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Glucose transporter type 2 (GLUT2), encoded by the SLC2A2 gene, is an essential component of glucose-stimulated insulin secretion in pancreatic islet β-cells. Like that of the gene encoding insulin, expression of the SLC2A2 gene expression is closely linked to β-cell functionality in rodents, but the mechanism by which β-cell-specific expression of SLC2A2 is controlled remains unclear. In this report, to identify putative enhancer elements of the mouse Slc2a2 gene, we examined evolutional conservation of the nucleotide sequence of its genomic locus, together with ChIP-seq data of histone modifications and various transcription factors published in previous studies. Using luciferase reporter assays, we found that an evolutionarily conserved region (ECR) located approximately 40 kbp downstream of the transcription start site of Slc2a2 functions as an active enhancer in the MIN6 β-cell line. We also found that three β-cell-enriched transcription factors, MafA, NeuroD1, and HNF1β, synergistically activate transcription through this 3’ downstream distal enhancer (ECR3’) and the proximal promoter region of the gene. Our data also indicate that the simultaneous binding of HNF1β to its target sites within the promoter and ECR3’ of Slc2a2 is indispensable for transcriptional activation, and that binding of MafA and NeuroD1 to their respective target sites within the ECR3’ enhances transcription. Co-immunoprecipitation experiments suggested that MafA, NeuroD1, and HNF1β interact with each other. Overall, these results suggest that promoter-enhancer communication through MafA, NeuroD1, and HNF1β is critical for Slc2a2 gene expression. These findings provide clues to help elucidate the mechanism of regulation of Slc2a2 gene expression in β-cells.

While apneas are associated with multiple pathological and fatal conditions, the underlying molecular mechanisms remain elusive. We report that a mutated form of the transcription factor Mafa (Mafa4A) that prevents phosphorylation of the Mafa protein leads to an abnormally high incidence of breath holding apneas and death in newborn Mafa4A/4A mutant mice. This apneic breathing is phenocopied by restricting the mutation to central GABAergic inhibitory neurons and by activation of inhibitory Mafa neurons while reversed by inhibiting GABAergic transmission centrally. We find that Mafa activates the Gad2 promoter in vitro and that this activation is enhanced by the mutation that likely results in increased inhibitory drives onto target neurons. We also find that Mafa inhibitory neurons are absent from respiratory, sensory (primary and secondary) and pontine structures but are present in the vicinity of the hypoglossal motor nucleus including premotor neurons that innervate the geniohyoid muscle, to control upper airway patency. Altogether, our data reveal a role for Mafa phosphorylation in regulation of GABAergic drives and suggest a mechanism whereby reduced premotor drives to upper airway muscles may cause apneic breathing at birth.

The large MAF transcription factor group is a group of transcription factors with an acidic region, a basic region, and a leucine zipper region. Four types of MAF, MAFA, MAFB, c-MAF, and NRL, have been identified in humans and mice. In order to elucidate the functions of the large MAF transcription factor group in vivo, our research group created genetically modified MAFA-, MAFB-, and c-MAF-deficient mice and analyzed their phenotypes. MAFA is expressed in pancreatic β cells and is essential for insulin transcription and secretion. MAFB is essential for the development of pancreatic endocrine cells, formation of inner ears, podocyte function in the kidneys, and functional differentiation of macrophages. c-MAF is essential for lens formation and osteoblast differentiation. Furthermore, a single-base mutation in genes encoding the large MAF transcription factor group causes congenital renal disease, eye disease, bone disease, diabetes, and tumors in humans. This review describes the functions of large MAF transcription factors in vivo and their relationships with human diseases.

Inadequate insulin secretion in response to glucose is an important factor for β-cell failure in type 2 diabetes (T2D). Although HMG-CoA reductase degradation 1 (HRD1), a subunit of the endoplasmic reticulum–associated degradation complex, plays a pivotal role in β-cell function, HRD1 elevation in a diabetic setting contributes to β-cell dysfunction. We report in this study the excessive HRD1 expression in islets from humans with T2D and T2D mice. Functional studies reveal that β-cell–specific HRD1 overexpression triggers impaired insulin secretion that will ultimately lead to severe hyperglycemia; by contrast, HRD1 knockdown improves glucose control and response in diabetic models. Proteomic analysis results reveal a large HRD1 interactome, which includes v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA), a master regulator of genes implicated in the maintenance of β-cell function. Furthermore, mechanistic assay results indicate that HRD1 is a novel E3 ubiquitin ligase that targets MafA for ubiquitination and degradation in diabetic β-cells, resulting in cytoplasmic accumulation of MafA and in the reduction of its biological function in the nucleus. Our results not only reveal the pathological importance of excessive HRD1 in β-cell dysfunction but also establish the therapeutic importance of targeting HRD1 in order to prevent MafA loss and suppress the development of T2D.

SummaryTranslational readthrough (TR) occurs when the ribosome decodes a stop codon as a sense codon, resulting in two protein isoforms synthesized from the same mRNA. TR is pervasive in eukaryotic organisms; however, its biological significance remains unclear. In this study, we quantify the TR potential of several candidate genes inDrosophila melanogasterand characterize the regulation of TR in the large Maf transcription factor Traffic jam (Tj). We used CRISPR/Cas9 generated mutant flies to show that the TR-generated Tj isoform is expressed in the nuclei of a subset of neural cells of the central nervous system and is excluded from the somatic cells of gonads, which express the short Tj isoform only. Translational control of TR is critical for preservation of neuronal integrity and maintenance of reproductive health. Fine-tuning of the gene regulatory functions of transcription factors by TR provides a new potential mechanism for cell-specific regulation of gene expression.HighlightsTj undergoes tissue-specific TR in neural cells of the central nervous system.Strict control of TR is crucial for neuroprotection and maintenance of reproductive capacity.TR selectively fine-tunes the gene regulatory functions of the transcription factor.TR in Tj links transcription and translation of tissue-specific control of gene expression.

Reduced expression of exocytotic genes is associated with functional defects in insulin exocytosis contributing to impaired insulin secretion and type 2 diabetes (T2D) development. MAFA and MAFB transcription factors regulate β-cell physiology, and their gene expression is reduced in T2D β cells. We investigate if loss of MAFA and MAFB in human β cells contributes to T2D progression by regulating genes required for insulin exocytosis.Three approaches were performed: (1) RNAseq analysis with the focus on exocytosis-related genes in MafAThe expression of 30 exocytosis-related genes was significantly downregulated in MafAOur data indicate that STXBP1 and STX1A are important MAFA/B-regulated exocytosis genes which may contribute to insulin exocytosis defects observed in MAFA-deficient human T2D β cells.

Diabetes is characterized by a loss of β-cell mass, and a greater understanding of the transcriptional mechanisms governing β-cell function is required for future therapies. Previously, we reported that a complex of the Islet-1 (Isl1) transcription factor and the co-regulator single-stranded DNA–binding protein 3 (SSBP3) regulates the genes necessary for β-cell function, but few proteins are known to interact with this complex in β-cells. To identify additional components, here we performed SSBP3 reverse–cross-linked immunoprecipitation (ReCLIP)- and MS-based experiments with mouse β-cell extracts and compared the results with those from our previous Isl1 ReCLIP study. Our analysis identified the E3 ubiquitin ligases ring finger protein 20 (RNF20) and RNF40, factors that in nonpancreatic cells regulate transcription through imparting monoubiquitin marks on histone H2B (H2Bub1), a precursor to histone H3 lysine 4 trimethylation (H3K4me3). We hypothesized that RNF20 and RNF40 regulate similar genes as those regulated by Isl1 and SSBP3 and are important for β-cell function. We observed that Rnf20 and Rnf40 depletion reduces β-cell H2Bub1 marks and uncovered several target genes, including glucose transporter 2 (Glut2), MAF BZIP transcription factor A (MafA), and uncoupling protein 2 (Ucp2). Strikingly, we also observed that Isl1 and SSBP3 depletion reduces H2Bub1 and H3K4me3 marks, suggesting that they have epigenetic roles. We noted that the RNF complex is required for glucose-stimulated insulin secretion and normal mitochondrial reactive oxygen species levels. These findings indicate that RNF20 and RNF40 regulate β-cell gene expression and insulin secretion and establish a link between Isl1 complexes and global cellular epigenetics.

Stem cell self-renewal and the daughter cell differentiation are tightly regulated by the respective niches, which produce extrinsic cues to support the proper development. In Drosophila ovary, Dpp is secreted from germline stem cell (GSC) niche and activates the BMP signaling in GSCs for their self-renewal. Escort cells (ECs) in differentiation niche restrict Dpp outside the GSC niche and extend protrusions to help with proper differentiation of the GSC daughter cells. Here we provide evidence that loss of large Maf transcriptional factor Traffic jam (Tj) blocks GSC progeny differentiation. Spatio-temporal specific knockdown experiments indicate that Tj is required in pre-adult EC lineage for germline differentiation control. Further molecular and genetic analyses suggest that the defective germline differentiation caused by tj-depletion is partly attributed to the elevated dpp in the differentiation niche. Moreover, our study reveals that tj-depletion induces ectopic En expression outside the GSC niche, which contributes to the upregulated dpp expression in ECs as well as GSC progeny differentiation defect. Alternatively, loss of EC protrusions and decreased EC number elicited by tj-depletion may also partially contribute to the germline differentiation defect. Collectively, our findings suggest that Tj in ECs regulates germline differentiation by controlling the differentiation niche characteristics.

Maf transcription factors are critical regulators of beta-cell function. We have previously shown that reduced MafA expression in human and mouse islets is associated with a pro-inflammatory gene signature. Here, we investigate if the loss of Maf transcription factors induced autoimmune processes in the pancreas. Transcriptomics analysis showed expression of pro-inflammatory as well as immune cell marker genes. However, clusters of CD4+ T and B220+ B cells were associated primarily with adult MafA−/−MafB+/−, but not MafA−/− islets. MafA expression was detected in the thymus, lymph nodes and bone marrow suggesting a novel role of MafA in regulating immune-cell function. Analysis of pancreatic lymph node cells showed activation of CD4+ T cells, but lack of CD8+ T cell activation which also coincided with an enrichment of naïve CD8+ T cells. Further analysis of T cell marker genes revealed a reduction of T cell receptor signaling gene expression in CD8, but not in CD4+ T cells, which was accompanied with a defect in early T cell receptor signaling in mutant CD8+ T cells. These results suggest that loss of MafA impairs both beta- and T cell function affecting the balance of peripheral immune responses against islet autoantigens, resulting in local inflammation in pancreatic islets.

Oxidative stress is a deteriorating factor for pancreatic β-cells under chronic hyperglycemia in diabetes. However, the molecular mechanism underlying the increase in oxidative stress in β-cells under diabetic conditions remains unclear. We demonstrated previously that the selective alleviation of glucotoxicity ameliorated the downregulation of several β-cell factors, including Cox6a2. Cox6a2 encodes a subunit of the respiratory chain complex IV in mitochondria. In this study, we analyzed the role of Cox6a2 in pancreatic β-cell function and its pathophysiological significance in diabetes mellitus. Cox6a2-knockdown experiments in MIN6-CB4 cells indicated an increased production of reactive oxygen species as detected by CellROX Deep Red reagent using flow cytometry. In systemic Cox6a2-knockout mice, impaired glucose tolerance was observed under a high-fat high-sucrose diet. However, insulin resistance was reduced when compared with control littermates. This indicates a relative insufficiency of β-cell function. To examine the transcriptional regulation of Cox6a2, ATAC-seq with islet DNA was performed and an open-chromatin area within the Cox6a2 enhancer region was detected. Reporter gene analysis using this area revealed that MafA directly regulates Cox6a2 expression. These findings suggest that the decreased expression of Cox6a2 increases the levels of reactive oxygen species and that Mafa is associated with decreased Cox6a2 expression under glucotoxic conditions.

Single cell RNA sequencing (scRNA-Seq) technologies have greatly enhanced our understanding of islet cell transcriptomes and have revealed the existence of β cell heterogeneity. However, comparison of scRNA-Seq datasets from different groups have highlighted inconsistencies in gene expression patterns, primarily due to variable detection of lower abundance transcripts. Furthermore, such analyses are unable to uncover the spatial organization of heterogeneous gene expression. Here we used fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) to quantify transcripts in single cells in mouse pancreatic islet sections. We compared the expression patterns of Insulin 2 (Ins2) with Mafa and Ucn3 – two genes expressed in β cells as they mature, as well as Rgs4 – a factor with variably reported expression in the islet. This approach accurately quantified transcripts across a wide range of expression levels - from single copies to over hundred copies per cell in one islet. Importantly, fliFISH allowed evaluation of transcript heterogeneity in the spatial context of an intact islet. These studies confirm the existence of a high degree of heterogeneous gene expression levels within the islet and highlight relative and radial expression patterns that likely reflect distinct β cell maturation states along the radial axis of the islet.

International audience; P43 is a truncated form of thyroid hormone receptor alpha localized in mitochondria, which stimulates mitochondrial respiratory chain activity. Previously, we showed that deletion of p43 led to reduction of pancreatic islet density and a loss of glucose-stimulated insulin secretion in adult mice. The present study was designed to determine whether p43 was involved in the processes of beta cell development and maturation. We used neonatal, juvenile, and adult p43-/- mice, and we analyzed the development of beta cells in the pancreas. Here, we show that p43 deletion affected only slightly beta cell proliferation during the postnatal period. However, we found a dramatic fall in p43-/- mice of MafA expression (V-Maf Avian Musculoaponeurotic Fibrosarcoma Oncogene Homolog A), a key transcription factor of beta-cell maturation. Analysis of the expression of antioxidant enzymes in pancreatic islet and 4-hydroxynonenal (4-HNE) (a specific marker of lipid peroxidation) staining revealed that oxidative stress occurred in mice lacking p43. Lastly, administration of antioxidants cocktail to p43-/- pregnant mice restored a normal islet density but failed to ensure an insulin secretion in response to glucose. Our findings demonstrated that p43 drives the maturation of beta cells via its induction of transcription factor MafA during the critical postnatal window.

The amplification of glucose-stimulated insulin secretion (GSIS) through incretin signaling is critical for maintaining physiological glucose levels. Incretins, like glucagon-like peptide 1 (GLP1), are a target of type 2 diabetes drugs aiming to enhance insulin secretion. Here we show that the protein phosphatase 1 inhibitor protein 1A (PPP1R1A), is expressed in β-cells and that its expression is reduced in dysfunctional β-cells lacking MafA and upon acute MafA knock down. MafA is a central regulator of GSIS and β-cell function. We observed a strong correlation of MAFA and PPP1R1A mRNA levels in human islets, moreover, PPP1R1A mRNA levels were reduced in type 2 diabetic islets and positively correlated with GLP1-mediated GSIS amplification. PPP1R1A silencing in INS1 (832/13) β-cells impaired GSIS amplification, PKA-target protein phosphorylation, mitochondrial coupling efficiency and also the expression of critical β-cell marker genes like MafA, Pdx1, NeuroD1 and Pax6. Our results demonstrate that the β-cell transcription factor MafA is required for PPP1R1A expression and that reduced β-cell PPP1R1A levels impaired β-cell function and contributed to β-cell dedifferentiation during type 2 diabetes. Loss of PPP1R1A in type 2 diabetic β-cells may explains the unresponsiveness of type 2 diabetic patients to GLP1R-based treatments.

Transcriptionally mature and immature β-cells co-exist within the adult islet. How such diversity contributes to insulin release remains poorly understood. Here we show that subtle differences in β-cell maturity, defined using PDX1 and MAFA expression, contribute to islet operation. Functional mapping of rodent and human islets containing proportionally more PDX1

Gamma-aminobutyric acid (GABA) administration attenuates streptozotocin (STZ)-induced diabetes in rodent models with unclear underlying mechanisms. We found that GABA and Sitagliptin possess additive effect on pancreatic β-cells, which prompted us to ask the existence of common or unique targets of GLP-1 and GABA in pancreatic β-cells. Effect of GABA on expression of thioredoxin-interacting protein (TxNIP) was assessed in the INS-1 832/13 (INS-1) cell line, WT and GLP-1R–/– mouse islets. GABA was also orally administrated in STZ-challenged WT or GLP-1R–/– mice, followed by immunohistochemistry assessment of pancreatic islets. Effect of GABA on Wnt pathway effector β-catenin (β-cat) was examined in INS-1 cells, WT and GLP-1R–/– islets. We found that GABA shares a common feature with GLP-1 on inhibiting TxNIP, while this function was attenuated in GLP-1R–/– islets. In WT mice with STZ challenge, GABA alleviated several ‘diabetic syndromes’, associated with increased β-cell mass. These features were virtually absent in GLP-1R–/– mice. Knockdown TxNIP in INS-1 cells increased GLP-1R, Pdx1, Nkx6.1 and Mafa levels, associated with increased responses to GABA or GLP-1 on stimulating insulin secretion. Cleaved caspase-3 level can be induced by high-glucose, dexamethasone, or STZ in INS-1 cell, while GABA treatment blocked the induction. Finally, GABA treatment increased cellular cAMP level and β-cat S675 phosphorylation in WT but not GLP-1R–/– islets. We, hence, identified TxNIP as a common target of GABA and GLP-1 and suggest that, upon STZ or other stress challenge, the GLP-1R-cAMP-β-cat signaling cascade also mediates beneficial effects of GABA in pancreatic β-cell, involving TxNIP reduction.

Limitation in the number of insulin-producing pancreatic β-cells is a typical feature of diabetes. It has been indicated that activating pancreatic transcription factors can promote the transformation of hepatocytes into insulin-secreting β-like cells, indicating that direct hepatocyte differentiation seems promising as a treatment for diabetes. Nevertheless, the reprogramming efficiency still remains low. Our previous study found that the expression of c-fos-induced growth factor (FIGF) was increased in the pancreatic tissues in partial pancreatectomy mice compared to that in normal mice. Here, we observed that treatment with Ad-FIGF was found to enhance MafA and Ngn3-induced reprogramming of BNL CL.2 cells to β-like cells with the ability of secreting insulin. And FIGF overexpression increased the levels of histone H3/H4 acetylation at MafA and Ngn3 promoter regions in BNL CL.2 cells. Importantly, in vivo study further confirmed that forced expression of FIGF facilitated the insulin expression and decreased the blood glucose levels in STZ mice. These results strengthen the possibility of developing cell-based therapies for diabetes through utilizing β-like cells derived from non-insulin-secreting cells.

β-Cell dysfunction and reduction in β-cell mass are hallmark events of diabetes mellitus. Here we show that β-cells express abundant Kindlin-2 and deleting its expression causes severe diabetes-like phenotypes without markedly causing peripheral insulin resistance. Kindlin-2, through its C-terminal region, binds to and stabilizes MafA, which activates insulin expression. Kindlin-2 loss impairs insulin secretion in primary human and mouse islets in vitro and in mice by reducing, at least in part, Ca2+ release in β-cells. Kindlin-2 loss activates GSK-3β and downregulates β-catenin, leading to reduced β-cell proliferation and mass. Kindlin-2 loss reduces the percentage of β-cells and concomitantly increases that of α-cells during early pancreatic development. Genetic activation of β-catenin in β-cells restores the diabetes-like phenotypes induced by Kindlin-2 loss. Finally, the inducible deletion of β-cell Kindlin-2 causes diabetic phenotypes in adult mice. Collectively, our results establish an important function of Kindlin-2 and provide a potential therapeutic target for diabetes., Beta cell dysfunction and reduction in beta cell mass are hallmark events in the pathogenesis of diabetes mellitus. We identify focal adhesion protein Kindlin-2 as a key factor that controls insulin synthesis and secretion and beta cell mass by modulating MafA and beta-catenin proteins in pancreatic beta cells.

The sustained expression of the MAFB transcription factor in human islet β-cells represents a distinct difference in mice. Moreover, mRNA expression of closely related and islet β-cell–enriched MAFA does not peak in humans until after 9 years of age. We show that the MAFA protein also is weakly produced within the juvenile human islet β-cell population and that MafB expression is postnatally restricted in mouse β-cells by de novo DNA methylation. To gain insight into how MAFB affects human β-cells, we developed a mouse model to ectopically express MafB in adult mouse β-cells using MafA transcriptional control sequences. Coexpression of MafB with MafA had no overt impact on mouse β-cells, suggesting that the human adult β-cell MAFA/MAFB heterodimer is functionally equivalent to the mouse MafA homodimer. However, MafB alone was unable to rescue the islet β-cell defects in a mouse mutant lacking MafA in β-cells. Of note, transgenic production of MafB in β-cells elevated tryptophan hydroxylase 1 mRNA production during pregnancy, which drives the serotonin biosynthesis critical for adaptive maternal β-cell responses. Together, these studies provide novel insight into the role of MAFB in human islet β-cells.

Zinc is integral for the normal function of pancreatic β-cells in glycaemic control. Large amounts of zinc are secreted from β-cells following insulin exocytosis and regulated replenishment is required, which is thought to be mediated by the ZIP family of zinc importer proteins. Within Type 2 Diabetic patients, β-cells are stressed through prolonged stimulation by hyperglycaemia and this is thought to be a major factor contributing to loss of β-cell identity and mass. However, the consequences for the β-cell zinc status remain largely unexplored. We used inductively coupled plasma mass spectrometry (ICP-MS) to show that 24 h treatment of MIN6 cells with potassium chloride, mimicking hyperglycaemic stimulation, reduces the total cellular zinc content 2.8-fold, and qPCR to show an increase in mRNA expression for metallothioneins (Mt1 and Mt2) following 4 and 24 h of stimulation, suggestive of an early rise in cytosolic zinc. To determine which ZIP paralogues may be responsible for zinc replenishment, we used immunocytochemistry, Western blot and qPCR to demonstrate initial ZIP1 protein upregulation proceeded by downregulation of mRNA coding for ZIP1, ZIP6, ZIP7 and ZIP14. To assign a biological significance to the decreased total cellular zinc content, we assessed expression of key β-cell markers to show downregulation of mRNA for MafA, Mnx-1, Nkx2.2 and Pax6. Our data suggest hyperglycaemia-induced zinc depletion may contribute to loss of β-cell markers and promote β-cell dedifferentiation through disrupting expression of key transcription factors.

Objective Pancreatic tissue, and islets in particular, are enriched in expression of the interleukin-1 receptor type I (IL-1R). Because of this enrichment, islet β-cells are exquisitely sensitive to the IL-1R ligands IL-1α and IL-1β, suggesting that signaling through this pathway regulates health and function of islet β-cells. Methods Herein, we report a targeted deletion of IL-1R in pancreatic tissue (IL-1RPdx1−/−) in C57BL/6J mice and in db/db mice on the C57 genetic background. Islet morphology, β-cell transcription factor abundance, and expression of the de-differentiation marker Aldh1a3 were analyzed by immunofluorescent staining. Glucose and insulin tolerance tests were used to examine metabolic status of these genetic manipulations. Glucose-stimulated insulin secretion was evaluated in vivo and in isolated islets ex vivo by perifusion. Results Pancreatic deletion of IL-1R leads to impaired glucose tolerance, a phenotype that is exacerbated by age. Crossing the IL-1RPdx1−/− with db/db mice worsened glucose tolerance without altering body weight. There were no detectable alterations in insulin tolerance between IL-1RPdx1−/− mice and littermate controls. However, glucose-stimulated insulin secretion was reduced in islets isolated from IL-1RPdx1−/− relative to control islets. Insulin output in vivo after a glucose challenge was also markedly reduced in IL-1RPdx1−/− mice when compared with littermate controls. Pancreatic islets from IL-1RPdx1−/− mice displayed elevations in Aldh1a3, a marker of de-differentiation, and reduction in nuclear abundance of the β-cell transcription factor MafA. Nkx6.1 abundance was unaltered. Conclusions There is an important physiological role for pancreatic IL-1R to promote glucose homeostasis by suppressing expression of Aldh1a3, sustaining MafA abundance, and supporting glucose-stimulated insulin secretion in vivo., Highlights • Pancreatic deletion of IL-1R impairs glucose tolerance in young and old male mice. • Pancreatic deletion of IL-1R worsens glucose tolerance in obese db/db mice. • Deletion of IL-1R triggers expression of the de-differentiation marker Aldh1a3. • IL-1 signaling in pancreatic tissue influences islet health and function.

Chronic exposure to arsenic, a potent environmental oxidative stressor, is associated with the incidence of diabetes. However, the mechanisms for arsenite-induced reduction of insulin remain largely unclear. After CD1 mice were treated with 20 or 40 ppm arsenite in the drinking water for 12 months, the mice showed reduced fasting insulin levels, a depression in glucose clearance, and lower insulin content in the pancreas. The levels of glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells isolated from arsenite-exposed mice were low compared with those for control mice. Immunohistochemistry studies showed that arsenite exposure resulted a reduction of insulin content in the pancreas of mice. Exposure of Min6 cells, a pancreatic beta cell line, to low levels of arsenite led to lower GSIS in a dose- and time-dependent fashion. Since microRNAs (miRNAs) are involved in pancreatic β-cell function and the pathogenesis of diabetes, we hypothesized that arsenite exposure activates miR-149, decreases insulin transcription factor v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (mafA), and induces an insulin synthesis and secretion disorder. In arsenite-exposed Min6 cells, mafA activity was lowered by the increase of its target miRNA, miR-149. Luciferase assays illustrated an interaction between miR-149 and the mafA 3' untranslated region. In Min6 cells transfected with an miR-149 inhibitor, arsenite did not regulate GSIS and mafA expression. In control cells, however, arsenite decreased GSIS or mafA expression. Our results suggest that low levels of arsenite affect β-cell function and regulate insulin synthesis and secretion by modulating mafA expression through miR-149.

Summary Human stem cells represent a potential source for implants that replace the depleted functional beta cell mass (FBM) in diabetes patients. Human embryonic stem cell-derived pancreatic endoderm (hES-PE) can generate implants with glucose-responsive beta cells capable of reducing hyperglycemia in mice. This study with device-encapsulated hES-PE (4 × 106 cells/mouse) determines the biologic characteristics at which implants establish metabolic control during a 50-week follow-up. A metabolically adequate FBM was achieved by (1) formation of a sufficient beta cell number (>0.3 × 106/mouse) at >50% endocrine purity and (2) their maturation to a functional state comparable with human pancreatic beta cells, as judged by their secretory responses during perifusion, their content in typical secretory vesicles, and their nuclear NKX6.1-PDX1-MAFA co-expression. Assessment of FBM in implants and its correlation with in vivo metabolic markers will guide clinical translation of stem cell-derived grafts in diabetes., Graphical Abstract, Highlights • Human stem cell-derived pancreatic precursors generate functional beta cell mass • Cellular markers identify metabolically adequate human stem cell-generated implants • Significance of determining beta cell number and maturation in implants • Functional implants differ in endocrine composition from endocrine pancreas, In this article, Pipeleers and colleagues demonstrate that subcutaneous implants of device-encapsulated human stem cell-derived pancreatic endoderm can generate a functional beta cell mass that establishes sustained glucose control in mice. They identified their biologic characteristics and correlation with in vivo outcome. Data and methods are expected to guide clinical translation to beta cell replacement therapy in diabetes.

Background/Aims: The islet is an important endocrine organ to secrete insulin to regulate the metabolism of glucose and maintain the stability of blood glucose. Long noncoding RNAs (lncRNAs) are involved in a variety of biological functions and play key roles in many diseases, including type 2 diabetes (T2D). The aim of this study was to determine whether lncRNA-p3134 is associated with glucose metabolism and insulin signaling in pancreatic β cells. Methods: LncRNA microarray technology was used to identify the differentially expressed circulating lncRNAs in T2D patients. RT-PCR analyses were performed to determine the expression of lncRNA-p3134 in 30 pairs of diabetic and non-diabetic patients. The correlation of lncRNA-p3134 to clinical data from T2D patients was analyzed. LncRNA-p3134 was overexpressed in Min6 cells and db/db mice by adenovirus-mediated technology. CCK-8, TUNEL, Western blot, glucose-stimulated insulin secretion (GSIS), ELISAs and immunochemistry were performed to determine the effect of lncRNA-p3134 on proliferation, apoptosis and insulin secretion both in vitro and vivo. Results: The circulating level of lncRNA-p3134 was higher in diabetic patients than in non-diabetic controls and was correlated with fasting blood glucose and HOMA-β levels. The lncRNA-p3134 had risen by 4 times in serum exosomes but nearly unchanged in exosome-free samples. The secretion of lncRNA-p3134 was dynamically modulated by glucose in both Min6 cells and isolated mouse islet cells. LncRNA-p3134 positively regulate GSIS through promoting of key regulators (Pdx-1, MafA, GLUT2 and Tcf7l2) in β cells. In addition, the overexpression of lncRNA-p3134 resulted in a decreased apoptosis ratio and partially reversed the glucotoxicity effects on GSIS function in Min6 cells. The restoration of insulin synthesis and secretion the increase of the insulin positive cells areas by upregulation of lncRNA-p3134 in db/db mice confirmed the compensatory role of lncRNA-p3134 to preserve β-cell function. Furthermore, a protective effect of lncRNA-p3134 on GSIS by positive modulation of PI3K/Akt/mTOR signaling was also confirmed. After blocking the PI3K/AKT signals with their specific inhibitor, the effect of overexpressed lncRNA-p3134 on insulin secretion was obviously attenuated. Conclusion: Taken together, the results of this study provide new insights into lncRNA-p3134 regulation in pancreatic β cells and provide a better understanding of novel mechanism of glucose homeostasis.

Background/Aims: The main pathogenic mechanism of diabetes is a decrease in the number of islet beta cells or a decline in their function. Recent studies have shown that pancreatic long noncoding RNAs (lncRNAs) have a high degree of tissue specificity and may be involved in the maintenance of islet cells function and the development of diabetes. The aim of this study was to investigate the molecular regulatory mechanism of mouse maternal expressed gene 3 (Meg3) in insulin biosynthesis in pancreatic islets. Methods: Chromatin immunoprecipitation–quantitative polymerase chain reaction (qPCR) and RNA immunoprecipitation–qPCR were used to investigate the molecular mechanism of lncRNA Meg3 in insulin biosynthesis by regulating v-Maf musculoaponeurotic fibrosarcoma oncogene family, protein A (MafA), a mature beta cell marker in the MIN6 beta cell line. Further, the expression levels of Meg3, Ezh2, MafA, Rad21, Smc3, and Sin3α were analyzed in vivo and in vitro by RT-PCR and western blotting. Results: Intranuclear lncRNA Meg3 can bind EZH2, a methyltransferase belonging to the Polycomb repressive complex-2, in pancreatic islet cells. In addition, knockdown of Ezh2 can also inhibit the expression of MafA and Ins2, while expression levels of Rad21, Smc3, and Sin3α are upregulated, by interfering with Ezh2 or Meg3 in pancreatic beta cells. Knockdown of Meg3 resulted in the loss of EZH2 binding and H3K27 trimethylation occupancy of Rad21, Smc3, and Sin3α promoter regions. The inhibition of Rad21, Smc3, or Sin3α, which directly act on the MafA promoter, leads to upregulated expression of MafA in both MIN6 cells and mouse islets. Moreover, the synthesis and secretion of insulin were increased by inhibition of these transcription factors. Conclusions: Pancreatic lncRNA Meg3 can epigenetically regulate the expression of Rad21, Smc3, and Sin3α via EZH2-driven H3K27 methylation. By inhibiting the expression of Rad21, Smc3, or Sin3α, Meg3 promotes the expression of MafA and affects the production of insulin.

A heterozygous missense mutation producing a variant of the islet β-cell-enriched MAFA transcription factor (Ser(S)64Phe(F) MAFA) was identified in humans who developed adult-onset, β-cell dysfunction (diabetes or insulinomatosis), with men more prone to diabetes. This mutation engenders increased stability to the normally unstable MAFA protein. To obtain insight into how this variant impacts β cell function, we developed a mouse model expressing S64F MafA and found sex-dependent phenotypes, with heterozygous mutant males displaying impaired glucose tolerance while females were slightly hypoglycemic with improved blood glucose clearance. Only heterozygous males showed transiently higher MafA protein levels preceding the onset of glucose intolerance and sex-dependent, differential expression of genes involved in calcium signaling, DNA damage, aging, and senescence. Functional changes in islet calcium handling and signs of islet aging and senescence processes were uniquely observed in male animals. In addition, S64F MAFA expression in human, male EndoC-βH2 β cells accelerated cellular senescence and increased production of senescence-associated secretory proteins compared to cells expressing wild-type MAFA. Together, these results implicate a conserved mechanism of accelerated islet aging and senescence in promoting diabetes in S64F MAFA carriers in a sex-dependent manner.

Cellular stress and proinflammatory cytokines induce phosphorylation of insulin receptor substrate (IRS) proteins at Ser sites that inhibit insulin and IGF-I signaling. We therefore examined the effects of mutation of five “inhibitory” Ser phosphorylation sites on IRS2 function in transgenic mice that overexpress, selectively in pancreatic β-cells, either wild-type (WT) or a mutated IRS2 protein (IRS25A). Islets size, number, and mRNA levels of catalase and superoxide dismutase were increased, whereas those of nitric oxide synthase were decreased, in 7- to 10-week-old IRS25A-β mice compared with IRS2WT-β mice. However, glucose homeostasis and insulin secretion in IRS25A-β mice were impaired when compared with IRS2WT-β mice or to nontransgenic mice. This was associated with reduced mRNA levels of Glut2 and islet β-cell transcription factors such as Nkx6.1 and MafA. Similarly, components mediating the unfolded protein response were decreased in islets of IRS25A-β mice in accordance with their decreased insulin secretion. The beneficial effects of IRS25A on β-cell proliferation and β-cell transcription factors were evident only in 5- to 8-day-old mice. These findings suggest that elimination of inhibitory Ser phosphorylation sites of IRS2 exerts short-term beneficial effects in vivo; however, their sustained elimination leads to impaired β-cell function.

Inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), statins, which are used to prevent cardiovascular diseases, are associated with a modest increase in the risk of new-onset diabetes. To investigate the role of HMGCR in the development of β-cells and glucose homeostasis, we deleted Hmgcr in a β-cell–specific manner by using the Cre-loxP technique. Mice lacking Hmgcr in β-cells (β-KO) exhibited hypoinsulinemic hyperglycemia as early as postnatal day 9 (P9) due to decreases in both β-cell mass and insulin secretion. Ki67-positive cells were reduced in β-KO mice at P9; thus, β-cell mass reduction was caused by proliferation disorder immediately after birth. The mRNA expression of neurogenin3 (Ngn3), which is transiently expressed in endocrine progenitors of the embryonic pancreas, was maintained despite a striking reduction in the expression of β-cell–associated genes, such as insulin, pancreatic and duodenal homeobox 1 (Pdx1), and MAF BZIP transcription factor A (Mafa) in the islets from β-KO mice. Histological analyses revealed dysmorphic islets with markedly reduced numbers of β-cells, some of which were also positive for glucagon. In conclusion, HMGCR plays critical roles not only in insulin secretion but also in the development of β-cells in mice.

Insulin plays a central role in glucose homeostasis and is produced exclusively by pancreatic islet β-cells. Insulin gene transcription is regulated by a set of β-cell-enriched transcription factors that bind to cis-regulatory elements within the promoter region, and regulation of the insulin gene promoter is closely linked to β-cell functionality. PIASy, a member of the PIAS family of SUMO E3 ligases, is thought to affect insulin gene transcription, but its mechanism of action is not fully understood. Here, we demonstrate that PIASy interacts with MafA and represses insulin gene promoter activity. MafA is a β-cell-restricted basic leucine-zipper transcriptional activator that binds to the C1 element of the insulin gene promoter. In line with previous studies showing the transactivator domain of MafA is SUMOylated, PIASy enhanced the SUMOylation of MafA. However, a SUMOylation-deficient mutant of MafA was still repressed by PIASy, indicating that this modification is dispensable for repression. Using a series of MafA and PIASy mutants, we found that the basic domain of MafA and the amino-terminal region of PIASy containing the SAP domain are necessary for their interaction. In addition, SUMO-interacting motif 1 (SIM1) at the carboxyl-terminal region of PIASy was required to repress the synergistic transactivation of MafA, Pdx1, and Beta2, transcription factors playing central roles in β-cell differentiation and function. The PINIT and SP-RING domains in the middle region of PIASy were dispensable. These findings suggest that PIASy binds to MafA through the SAP domain and negatively regulates the insulin gene promoter through a novel SIM1-dependent mechanism.

Imatinib (Gleevec) reverses type 1 diabetes (T1D) in NOD mice and is currently in clinical trials in individuals with recent-onset disease. While research has demonstrated that imatinib protects islet β cells from the harmful effects of ER stress, the role the immune system plays in its reversal of T1D has been less well understood, and specific cellular immune targets have not been identified. In this study, we demonstrate that B lymphocytes, an immune subset that normally drives diabetes pathology, are unexpectedly required for reversal of hyperglycemia in NOD mice treated with imatinib. In the presence of B lymphocytes, reversal was linked to an increase in serum insulin concentration, but not an increase in islet β cell mass or proliferation. However, improved β cell function was reflected by a partial recovery of MafA transcription factor expression, a sensitive marker of islet β cell stress that is important to adult β cell function. Imatinib treatment was found to increase the antioxidant capacity of B lymphocytes, improving reactive oxygen species (ROS) handling in NOD islets. This study reveals a novel mechanism through which imatinib enables B lymphocytes to orchestrate functional recovery of T1D β cells.

The pancreatic-islet-enriched transcription factors MafA and MafB have unique expression patterns in β cells in rodents. MafA is specifically expressed in β cells and is a key regulatory factor for maintaining adult β-cell function, whereas MafB plays an essential role in β-cell development during embryogenesis, and its expression in β cells gradually decreases and is restricted to α cells after birth in rodents., The pancreatic-islet-enriched transcription factors MafA and MafB have unique expression patterns in β cells in rodents. MafA is specifically expressed in β cells and is a key regulatory factor for maintaining adult β-cell function, whereas MafB plays an essential role in β-cell development during embryogenesis, and its expression in β cells gradually decreases and is restricted to α cells after birth in rodents. However, it was previously observed that MafB started to be reexpressed in insulin-positive (insulin+) β cells in MafA-deficient adult mice. To elucidate how MafB functions in the adult β cell under MafA-deficient conditions, we generated MafA and MafB double-knockout (A0B0) mice in which MafB was specifically deleted from β cells. As a result, the A0B0 mice became more vulnerable to diabetes under a high-fat diet (HFD) treatment, with impaired islet formation and a decreased number of insulin+ β cells because of increased β-cell apoptosis, indicating MafB can take part in the maintenance of adult β cells under certain pathological conditions.

Interneurons (INs) coordinate motoneuron activity to generate appropriate patterns of muscle contractions, providing animals with the ability to adjust their body posture and to move over a range of speeds. In Drosophila larvae several IN subtypes have been morphologically described and their function well documented. However, the general lack of molecular characterization of those INs prevents the identification of evolutionary counterparts in other animals, limiting our understanding of the principles underlying neuronal circuit organization and function. Here we characterize a restricted subset of neurons in the nerve cord expressing the Maf transcription factor Traffic Jam (TJ). We found that TJ+ neurons are highly diverse and selective activation of these different subtypes disrupts larval body posture and induces specific locomotor behaviors. Finally, we show that a small subset of TJ+ GABAergic INs, singled out by the expression of a unique transcription factors code, controls larval crawling speed., Spinal interneurons (IN) coordinate motoneuron activity to modulate locomotion behavior. Here, the authors characterize a subset of IN subtypes expressing the Maf transcription factor Traffic Jam (TJ) and report the distinct effects of their activation on body posture and locomotion in Drosophila larvae.

The glucose-6-phosphatase catalytic subunit 2 (G6PC2) gene encodes an islet-specific glucose-6-phosphatase catalytic subunit. G6PC2 forms a substrate cycle with glucokinase that determines the glucose sensitivity of insulin secretion. Consequently, deletion of G6pc2 lowers fasting blood glucose (FBG) without affecting fasting plasma insulin. Although chronic elevation of FBG is detrimental to health, glucocorticoids induce G6PC2 expression, suggesting that G6PC2 evolved to transiently modulate FBG under conditions of glucocorticoid-related stress. We show, using competition and mutagenesis experiments, that the synthetic glucocorticoid dexamethasone (Dex) induces G6PC2 promoter activity through a mechanism involving displacement of the islet-enriched transcription factor MafA by the glucocorticoid receptor. The induction of G6PC2 promoter activity by Dex is modulated by a single nucleotide polymorphism, previously linked to altered FBG in humans, that affects FOXA2 binding. A 5-day repeated injection paradigm was used to examine the chronic effect of Dex on FBG and glucose tolerance in wild-type (WT) and G6pc2 knockout mice. Acute Dex treatment only induces G6pc2 expression in 129SvEv but not C57BL/6J mice, but this chronic treatment induced G6pc2 expression in both. In 6-hour fasted C57BL/6J WT mice, Dex treatment lowered FBG and improved glucose tolerance, with G6pc2 deletion exacerbating the decrease in FBG and enhancing the improvement in glucose tolerance. In contrast, in 24-hour fasted C57BL/6J WT mice, Dex treatment raised FBG but still improved glucose tolerance, with G6pc2 deletion limiting the increase in FBG and enhancing the improvement in glucose tolerance. These observations demonstrate that G6pc2 modulates the complex effects of Dex on both FBG and glucose tolerance.

β-Cells rely on the islet microenvironment for their functionality and mass. Pericytes, along with endothelial cells, make up the dense islet capillary network. However, although the role of endothelial cells in supporting β-cell homeostasis has been vastly investigated, the role of pericytes remains largely unknown. Here, we focus on contribution of pericytes to β-cell function. To this end, we used a transgenic mouse system that allows diphtheria toxin–based depletion of pericytes. Our results indicate that islets depleted of their pericytes have reduced insulin content and expression. Additionally, isolated islets displayed impaired glucose-stimulated insulin secretion, accompanied by a reduced expression of genes associated with β-cell function. Importantly, reduced levels of the transcription factors MafA and Pdx1 point to β-cell dedifferentiation in the absence of pericytes. Ex vivo depletion of pericytes in isolated islets resulted in a similar impairment of gene expression, implicating their direct, blood flow–independent role in maintaining β-cell maturity. To conclude, our findings suggest that pericytes are pivotal components of the islet niche, which are required for β-cell maturity and functionality. Abnormalities of islet pericytes, as implicated in type 2 diabetes, may therefore contribute to β-cell dysfunction and disease progression.

Type 2 diabetes (T2D) is associated with pancreatic β-cell dysfunction, manifested by reduced glucose-stimulated insulin secretion (GSIS). Several transcription factors enriched in β-cells, such as MafA, control β-cell function by organizing genes involved in GSIS. Here we demonstrate that nardilysin (N-arginine dibasic convertase; Nrd1 and NRDc) critically regulates β-cell function through MafA. Nrd1−/− mice showed glucose intolerance and severely decreased GSIS. Islets isolated from Nrd1−/− mice exhibited reduced insulin content and impaired GSIS in vitro. Moreover, β-cell-specific NRDc-deficient (Nrd1delβ) mice showed a diabetic phenotype with markedly reduced GSIS. MafA was specifically downregulated in islets from Nrd1delβ mice, whereas overexpression of NRDc upregulated MafA and insulin expression in INS832/13 cells. Chromatin immunoprecipitation assay revealed that NRDc is associated with Islet-1 in the enhancer region of MafA, where NRDc controls the recruitment of Islet-1 and MafA transcription. Our findings demonstrate that NRDc controls β-cell function via regulation of the Islet-1–MafA pathway.

SUMMARY Monoamine and acetylcholine neurotransmitters from the autonomic nervous system (ANS) regulate insulin secretion in pancreatic islets. The molecular mechanisms controlling neurotransmitter signaling in islet β cells and their impact on diabetes development are only partially understood. Using a glucose-intolerant, MafA-deficient mouse model, we demonstrate that MAFA controls ANS-mediated insulin secretion by activating the transcription of nicotinic (ChrnB2 and ChrnB4) and adrenergic (Adra2A) receptor genes, which are integral parts of acetylcholine-and monoamine-signaling pathways. We show that acetylcholine-mediated insulin secretion requires nicotinic signaling and that nicotinic receptor expression is positively correlated with insulin secretion and glycemic control in human donor islets. Moreover, polymorphisms spanning MAFA-binding regions within the human CHRNB4 gene are associated with type 2 diabetes. Our data show that MAFA transcriptional activity is required for establishing β cell sensitivity to neurotransmitter signaling and identify nicotinic signaling as a modulator of insulin secretion impaired in type 2 diabetes., Graphical abstract

Diabetes mellitus is an increasingly severe chronic metabolic disease that is occurring at an alarming rate worldwide. Various diabetic models, including non-obese diabetic mice and chemically induced diabetic models, are used to characterize and explore the mechanism of the disease&rsquo, s pathophysiology, in hopes of detecting and identifying novel potential therapeutic targets. However, this is accompanied by disadvantages, such as specific conditions for maintaining the incidence, nonstable hyperglycemia induction, and potential toxicity to other organs. Murine MAFA and MAFB, two closely-linked islet-enriched transcription factors, play fundamental roles in glucose sensing and insulin secretion, and maintenance of pancreatic &beta, cell, respectively, which are highly homologous to human protein orthologs. Herein, to induce the diabetes mellitus model at a specific time point, we generated Pdx1-dependent Mafb-deletion mice under Mafa knockout condition (A0B&Delta, panc), via tamoxifen-inducible Cre-loxP system. After 16 weeks, metabolic phenotypes were characterized by intraperitoneal glucose tolerance test (IPGTT), urine glucose test, and metabolic parameters analysis. The results indicated that male A0B&Delta, panc mice had obvious impaired glucose tolerance, and high urine glucose level. Furthermore, obvious renal lesions, impaired islet structure and decreased proportion of insulin positive cells were observed. Collectively, our results indicate that A0B&Delta, panc mice can be an efficient inducible model for diabetes research.

To investigate the effect of long non-coding RNA (lncRNA) HOTTIP on islet β cells and its underlying mechanism.The expressions of HOTTIP in different organs of db/db mice and C57BL/6J mice were detected by quantitative Real-time polymerase chain reaction (qRT-PCR). Effects of HOTTIP on the proliferation, insulin secretion and apoptosis of islet β cells transfected with lentivirus were detected by cell counting kit-8 (CCK-8) assay, enzyme-linked immunosorbent assay (ELISA) and flow cytometry, respectively. We also assessed the protein expressions of key genes in MEK/ERK pathway by using Western blot.HOTTIP was upregulated in normal islet tissues of C57BL/6J mice but downregulated in islet tissues of diabetic mice. Inhibition of HOTTIP attenuated insulin secretion and reduced expressions of Pdx1 and MafA. Downregulation of HOTTIP also inhibited cell proliferation and reduced expressions of CyclinDl, CyclinD2, CyclinE1 and CyclinE2. Moreover, islet β cells were arrested in G0/G1 phase after HOTTIP knockdown. Our data showed that the biological function of HOTTIP in regulating insulin secretion and cell cycle in islet β cells might be related to the MEK/ERK pathway.Downregulation of HOTTIP inhibits insulin secretion and cell cycle in islet β cells via MEK/ERK pathway.

Objective: To investigate the effect of dapagliflozin on intestinal microflora in MafA-deficient mice using an animal model of diabetes. Methods: Male MafA-deficient mice were administered dapagliflozin (1.0 mg/kg/d) intragastrically for 6 weeks. Mouse body weights and fasting blood glucose levels were measured, and intestinal short-chain fatty acids were measured by gas chromatography. A series of methods was used to analyse the number of primary harmful bacteria in the faeces, and high-throughput sequencing was used to sequence the changes in intestinal flora. Results: The weight of the mice decreased after dapagliflozin gavage, and fasting blood glucose was significantly lower than that in the control group (P < 0.001). Acetic acid and butyric acid contents in the intestinal tracts of the mice increased, and the growth of harmful microorganisms, such as Clostridium perfringens, enterococci, Enterobacteriaceae, and intestinal enterococci, was inhibited. Blautia is a species found in the experimental group and was significantly different from the control and blank groups as determined by the LDA score from highthroughput sequencing. Conclusion: Dapagliflozin can reduce fasting blood glucose, decrease body weight, increase short-chain fatty acid content, regulate the intestinal microecological balance of the body and promote blood glucose and energy homeostasis.

Summary Generation of functional β cells from pluripotent sources would accelerate diagnostic and therapeutic applications for diabetes research and therapy. However, it has been challenging to generate competent β cells with dynamic insulin-secretory capacity to glucose and incretin stimulations. We introduced transcription factors, critical for β-cell development and function, in differentiating human induced pluripotent stem cells (PSCs) and assessed the impact on the functionality of derived β-cell (psBC) progeny. A perifusion system revealed stepwise transduction of the PDX1, NEUROG3, and MAFA triad (PNM) enabled in vitro generation of psBCs with glucose and GLP-1 responsiveness within 3 weeks. PNM transduction upregulated genes associated with glucose sensing, insulin secretion, and β-cell maturation. In recipient diabetic mice, PNM-transduced psBCs showed glucose-responsive insulin secretion as early as 1 week post transplantation. Thus, enhanced pre-emptive β-cell specification of PSCs by PNM drives generation of glucose- and incretin-responsive psBCs in vitro, offering a competent tissue-primed biotherapy., Highlights • PNM transduction facilitates generation of glucose- and GLP1-responsive β cells • Stepwise PNM improves glucose sensing, insulin secretion, and β-cell maturation • PNM-transduced psBCs showed glucose-responsive insulin secretion in diabetic mice, In this article, Ikeda Yasuhiro and colleagues show that stepwise transduction of the triad of transcription factors PDX1, NEUROG3, and MAFA (PNM) enabled in vitro generation of glucose- and GLP-1-responsive β cells from iPSCs within 3 weeks. PNM transduction improves glucose sensing, insulin secretion, and β-cell maturation of generated cells. PNM-transduced β cells showed glucose-responsive insulin secretion as early as 1 week post transplantation in diabetic mice.

To examine the effects of luseogliflozin, a sodium–glucose cotransporter 2 inhibitor, on pancreatic beta cell mass in db/db mice of different ages. db/db mice aged 6, 10, 14 and 24 weeks old were fed either standard chow (control group) or standard chow containing 0.01% luseogliflozin (luseo group). After 4 weeks, immunohistochemistry and gene expression tests were conducted. In 6-week-old db/db mice, immunohistochemistry revealed a significant increase in beta cell mass in the luseo group compared with the control group after 4 weeks of treatment. Gene expression profiling of isolated islets showed upregulation Mafa, Pdx1, Ki67 and Ccnd2 in the luseo group. Beta cell mass decreased with age in db/db mice in the control group. Beta cell mass in the luseo group significantly increased compared with the control group regardless of age, although beta cell mass in the 28-week-old luseo group (4 weeks of treatment in 24-week-old db/db mice) was significantly lower than in the 10-week-old luseo group (4 weeks of treatment in 6-week-old db/db mice). Luseogliflozin preserved beta cell mass in db/db mice. The protective effect was more evident in the earlier phase of diabetes.

Direct lineage reprogramming can convert readily available cells in the body into desired cell types for cell replacement therapy. This is usually achieved through forced activation or repression of lineage-defining factors or pathways. In particular, reprogramming toward the pancreatic β cell fate has been of great interest in the search for new diabetes therapies. It has been suggested that cells from various endodermal lineages can be converted to β-like cells. However, it is unclear how closely induced cells resemble endogenous pancreatic β cells and whether different cell types have the same reprogramming potential. Here, we report in vivo reprogramming of pancreatic ductal cells through intra-ductal delivery of an adenoviral vector expressing the transcription factors Pdx1, Neurog3, and Mafa. Induced β-like cells are mono-hormonal, express genes essential for β cell function, and correct hyperglycemia in both chemically and genetically induced diabetes models. Compared with intrahepatic ducts and hepatocytes treated with the same vector, pancreatic ducts demonstrated more rapid activation of β cell transcripts and repression of donor cell markers. This approach could be readily adapted to humans through a commonly performed procedure, endoscopic retrograde cholangiopancreatography (ERCP), and provides potential for cell replacement therapy in type 1 diabetes patients.

Previously, we showed that thyroid hormone (TH) triiodothyronine (T(3)) enhanced β-cell functional maturation through induction of Mafa. High levels of T(3) have been linked to decreased life span in mammals and low levels to lengthened life span, suggesting a relationship between TH and aging. Here, we show that T(3) increased p16(Ink4a) (a β-cell senescence marker and effector) mRNA in rodent and human β-cells. The kinetics of Mafa and p16(Ink4a) induction suggested both genes as targets of TH via TH receptors (THRs) binding to specific response elements. Using specific agonists CO23 and GC1, we showed that p16(Ink4a) expression was controlled by THRA and Mafa by THRB. Using chromatin immunoprecipitation and a transient transfection yielding biotinylated THRB1 or THRA isoforms to achieve specificity, we determined that THRA isoform bound to p16(Ink4a), whereas THRB1 bound to Mafa but not to p16(Ink4a). On a cellular level, T(3) treatment accelerated cell senescence as shown by increased number of β-cells with acidic β-galactosidase activity. Our data show that T(3) can simultaneously induce both maturation (Mafa) and aging (p16(Ink4a)) effectors and that these dichotomous effects are mediated through different THR isoforms. These findings may be important for further improving stem cell differentiation protocols to produce functional β-cells for replacement therapies in diabetes.

Glucose metabolism is regulated by insulin, which is produced from β-cells in the pancreas. Because insulin is secreted into vessels in response to blood glucose, vascular structures of the pancreas, especially the relationship between vessels and β-cells, are important for physiological and pathological glucose metabolism. Here, we developed a system to visualize vessels surrounding mature β-cells expressing transcription factor MafA in a three-dimensional manner. Optical clearing of the pancreas prevented light scattering of fluorescence driven by the bacterial artificial chromosome (BAC)-mafA promoter in β-cells. Reconstruction of confocal images demonstrated mature β-cells and the glomerular-like structures of β-cell vasculatures labeled with DyLight 488-conjugated lectin in normal mice as well as in low-dose streptozotocin-injected diabetes model mice with reduced β-cell mass. This technological innovation of organ imaging can be used to investigate morphological changes in vascular structures during transplantation, regeneration and diabetes development.

Insulin produced by islet β-cells plays a critical role in glucose homeostasis, with type 1 and type 2 diabetes both resulting from inactivation and/or loss of this cell population. Islet-enriched transcription factors regulate β-cell formation and function, yet little is known about the molecules recruited to mediate control. An unbiased in-cell biochemical and mass spectrometry strategy was used to isolate MafA transcription factor–binding proteins. Among the many coregulators identified were all of the subunits of the mixed-lineage leukemia 3 (Mll3) and 4 (Mll4) complexes, with histone 3 lysine 4 methyltransferases strongly associated with gene activation. MafA was bound to the ∼1.5 MDa Mll3 and Mll4 complexes in size-fractionated β-cell extracts. Likewise, closely related human MAFB, which is important to β-cell formation and coproduced with MAFA in adult human islet β-cells, bound MLL3 and MLL4 complexes. Knockdown of NCOA6, a core subunit of these methyltransferases, reduced expression of a subset of MAFA and MAFB target genes in mouse and human β-cell lines. In contrast, a broader effect on MafA/MafB gene activation was observed in mice lacking NCoA6 in islet β-cells. We propose that MLL3 and MLL4 are broadly required for controlling MAFA and MAFB transactivation during development and postnatally.

The murine Mafa transcription factor is a key regulator of postnatal islet β-cell activity, affecting insulin transcription, insulin secretion, and β-cell mass. Human MAFA expression is also markedly decreased in islet β-cells of type 2 diabetes mellitus (T2DM) patients. Moreover, levels are profoundly reduced in db/db islet β-cells, a mouse model of T2DM. To examine the significance of this key islet β-cell-enriched protein to glycemic control under diabetic conditions, we generated transgenic mice that conditionally and specifically produced Mafa in db/db islet β-cells. Sustained expression of Mafa resulted in significantly lower plasma glucose levels, higher plasma insulin, and augmented islet β-cell mass. In addition, there was increased expression of insulin, Slc2a2, and newly identified Mafa-regulated genes involved in reducing β-cell stress, like Gsta1 and Gckr. Importantly, the levels of human GSTA1 were also compromised in T2DM islets. Collectively, these results illustrate how consequential the reduction in Mafa activity is to islet β-cell function under pathophysiological conditions.

SummaryAlthough persistent elevations in circulating glucose concentrations promote compensatory increases in pancreatic islet mass, unremitting insulin resistance causes deterioration in beta cell function that leads to the progression to diabetes. Here, we show that mice with a knockout of the CREB coactivator CRTC2 in beta cells have impaired oral glucose tolerance due to decreases in circulating insulin concentrations. CRTC2 was found to promote beta cell function in part by stimulating the expression of the transcription factor MafA. Chronic hyperglycemia disrupted cAMP signaling in pancreatic islets by activating the hypoxia inducible factor (HIF1)-dependent induction of the protein kinase A inhibitor beta (PKIB), a potent inhibitor of PKA catalytic activity. Indeed, disruption of the PKIB gene improved islet function in the setting of obesity. These results demonstrate how crosstalk between nutrient and hormonal pathways contributes to loss of pancreatic islet function.

The members of the MAF family of transcription factors are homologs of v-Maf -the oncogenic component of the avian retrovirus AS42. The MAF family is subdivided into 2 groups, small and large MAFs. To elucidate the role of the large MAF transcription factors in the endocrine pancreas, we analyzed large MAF gene knockout mice. It has been shown that Mafa(-/-) mice develop phenotypes including abnormal islet structure soon after birth. This study revealed that Ins1 and Ins2 transcripts and the protein contents were significantly reduced in Mafa(-/-) mice at embryonic day 18.5. In addition, Mafa(-/-);Mafb(-/-) mice contained less than 10% of the insulin transcript and protein of those of wild-type mice, suggesting that Mafa and Mafb cooperate to maintain insulin levels at the embryonic stage. On the other hand, the number of insulin-positive cells in Mafa(-/-) mice was comparable to that of wild-type mice, and even under a Mafb-deficient background the number of insulin-positive cells was not decreased, suggesting that Mafb plays a dominant role in embryonic β-cell development. We also found that at 20 weeks of age Mafa(-/-);Mafb(+/-) mice showed a higher fasting blood glucose level than single Mafa(-/-) mice. In summary, our results indicate that Mafa is necessary for the maintenance of normal insulin levels even in embryos and that Mafb is important for the maintenance of fasting blood glucose levels in the Mafa-deficient background in adults.

MafA and MafB are basic leucine zipper transcription factors expressed in mature pancreatic β- and α-cells, respectively. MafA is not only an insulin gene transcription factor but is also critical for the maturation and maintenance of β-cell function, whereas MafB is expressed in immature β-cells during development and in compromised β-cells in diabetes. In this study, we developed a mouse model to easily trace the promoter activity of MafA in β-cells as a tool for studying β-cell differentiation, maturation, regeneration and function using the expression of the fluorescent protein Kusabira Orange (KOr) driven by the BAC-mafA promoter. The expression of KOr was highly restricted to β-cells in the transgenic pancreas. By crossing MafA-KOr mice with MafB(GFP/+) reporter mice, simultaneous monitoring of MafA and MafB expressions in the isolated islets was successfully performed. This system can be a useful tool for examining dynamic changes in the differentiation and function of pancreatic islets by visualizing the expressions of MafA and MafB.

Transcription factors regulate gene expression through binding to specific enhancer sequences. Pancreas/duodenum homeobox protein 1 (PDX1), Neurogenin-3 (NEUROG3), and V-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MAFA) are transcription factors critical for beta cell development and maturation. NEUROG3 is expressed in endocrine progenitor cells and controls islet differentiation and regeneration. PDX1 is essential for the development of pancreatic exocrine and endocrine cells including beta cells. PDX1 also binds to the regulatory elements and increases insulin gene transcription. Likewise, MAFA binds to the enhancer/promoter region of the insulin gene and drives insulin expression in response to glucose. In addition to those natural roles in beta cell development and maturation, ectopic expression of PDX1, NEUROG3, and/or MAFA has been successfully used to reprogram various cell types into insulin-producing cells in vitro and in vivo, such as pancreatic exocrine cells, hepatocytes, and pluripotent stem cells. Here, we review biological properties of PDX1, NEUROG3, and MAFA, and their applications and limitations for beta cell regenerative approaches. The primary source literature for this review was acquired using a PubMed search for articles published between 1990 and 2017. Search terms include diabetes, insulin, trans-differentiation, stem cells, and regenerative medicine.

Adenoviral gene transfer of key β cell developmental regulators including Pdx1, Neurod1, and Mafa (PDA) has been reported to generate insulin-producing cells in the liver. However, PDA insulin secretion is transient and glucose unresponsive. Here, we report that an additional β cell developmental regulator, insulin gene enhancer binding protein splicing variant (Isl1β), improved insulin production and glucose-responsive secretion in PDA mice. Microarray gene expression analysis suggested that adenoviral PDA transfer required an additional element for mature β cell generation, such as Isl1 and Elf3 in the liver. In vitro promoter analysis indicated that splicing variant Isl1, or Isl1β, is an important factor for transcriptional activity of the insulin gene. In vivo bioluminescence monitoring using insulin promoter-luciferase transgenic mice verified that adenoviral PDA + Isl1β transfer produced highly intense luminescence from the liver, which peaked at day 7 and persisted for more than 10 days. Using insulin promoter-GFP transgenic mice, we further confirmed that Isl1β supplementation to PDA augmented insulin-producing cells in the liver, insulin production and secretion, and β cell‒related genes. Finally, the PDA + Isl1β combination ameliorated hyperglycemia in diabetic mice for 28 days and enhanced glucose tolerance and responsiveness. Thus, our results suggest that Isl1β is a key additional transcriptional factor for advancing the generation of insulin-producing cells in the liver in combination with PDA.