insulin-positive cells was lower in fetuin-A-treated NICCs than in control NICCs (14.4 ± 1.2% and 22.3 ± 1.1%, respectively). In adult human islets fetuin-A abolished glucose responsiveness, i.e. 1.7- and 1.1-fold change over 2.8 mmol/l glucose in control- and fetuin-A-cultured islets, respectively. In addition, fetuin-A reduced SMAD2/3 phosphorylation and suppressed expression of proliferative genes. Of note, in non-diabetic humans, plasma fetuin-A was negatively correlated (p = 0.013) with islet beta cell area.Conclusions/interpretationOur results suggest that the perinatal decline of fetuin-A relieves TGFBR signalling in islets, a process that facilitates functional maturation of neonatal beta cells. Functional maturity remains revocable in later life, and the occurrence of a metabolically unhealthy milieu, such as liver steatosis and elevated plasma fetuin-A, can impair both function and adaptive proliferation of beta cells.Data availabilityThe RNAseq datasets and computer code produced in this study are available in the Gene Expression Omnibus (GEO): GSE144950; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE144950Graphical abstract

Positive Effects of NPY1 Receptor Activation on Islet Structure Are Driven by Pancreatic Alpha- and Beta-Cell Transdifferentiation in Diabetic Mice.

Enzymatically stable and specific neuropeptide Y1 receptor (NPYR1) agonists, such as sea lamprey PYY(1-36) (SL-PYY(1-36)), are believed to improve glucose regulation in diabetes by targeting pancreatic islets. In this study, streptozotocin (STZ) diabetic transgenic GluCreERT2;ROSA26-eYFP and Ins1 Cre/+;Rosa26-eYFP mouse models have been used to study effects of sustained NPYR1 activation on islet cell composition and alpha- and beta-cell lineage transitioning. STZ induced a particularly severe form of diabetes in Ins1 Cre/+;Rosa26-eYFP mice, but twice-daily administration (25 nmol/kg) of SL-PYY(1-36) for 11 days consistently improved metabolic status. Blood glucose was decreased (p < 0.05 - p < 0.001) and both fasted plasma and pancreatic insulin significantly increased by SL-PYY(1-36). In both GluCreERT2;ROSA26-eYFP and Ins1 Cre/+; Rosa26-eYFP mice, STZ provoked characteristic losses (p < 0.05 - p < 0.001) of islet numbers, beta-cell and pancreatic islet areas together with increases in area and central islet location of alpha-cells. With exception of alpha-cell area, these morphological changes were fully, or partially, returned to non-diabetic control levels by SL-PYY(1-36). Interestingly, STZ apparently triggered decreased (p < 0.001) alpha- to beta-cell transition in GluCreERT2;ROSA26-eYFP mice, together with increased loss of beta-cell identity in Ins1 Cre/+;Rosa26-eYFP mice, but both effects were significantly (p < 0.001) reversed by SL-PYY(1-36). SL-PYY(1-36) also apparently reduced (p < 0.05) beta- to alpha-cell conversion in Ins1 Cre/+;Rosa26-eYFP mice and glucagon expressing alpha-cells in GluCreERT2;ROSA26-eYFP mice. These data indicate that islet benefits of prolonged NPY1R activation, and especially restoration of beta-cell mass, are observed irrespective of diabetes status, being linked to cell lineage alterations including transdifferentiation of alpha- to beta-cells.