NRF2 is required for neonatal mouse beta cell growth by maintaining redox balance and promoting mitochondrial biogenesis and function.

AIMS/HYPOTHESIS: All forms of diabetes result from insufficient functional beta cell mass. Due to the relatively limited expression of several antioxidant enzymes, beta cells are highly vulnerable to pathological levels of reactive oxygen species (ROS), which can lead to the reduction of functional beta cell mass. During early postnatal ages, both human and rodent beta cells go through a burst of proliferation that quickly declines with age. The exact mechanisms that account for neonatal beta cell proliferation are understudied but mitochondrial release of moderated ROS levels has been suggested as one of the main drivers. We previously showed that, apart from its conventional role in protecting beta cells from oxidative stress, the nuclear factor erythroid 2-related factor 2 (NRF2) is also essential for beta cell proliferation. We therefore hypothesised that NRF2, which is activated by ROS, plays an essential role in beta cell proliferation at early postnatal ages. METHODS: Beta cell NRF2 levels and beta cell proliferation were measured in pancreatic sections from non-diabetic human cadaveric donors at different postnatal ages, childhood and adulthood. Pancreatic sections from 1-, 7-, 14- and 28-day-old beta cell-specific Nrf2 (also known as Nfe2l2)-knockout mice (ßNrf2KO) or control (Nrf2lox/lox) mice were assessed for beta cell NRF2 levels, beta cell proliferation, beta cell oxidative stress, beta cell death, nuclear beta cell pancreatic duodenal homeobox protein 1 (PDX1) levels and beta cell mass. Seven-day-old ßNrf2KO and Nrf2lox/lox mice were injected daily with N-acetylcysteine (NAC) or saline (154 mmol/l NaCl) to explore the potential contribution of oxidative stress to the phenotypes seen in ßNrf2KO mice at early postnatal ages. RNA-seq was performed on 7-day-old ßNrf2KO and Nrf2lox/lox mice to investigate the mechanisms by which NRF2 stimulates beta cell proliferation at early postnatal ages. Mitochondrial biogenesis and function were determined using dispersed islets from 7-day-old ßNrf2KO and Nrf2lox/lox mice by measuring MitoTracker intensity, mtDNA/gDNA ratio and ATP/ADP ratio. To study the effect of neonatal beta cell-specific Nrf2 deletion on glucose homeostasis in adulthood, blood glucose, plasma insulin and insulin secretion were determined and a GTT was performed on 3-month-old ßNrf2KO and Nrf2lox/lox mice fed on regular diet (RD) or high-fat diet (HFD). RESULTS: The expression of the master antioxidant regulator NRF2 was increased at early postnatal ages in both human (1 day to 19 months old, 31%) and mouse (7 days old, 57%) beta cells, and gradually declined with age (8% in adult humans, 3.77% in adult mice). A significant correlation (R2=0.568; p=0.001) was found between beta cell proliferation and NRF2 levels in human beta cells. Seven-day-old ßNrf2KO mice showed reduced beta cell proliferation (by 65%), beta cell nuclear PDX1 levels (by 23%) and beta cell mass (by 67%), and increased beta cell oxidative stress (threefold) and beta cell death compared with Nrf2lox/lox control mice. NAC injections increased beta cell proliferation in 7-day-old ßNrf2KO mice (3.4-fold) compared with saline-injected ßNrf2KO mice. Interestingly, RNA-seq of islets isolated from 7-day-old ßNrf2KO mice revealed reduced expression of mitochondrial RNA genes and genes involved in the electron transport chain. Islets isolated from 7-day old ßNrf2KO mice presented reduced MitoTracker intensity (by 47%), mtDNA/gDNA ratio (by 75%) and ATP/ADP ratio (by 68%) compared with islets from Nrf2lox/lox littermates. Lastly, HFD-fed 3-month-old ßNrf2KO male mice displayed a significant reduction in beta cell mass (by 35%), a mild increase in non-fasting blood glucose (1.2-fold), decreased plasma insulin (by 14%), and reduced glucose tolerance (1.3-fold) compared with HFD-fed Nrf2lox/lox mice. CONCLUSIONS/INTERPRETATION: Our study highlights NRF2 as an essential transcription factor for maintaining neonatal redox balance, mitochondrial biogenesis and function and beta cell growth, and for preserving functional beta cell mass in adulthood under metabolic stress. DATA AVAILABILITY: Sequencing data are available in the NCBI Gene Expression Omnibus, accession number GSE242718 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE242718 ).

Adaptive and maladaptive roles for ChREBP in the liver and pancreatic islets.

Excessive sugar consumption is a contributor to the worldwide epidemic of cardiometabolic disease. Understanding mechanisms by which sugar is sensed and regulates metabolic processes may provide new opportunities to prevent and treat these epidemics. Carbohydrate Responsive-Element Binding Protein (ChREBP) is a sugar-sensing transcription factor that mediates genomic responses to changes in carbohydrate abundance in key metabolic tissues. Carbohydrate metabolites activate the canonical form of ChREBP, ChREBP-alpha, which stimulates production of a potent, constitutively active ChREBP isoform called ChREBP-beta. Carbohydrate metabolites and other metabolic signals may also regulate ChREBP activity via posttranslational modifications including phosphorylation, acetylation, and O-GlcNAcylation that can affect ChREBP's cellular localization, stability, binding to cofactors, and transcriptional activity. In this review, we discuss mechanisms regulating ChREBP activity and highlight phenotypes and controversies in ChREBP gain- and loss-of-function genetic rodent models focused on the liver and pancreatic islets.

The many lives of Myc in the pancreatic ß-cell.

Diabetes results from insufficient numbers of functional pancreatic ß-cells. Thus, increasing the number of available functional ß-cells ex vivo for transplantation, or regenerating them in situ in diabetic patients, is a major focus of diabetes research. The transcription factor, Myc, discovered decades ago lies at the nexus of most, if not all, known proliferative pathways. Based on this, many studies in the 1990s and early 2000s explored the potential of harnessing Myc expression to expand ß-cells for diabetes treatment. Nearly all these studies in ß-cells used pathophysiological or supraphysiological levels of Myc and reported enhanced ß-cell death, dedifferentiation, or the formation of insulinomas if cooverexpressed with Bcl-xL, an inhibitor of apoptosis. This obviously reduced the enthusiasm for Myc as a therapeutic target for ß-cell regeneration. However, recent studies indicate that "gentle" induction of Myc expression enhances ß-cell replication without induction of cell death or loss of insulin secretion, suggesting that appropriate levels of Myc could have therapeutic potential for ß-cell regeneration. Furthermore, although it has been known for decades that Myc is induced by glucose in ß-cells, very little is known about how this essential anabolic transcription factor perceives and responds to nutrients and increased insulin demand in vivo. Here we summarize the previous and recent knowledge of Myc in the ß-cell, its potential for ß-cell regeneration, and its physiological importance for neonatal and adaptive ß-cell expansion.

Aging of Antiviral CD8+ Memory T Cells Fosters Increased Survival, Metabolic Adaptations, and Lymphoid Tissue Homing.

Aging of established antiviral T cell memory can foster a series of progressive adaptations that paradoxically improve rather than compromise protective CD8+ T cell immunity. We now provide evidence that this gradual evolution, the pace of which is contingent on the precise context of the primary response, also impinges on the molecular mechanisms that regulate CD8+ memory T cell (TM) homeostasis. Over time, CD8+ TM generated in the wake of an acute infection with the natural murine pathogen lymphocytic choriomeningitis virus become more resistant to apoptosis and acquire enhanced cytokine responsiveness without adjusting their homeostatic proliferation rates; concurrent metabolic adaptations promote increased CD8+ TM quiescence and fitness but also impart the reacquisition of a partial effector-like metabolic profile; and a gradual redistribution of aging CD8+ TM from blood and nonlymphoid tissues to lymphatic organs results in CD8+ TM accumulations in bone marrow, splenic white pulp, and, particularly, lymph nodes. Altogether, these data demonstrate how temporal alterations of fundamental homeostatic determinants converge to render aged CD8+ TM poised for greater recall responses.

Transcriptional activation of the Myc gene by glucose in ß-cells requires a ChREBP-dependent 3-D chromatin interaction between the Myc and Pvt1 genes.

OBJECTIVE: All forms of diabetes result from insufficient functional ß-cell mass. Thus, achieving the therapeutic goal of expanding ß-cell mass requires a better mechanistic understanding of how ß-cells proliferate. Glucose is a natural ß-cell mitogen that mediates its effects in part through the glucose-responsive transcription factor, carbohydrate response element binding protein (ChREBP) and the anabolic transcription factor, MYC. However, mechanistic details by which glucose activates Myc at the transcriptional level are poorly understood. METHODS: Here, siRNA was used to test the role of ChREBP in the glucose response of MYC, ChIP and ChIPseq to identify potential regulatory binding sites, chromatin conformation capture to identify DNA/DNA interactions, and an adenovirus was constructed to expresses x-dCas9 and an sgRNA that specifically disrupts the recruitment of ChREBP to a specific targeted ChoRE. RESULTS: We found that ChREBP is essential for glucose-mediated transcriptional induction of Myc, and for increases in Myc mRNA and protein abundance. Further, ChIPseq revealed that the carbohydrate response element (ChoRE) nearest to the Myc transcriptional start site (TSS) is immediately upstream of the gene encoding the lncRNA, Pvt1, 60,000 bp downstream of the Myc gene. Chromatin Conformation Capture (3C) confirmed a glucose-dependent interaction between these two sites. Transduction with an adenovirus expressing x-dCas9 and an sgRNA specifically targeting the highly conserved Pvt1 ChoRE, attenuates ChREBP recruitment, decreases Myc-Pvt1 DNA/DNA interaction, and decreases expression of the Pvt1 and Myc genes in response to glucose. Importantly, isolated and dispersed rat islet cells transduced with the ChoRE-disrupting adenovirus also display specific decreases in ChREBP-dependent, glucose-mediated expression of Pvt1 and Myc, as well as decreased glucose-stimulated ß-cell proliferation. CONCLUSIONS: The mitogenic glucose response of Myc is mediated via glucose-dependent recruitment of ChREBP to the promoter of the Pvt1 gene and subsequent DNA looping with the Myc promoter.

Molecular glues of the regulatory ChREBP/14-3-3 complex protect beta cells from glucolipotoxicity.

The Carbohydrate Response Element Binding Protein (ChREBP) is a glucose-responsive transcription factor (TF) that is characterized by two major splice isoforms (α and ß). In acute hyperglycemia, both ChREBP isoforms regulate adaptive ß-expansion; however, during chronic hyperglycemia and glucolipotoxicity, ChREBPß expression surges, leading to ß-cell dedifferentiation and death. 14-3-3 binding to ChREBPα results in its cytoplasmic retention and concomitant suppression of transcriptional activity, suggesting that small molecule-mediated stabilization of this protein-protein interaction (PPI) via molecular glues may represent an attractive entry for the treatment of metabolic disease. Here, we show that structure-based optimizations of a molecular glue tool compound led not only to more potent ChREBPα/14-3-3 PPI stabilizers but also for the first time cellular active compounds. In primary human ß-cells, the most active compound stabilized the ChREBPα/14-3-3 interaction and thus induced cytoplasmic retention of ChREBPα, resulting in highly efficient ß-cell protection from glucolipotoxicity while maintaining ß-cell identity. This study may thus not only provide the basis for the development of a unique class of compounds for the treatment of Type 2 Diabetes but also showcases an alternative 'molecular glue' approach for achieving small molecule control of notoriously difficult targetable TFs.

Select DYRK1A Inhibitors Enhance Both Proliferation and Differentiation in Human Pancreatic Beta Cells.

The small molecule DYRK1A inhibitor, harmine, induces human beta cell proliferation, expands beta cell mass, enhances expression of beta cell phenotypic genes, and improves human beta cell function i n vitro and in vivo . It is unknown whether the "pro-differentiation effect" is a DYRK1A inhibitor class-wide effect. Here we compare multiple commonly studied DYRK1A inhibitors. Harmine, 2-2c and 5-IT increase expression of PDX1, MAFA, NKX6.1, SLC2A2, PCSK1, MAFB, SIX2, SLC2A2, SLC30A8, ENTPD3 in normal and T2D human islets. Unexpectedly, GNF4877, CC-401, INDY, CC-401 and Leucettine fail to induce expression of these essential beta cell molecules. Remarkably, the pro-differentiation effect is independent of DYRK1A inhibition: although silencing DYRK1A induces human beta cell proliferation, it has no effect on differentiation; conversely, harmine treatment enhances beta cell differentiation in DYRK1A-silenced islets. A careful screen of multiple DYRK1A inhibitor kinase candidate targets was unable to identify pro-differentiation pathways. Overall, harmine, 2-2c and 5-IT are unique among DYRK1A inhibitors in their ability to enhance both beta cell proliferation and differentiation. While beta cell proliferation is mediated by DYRK1A inhibition, the pro-differentiation effects of harmine, 2-2c and 5-IT are distinct, and unexplained in mechanistic terms. These considerations have important implications for DYRK1A inhibitor pharmaceutical development.

²H enrichment distribution of hepatic glycogen from ²H2O reveals the contribution of dietary fructose to glycogen synthesis.

Dietary fructose can benefit or hinder glycemic control, depending on the quantity consumed, and these contrasting effects are reflected by alterations in postprandial hepatic glycogen synthesis. Recently, we showed that ²H enrichment of glycogen positions 5 and 2 from deuterated water (²H2O) informs direct and indirect pathway contributions to glycogenesis in naturally feeding rats. Inclusion of position 6(S) ²H enrichment data allows indirect pathway sources to be further resolved into triose phosphate and Krebs cycle precursors. This analysis was applied to six rats that had fed on standard chow (SC) and six rats that had fed on SC plus 35% sucrose in their drinking water (HS). After 2 wk, hepatic glycogenesis sources during overnight feeding were determined by ²H2O administration and postmortem analysis of glycogen ²H enrichment at the conclusion of the dark period. Net overnight hepatic glycogenesis was similar between SC and HS rodents. Whereas direct pathway contributions were similar (403 ± 71 µmol/g dry wt HS vs. 578 ± 76 µmol/g dry wt SC), triose phosphate contributions were significantly higher for HS compared with SC (382 ± 61 vs. 87 ± 24 µmol/g dry wt, P < 0.01) and Krebs cycle inputs lower for HS compared with SC (110 ± 9 vs. 197 ± 32 µmol/g dry wt, P < 0.05). Analysis of plasma glucose ²H enrichments at the end of the feeding period also revealed a significantly higher fractional contribution of triose phosphate to plasma glucose levels in HS vs. SC. Hence, the ²H enrichment distributions of hepatic glycogen and glucose from ²H2O inform the contribution of dietary fructose to hepatic glycogen and glucose synthesis.

ß-catenin is essential for ethanol metabolism and protection against alcohol-mediated liver steatosis in mice.

UNLABELLED: The liver plays a central role in ethanol metabolism, and oxidative stress is implicated in alcohol-mediated liver injury. ß-Catenin regulates hepatic metabolic zonation and adaptive response to oxidative stress. We hypothesized that ß-catenin regulates the hepatic response to ethanol ingestion. Female liver-specific ß-catenin knockout (KO) mice and wild-type (WT) littermates were fed the Lieber-Decarli liquid diet (5% ethanol) in a pairwise fashion. Liver histology, biochemistry, and gene-expression studies were performed. Plasma alcohol and ammonia levels were measured using standard assays. Ethanol-fed (EtOH) KO mice exhibited systemic toxicity and early mortality. KO mice exhibited severe macrovesicular steatosis and 5 to 6-fold higher serum alanine aminotransferase and aspartate aminotransferase levels. KO mice had a modest increase in hepatic oxidative stress, lower expression of mitochondrial superoxide dismutase (SOD2), and lower citrate synthase activity, the first step in the tricarboxylic acid cycle. N-Acetylcysteine did not prevent ethanol-induced mortality in KO mice. In WT livers, ß-catenin was found to coprecipitate with forkhead box O3, the upstream regulator of SOD2. Hepatic alcohol dehydrogenase and aldehyde dehydrogenase activities and expression were lower in KO mice. Hepatic cytochrome P450 2E1 protein levels were up-regulated in EtOH WT mice, but were nearly undetectable in KO mice. These changes in ethanol-metabolizing enzymes were associated with 30-fold higher blood alcohol levels in KO mice. CONCLUSION: ß-Catenin is essential for hepatic ethanol metabolism and plays a protective role in alcohol-mediated liver steatosis. Our results strongly suggest that integration of these functions by ß-catenin is critical for adaptation to ethanol ingestion in vivo.

NRF2 Dysregulation in Mice Leads to Inadequate Beta-Cell Mass Expansion during Pregnancy and Gestational Diabetes.

The late stages of the mammalian pregnancy are accompanied with increased insulin resistance due to the increased glucose demand of the growing fetus. Therefore, as a compensatory response to maintain the maternal normal blood glucose levels, maternal beta-cell mass expands leading to increased insulin release. Defects in beta-cell adaptive expansion during pregnancy can lead to gestational diabetes mellitus (GDM). Although the exact mechanisms that promote GDM are poorly understood, GDM strongly associates with impaired beta-cell proliferation and with increased levels of reactive oxygen species (ROS). Here, we show that NRF2 levels are upregulated in mouse beta-cells at gestation day 15 (GD15) concomitant with increased beta-cell proliferation. Importantly, mice with tamoxifen-induced beta-cell-specific NRF2 deletion display inhibition of beta-cell proliferation, increased beta-cell oxidative stress and elevated levels of beta-cell death at GD15. This results in attenuated beta-cell mass expansion and disturbed glucose homeostasis towards the end of pregnancy. Collectively, these results highlight the importance of NRF2-oxidative stress regulation in beta-cell mass adaptation to pregnancy and suggest NRF2 as a potential therapeutic target for treating GDM.

Single-nucleus RNA sequencing of human pancreatic islets identifies novel gene sets and distinguishes ß-cell subpopulations with dynamic transcriptome profiles.

BACKGROUND: Single-cell RNA sequencing (scRNA-seq) provides valuable insights into human islet cell types and their corresponding stable gene expression profiles. However, this approach requires cell dissociation that complicates its utility in vivo. On the other hand, single-nucleus RNA sequencing (snRNA-seq) has compatibility with frozen samples, elimination of dissociation-induced transcriptional stress responses, and affords enhanced information from intronic sequences that can be leveraged to identify pre-mRNA transcripts. METHODS: We obtained nuclear preparations from fresh human islet cells and generated snRNA-seq datasets. We compared these datasets to scRNA-seq output obtained from human islet cells from the same donor. We employed snRNA-seq to obtain the transcriptomic profile of human islets engrafted in immunodeficient mice. In both analyses, we included the intronic reads in the snRNA-seq data with the GRCh38-2020-A library. RESULTS: First, snRNA-seq analysis shows that the top four differentially and selectively expressed genes in human islet endocrine cells in vitro and in vivo are not the canonical genes but a new set of non-canonical gene markers including ZNF385D, TRPM3, LRFN2, PLUT (ß-cells); PTPRT, FAP, PDK4, LOXL4 (α-cells); LRFN5, ADARB2, ERBB4, KCNT2 (δ-cells); and CACNA2D3, THSD7A, CNTNAP5, RBFOX3 (γ-cells). Second, by integrating information from scRNA-seq and snRNA-seq of human islet cells, we distinguish three ß-cell sub-clusters: an INS pre-mRNA cluster (ß3), an intermediate INS mRNA cluster (ß2), and an INS mRNA-rich cluster (ß1). These display distinct gene expression patterns representing different biological dynamic states both in vitro and in vivo. Interestingly, the INS mRNA-rich cluster (ß1) becomes the predominant sub-cluster in vivo. CONCLUSIONS: In summary, snRNA-seq and pre-mRNA analysis of human islet cells can accurately identify human islet cell populations, subpopulations, and their dynamic transcriptome profile in vivo.

Structure-Function Analysis of p57KIP2 in the Human Pancreatic Beta Cell Reveals a Bipartite Nuclear Localization Signal.

Mutations in CDKN1C, encoding p57KIP2, a canonical cell cycle inhibitor, underlie multiple pediatric endocrine syndromes. Despite this central role in disease, little is known about the structure and function of p57KIP2 in the human pancreatic beta cell. Since p57KIP2 is predominantly nuclear in human beta cells, we hypothesized that disease-causing mutations in its nuclear localization sequence (NLS) may correlate with abnormal phenotypes. We prepared RIP1 insulin promoter-driven adenoviruses encoding deletions of multiple disease-associated but unexplored regions of p57KIP2 and performed a comprehensive structure-function analysis of CDKN1C/p57KIP2. Real-time polymerase chain reaction and immunoblot analyses confirmed p57KIP2 overexpression, construct size, and beta cell specificity. By immunocytochemistry, wild-type (WT) p57KIP2 displayed nuclear localization. In contrast, deletion of a putative NLS at amino acids 278-281 failed to access the nucleus. Unexpectedly, we identified a second downstream NLS at amino acids 312-316. Further analysis showed that each individual NLS is required for nuclear localization, but neither alone is sufficient. In summary, p57KIP2 contains a classical bipartite NLS characterized by 2 clusters of positively charged amino acids separated by a proline-rich linker region. Variants in the sequences encoding these 2 NLS sequences account for functional p57KIP2 loss and beta cell expansion seen in human disease.

Human Pancreatic α-Cell Heterogeneity and Trajectory Inference Analysis Using Integrated Single Cell- and Single Nucleus-RNA Sequencing Platforms.

Prior studies have shown that pancreatic α-cells can transdifferentiate into ß-cells, and that ß-cells de-differentiate and are prone to acquire an α-cell phenotype in type 2 diabetes (T2D). However, the specific human α-cell and ß-cell subtypes that are involved in α-to-ß-cell and ß-to-α-cell transitions are unknown. Here, we have integrated single cell RNA sequencing (scRNA-seq) and single nucleus RNA-seq (snRNA-seq) of isolated human islets and human islet grafts and provide additional insight into α-ß cell fate switching. Using this approach, we make seven novel observations. 1) There are five different GCG -expressing human α-cell subclusters [α1, α2, α-ß-transition 1 (AB-Tr1), α-ß-transition 2 (AB-Tr2), and α-ß (AB) cluster] with different transcriptome profiles in human islets from non-diabetic donors. 2) The AB subcluster displays multihormonal gene expression, inferred mostly from snRNA-seq data suggesting identification by pre-mRNA expression. 3) The α1, α2, AB-Tr1, and AB-Tr2 subclusters are enriched in genes specific for α-cell function while AB cells are enriched in genes related to pancreatic progenitor and ß-cell pathways; 4) Trajectory inference analysis of extracted α- and ß-cell clusters and RNA velocity/PAGA analysis suggests a bifurcate transition potential for AB towards both α- and ß-cells. 5) Gene commonality analysis identifies ZNF385D, TRPM3, CASR, MEG3 and HDAC9 as signature for trajectories moving towards ß-cells and SMOC1, PLCE1, PAPPA2, ZNF331, ALDH1A1, SLC30A8, BTG2, TM4SF4, NR4A1 and PSCK2 as signature for trajectories moving towards α-cells. 6) Remarkably, in contrast to the events in vitro , the AB subcluster is not identified in vivo in human islet grafts and trajectory inference analysis suggests only unidirectional transition from α-to-ß-cells in vivo . 7) Analysis of scRNA-seq datasets from adult human T2D donor islets reveals a clear unidirectional transition from ß-to-α-cells compatible with dedifferentiation or conversion into α-cells. Collectively, these studies show that snRNA-seq and scRNA-seq can be leveraged to identify transitions in the transcriptional status among human islet endocrine cell subpopulations in vitro , in vivo , in non-diabetes and in T2D. They reveal the potential gene signatures for common trajectories involved in interconversion between α- and ß-cells and highlight the utility and power of studying single nuclear transcriptomes of human islets in vivo . Most importantly, they illustrate the importance of studying human islets in their natural in vivo setting.

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.

T3 and glucose increase expression of phosphoenolpyruvate carboxykinase (PCK1) leading to increased ß-cell proliferation.

OBJECTIVES: Thyroid hormone (T3) and high glucose concentrations are critical components of ß-cell maturation and function. In the present study, we asked whether T3 and glucose signaling pathways coordinately regulate transcription of genes important for ß-cell function and proliferation. METHODS: RNA-seq analysis was performed on cadaveric human islets from five different donors in response to low and high glucose concentrations and in the presence or absence of T3. Gene expression was also studies in sorted human ß-cells, mouse islets and Ins-1 cells by RT-qPCR. Silencing of the thyroid hormone receptors (THR) was conducted using lentiviruses. Proliferation was assessed by ki67 immunostaining in primary human/mouse islets. Chromatin immunoprecipitation and proximity ligation assay were preformed to validate interactions of ChREBP and THR. RESULTS: We found glucose-mediated expression of carbohydrate response element binding protein alpha and beta (ChREBPα and ChREBPß) mRNAs and their target genes are highly dependent on T3 concentrations in rodent and human ß-cells. In ß-cells, T3 and glucose coordinately regulate the expression of ChREBPß and PCK1 (phosphoenolpyruvate carboxykinase-1) among other important genes for ß-cell maturation. Additionally, we show the thyroid hormone receptor (THR) and ChREBP interact, and their relative response elements are located near to each other on mutually responsive genes. In FACS-sorted adult human ß-cells, we found that high concentrations of glucose and T3 induced the expression of PCK1. Next, we show that overexpression of Pck1 together with dimethyl malate (DMM), a substrate precursor, significantly increased ß-cell proliferation in human islets. Finally, using a Cre-Lox approach, we demonstrated that ChREBPß contributes to Pck1-dependent ß-cell proliferation in mouse ß-cells. CONCLUSIONS: We conclude that T3 and glucose act together to regulate ChREBPß, leading to increased expression and activity of Pck1, and ultimately increased ß-cell proliferation.

The c-Myc Oncogene Maintains Corneal Epithelial Architecture at Homeostasis, Modulates p63 Expression, and Enhances Proliferation During Tissue Repair.

Purpose: The transcription factor c-Myc (Myc) plays central regulatory roles in both self-renewal and differentiation of progenitors of multiple cell lineages. Here, we address its function in corneal epithelium (CE) maintenance and repair. Methods: Myc ablation in the limbal-corneal epithelium was achieved by crossing a floxed Myc mouse allele (Mycfl/fl) with a mouse line expressing the Cre recombinase gene under the keratin (Krt) 14 promoter. CE stratification and protein localization were assessed by histology of paraffin and plastic sections and by immunohistochemistry of frozen sections, respectively. Protein levels and gene expression were determined by western blot and real-time quantitative PCR, respectively. CE wound closure was tracked by fluorescein staining. Results: At birth, mutant mice appeared indistinguishable from control littermates; however, their rates of postnatal weight gain were 67% lower than those of controls. After weaning, mutants also exhibited spontaneous skin ulcerations, predominantly in the tail and lower lip, and died 45 to 60 days after birth. The mutant CE displayed an increase in stratal thickness, increased levels of Krt12 in superficial cells, and decreased exfoliation rates. Accordingly, the absence of Myc perturbed protein and mRNA levels of genes modulating differentiation and proliferation processes, including ΔNp63ß, Ets1, and two Notch target genes, Hey1 and Maml1. Furthermore, Myc promoted CE wound closure and wound-induced hyperproliferation. Conclusions: Myc regulates the balance among CE stratification, differentiation, and surface exfoliation and promotes the transition to the hyperproliferative state during wound healing. Its effect on this balance may be exerted through the control of multiple regulators of cell fate, including isoforms of tumor protein p63.

Nrf2 Regulates ß-Cell Mass by Suppressing ß-Cell Death and Promoting ß-Cell Proliferation.

Finding therapies that can protect and expand functional ß-cell mass is a major goal of diabetes research. Here, we generated ß-cell-specific conditional knockout and gain-of-function mouse models and used human islet transplant experiments to examine how manipulating Nrf2 levels affects ß-cell survival, proliferation, and mass. Depletion of Nrf2 in ß-cells results in decreased glucose-stimulated ß-cell proliferation ex vivo and decreased adaptive ß-cell proliferation and ß-cell mass expansion after a high-fat diet in vivo. Nrf2 protects ß-cells from apoptosis after a high-fat diet. Nrf2 loss of function decreases Pdx1 abundance and insulin content. Activating Nrf2 in a ß-cell-specific manner increases ß-cell proliferation and mass and improves glucose tolerance. Human islets transplanted under the kidney capsule of immunocompromised mice and treated systemically with bardoxolone methyl, an Nrf2 activator, display increased ß-cell proliferation. Thus, by managing reactive oxygen species levels, Nrf2 regulates ß-cell mass and is an exciting therapeutic target for expanding and protecting ß-cell mass in diabetes.

Disrupting the DREAM complex enables proliferation of adult human pancreatic ß cells.

Resistance to regeneration of insulin-producing pancreatic ß cells is a fundamental challenge for type 1 and type 2 diabetes. Recently, small molecule inhibitors of the kinase DYRK1A have proven effective in inducing adult human ß cells to proliferate, but their detailed mechanism of action is incompletely understood. We interrogated our human insulinoma and ß cell transcriptomic databases seeking to understand why ß cells in insulinomas proliferate, while normal ß cells do not. This search reveals the DREAM complex as a central regulator of quiescence in human ß cells. The DREAM complex consists of a module of transcriptionally repressive proteins that assemble in response to DYRK1A kinase activity, thereby inducing and maintaining cellular quiescence. In the absence of DYRK1A, DREAM subunits reassemble into the pro-proliferative MMB complex. Here, we demonstrate that small molecule DYRK1A inhibitors induce human ß cells to replicate by converting the repressive DREAM complex to its pro-proliferative MMB conformation.

Maladaptive positive feedback production of ChREBPß underlies glucotoxic ß-cell failure.

Preservation and expansion of ß-cell mass is a therapeutic goal for diabetes. Here we show that the hyperactive isoform of carbohydrate response-element binding protein (ChREBPß) is a nuclear effector of hyperglycemic stress occurring in ß-cells in response to prolonged glucose exposure, high-fat diet, and diabetes. We show that transient positive feedback induction of ChREBPß is necessary for adaptive ß-cell expansion in response to metabolic challenges. Conversely, chronic excessive ß-cell-specific overexpression of ChREBPß results in loss of ß-cell identity, apoptosis, loss of ß-cell mass, and diabetes. Furthermore, ß-cell "glucolipotoxicity" can be prevented by deletion of ChREBPß. Moreover, ChREBPß-mediated cell death is mitigated by overexpression of the alternate CHREBP gene product, ChREBPα, or by activation of the antioxidant Nrf2 pathway in rodent and human ß-cells. We conclude that ChREBPß, whether adaptive or maladaptive, is an important determinant of ß-cell fate and a potential target for the preservation of ß-cell mass in diabetes.

Nrf2: The Master and Captain of Beta Cell Fate.

Prolonged hyperglycemia is toxic to pancreatic ß cells, generating excessive reactive oxygen species, defective glucose-stimulated insulin secretion, decreased insulin production, and eventually ß cell death and diabetes. Nrf2 is a master regulator of cellular responses to counteract dangerous levels of oxidative stress. Maintenance of ß cell mass depends on Nrf2 to promote the survival, function, and proliferation of ß cells. Indeed, Nrf2 activation decreases inflammation, increases insulin sensitivity, reduces body weight, and preserves ß cell mass. Therefore, numerous pharmacological activators of Nrf2 are being tested in clinical trials for the treatment of diabetes and diabetic complications. Modulating Nrf2 activity in ß cells is a promising therapeutic approach for the treatment of diabetes.