PDX1 Methylation, NGN3 and Pax6 Expression Levels in Pregnant Women with Gestational Diabetes Mellitus and their association with neonatal blood sugars and birth weight.

Objective: To investigate the correlation of maternal PDX1 methylation, NGN3 and Pax6 expression levels with neonatal blood sugars and birth weight in pregnant women with GDM and non GDM. Methods: This was a prospective cohort study. Total 80 pregnant women who were examined and delivered in the Department of Obstetrics of Affiliated Hospital of Hebei University from January 2019 to June 2022 were recruited and divided into two groups according to the results of oral glucose tolerance test (OGTT): the control group and the observation group, with 40 cases in each group. PDXl methylation rate was measured by the methylation-specific PCR method, whereas gene expression levels of PDX1, NGN3 and Pax6 were measured by RT-PCR meanwhile, neonatal blood glucose and hemoglobin A1c (HbA1c) levels were also measured. Results: In comparison with the control group, the observation group had higher levels of FBG, 2-hour postprandial blood glucose (2hPBG) and HbA1c (P<0.05). Neonatal birth weight and insulin levels in the observation group were significantly higher than those in the control group, while Apgar scores and blood glucose were lower than those in the control group(P<0.05). Moreover, the observation group had significantly lower gene expression levels of PDX1, NGN3 and Pax6, and a higher PDX1 methylation rate than the control group (P<0.05). Correlation analysis revealed a negative correlation between neonatal blood glucose levels and PDX1, NGN3 and Pax6 levels in the observation group, with statistical significance (P<0.05). Conclusion: Changes in maternal PDX1 methylation, NGN3 and Pax6 expression levels may lead to abnormal glucose metabolism in neonates, which has a close bearing on neonatal hypoglycemia and blood glucose levels caused by GDM.

FTZ promotes islet ß-cell regeneration in T1DM mice via the regulation of nuclear proliferation factors.

ETHNOPHARMACOLOGICAL RELEVANCE: Fufang-Zhenzhu-Tiaozhi capsule (FTZ), a Traditional Chinese Medicine (TCM) patent prescription commonly used in clinical practice, has a significant curative effect on hyperglycemia and hyperlipidemia. Previous studies have shown that FTZ can treat diabetes, but the effect of FTZ on ß-cell regeneration needs to be further explored in T1DM mice. AIM OF THE STUDY: The aim is to investigate the role of FTZ in promoting ß-cell regeneration in T1DM mice, and to further explore its mechanism. MATERIALS AND METHODS: C57BL/6 mice were used as control. NOD/LtJ mice were divided into the Model group and FTZ group. Oral glucose tolerance, fasting blood glucose, and fasting insulin level were measured. Immunofluorescence staining was used to detect the level of ß-cell regeneration and the composition of α-cells and ß-cells in islets. Hematoxylin and eosin staining was used to detect the infiltration degree of inflammatory cells. The apoptosis of islet cells was detected by terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling. Western blotting was used to detect the expression levels of Pancreas/duodenum homeobox protein 1 (PDX-1), V-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MAFA), and Neurogenin-3 (NGN3). RESULTS: FTZ could increase insulin levels and reduce the glucose level of T1DM mice and promote ß-cell regeneration. FTZ also inhibited the invasion of inflammatory cells and the islet cell apoptosis, and maintained the normal composition of islet cells, thus preserving the quantity and quality of ß-cells. Furthermore, FTZ promoting ß-cell regeneration was accompanied by increasing the expression of PDX-1, MAFA, and NGN3. CONCLUSION: FTZ can restore the insulin-secreting function of the impaired pancreatic islet, improve blood glucose level, possibly via the enhancing ß cell regeneration via upregulation of PDX-1, MAFA, and NGN3 in T1DM mice, and may be a potential therapeutic drug for T1DM.

Kif21B mediates the effect of estradiol on the morphological plasticity of mouse hippocampal neurons.

Introduction: Neurons are polarized cells, and their ability to change their morphology has a functional implication in the development and plasticity of the nervous system in order to establish new connections. Extracellular factors strongly influence neuronal shape and connectivity. For instance, the developmental actions of estradiol on hippocampal neurons are well characterized, and we have demonstrated in previous studies that Ngn3 mediates these actions. On the other hand, Kif21B regulates microtubule dynamics and carries out retrograde transport of the TrkB/brain-derived neurotrophic factor (BDNF) complex, essential for neuronal development. Methods: In the present study, we assessed the involvement of kinesin Kif21B in the estradiol-dependent signaling mechanisms to regulate neuritogenesis through cultured mouse hippocampal neurons. Results: We show that estradiol treatment increases BDNF expression, and estradiol and BDNF modify neuron morphology through TrkB signaling. Treatment with K252a, a TrkB inhibitor, decreases dendrite branching without affecting axonal length, whereas. Combined with estradiol or BDNF, it blocks their effects on axons but not dendrites. Notably, the downregulation of Kif21B abolishes the actions of estradiol and BDNF in both the axon and dendrites. In addition, Kif21B silencing also decreases Ngn3 expression, and downregulation of Ngn3 blocks the effect of BDNF on neuron morphology. Discussion: These results suggest that Kif21B is required for the effects of estradiol and BDNF on neuronal morphology, but phosphorylation-mediated activation of TrkB is essential only for axonal growth. Our results show that the Estradiol/BDNF/TrkB/Kif21B/Ngn3 is a new and essential pathway mediating hippocampal neuron development.

Fufang-zhenzhu-tiaozhi formula protects islet against injury and promotes ß cell regeneration in diabetic mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Fufang-zhenzhu-tiaozhi formula (FTZ) is a patented preparation of traditional Chinese medicine that has been used to treat hyperglycemia and hyperlipidemia in the clinic for almost 10 years. Our previous study had demonstrated that FTZ can protect islet ß cell injury in vitro. However, the efficacy of FTZ on ß cell regeneration in vivo and the involved anti-diabetic mechanism remains unknown. AIM OF THE STUDY: We aim to investigate the effects of FTZ as a good remedy for islet protection and ß cell regeneration, and to reveal the underlying mechanism. MATERIALS AND METHODS: C57BL/6 mice were fed with high-fat diet for 3 weeks and then intraperitoneally injected with streptozotocin (90 mg/kg/d × 1 d) to establish type 2 diabetes (T2D) models. Mice in each group were divided into three batches that sacrificed after 3, 7 and 28 days of FTZ administration. Body weight, blood glucose, and oral glucose tolerance test were measured at indicated time points. Fasting insulin was determined by enzyme-linked immunosorbent assay (ELISA) kit. Neonatal ß cell was assessed by insulin & PCNA double immunofluorescence staining, and the underlying mechanisms related to ß cell regeneration were further performed by hematoxylin-eosin staining, insulin & glucagon double immunofluorescence staining and Western blot. RESULTS: FTZ and metformin can significantly help with the symptoms of DM, such as alleviating weight loss, reducing blood glucose, improving the level of insulin in vivo, and relieving insulin resistance, suggesting FTZ and metformin treatment maintained the normal morphological function of islet. Notably, ß cell regeneration, which is indicated by insulin and PCNA double-positive cells, was promoted by FTZ, whereas few neonatal ß cells were observed in metformin group. Hematoxylin-eosin staining, and its quantification results showed that FTZ effectively prevented the invasion of inflammatory cells into the islets in diabetic mice. Most ß cells in the islets of diabetic model mice were devoid, and the islets were almost all α cells, while the diabetic mice administered FTZ could still maintain about half of the ß cells in the islet. Furthermore, FTZ upregulated the expression of critical transcription factors during ß cell development and maturation (such as PDX-1, MAFA and NGN3) in diabetic mice. CONCLUSIONS: FTZ can alleviate diabetes symptoms and promote ß cell regeneration in diabetic mice. Moreover, FTZ promotes ß cell regeneration by preserving islet (resisting inflammatory cells invading islets), maintaining the number of ß cells in islets, and increasing the expression of PDX-1, MAFA and NGN3.

Epigenetic modifier Kdm6a/Utx controls the specification of hypothalamic neuronal subtypes in a sex-dependent manner.

Kdm6a is an X-chromosome-linked H3K27me2/3 demethylase that promotes chromatin accessibility and gene transcription and is critical for tissue/cell-specific differentiation. Previous results showed higher Kdm6a levels in XX than in XY hypothalamic neurons and a female-specific requirement for Kdm6a in mediating increased axogenesis before brain masculinization. Here, we explored the sex-specific role of Kdm6a in the specification of neuronal subtypes in the developing hypothalamus. Hypothalamic neuronal cultures were established from sex-segregated E14 mouse embryos and transfected with siRNAs to knockdown Kdm6a expression (Kdm6a-KD). We evaluated the effect of Kdm6a-KD on Ngn3 expression, a bHLH transcription factor regulating neuronal sub-specification in hypothalamus. Kdm6a-KD decreased Ngn3 expression in females but not in males, abolishing basal sex differences. Then, we analyzed Kdm6a-KD effect on Ascl1, Pomc, Npy, Sf1, Gad1, and Th expression by RT-qPCR. While Kdm6a-KD downregulated Ascl1 in both sexes equally, we found sex-specific effects for Pomc, Npy, and Th. Pomc and Th expressed higher in female than in male neurons, and Kdm6a-KD reduced their levels only in females, while Npy expressed higher in male than in female neurons, and Kdm6a-KD upregulated its expression only in females. Identical results were found by immunofluorescence for Pomc and Npy neuropeptides. Finally, using ChIP-qPCR, we found higher H3K27me3 levels at Ngn3, Pomc, and Npy promoters in male neurons, in line with Kdm6a higher expression and demethylase activity in females. At all three promoters, Kdm6a-KD induced an enrichment of H3K27me3 only in females. These results indicate that Kdm6a plays a sex-specific role in controlling the expression of transcription factors and neuropeptides critical for the differentiation of hypothalamic neuronal populations regulating food intake and energy homeostasis.

X-linked histone H3K27 demethylase Kdm6a regulates sexually dimorphic differentiation of hypothalamic neurons.

Several X-linked genes are involved in neuronal differentiation and may contribute to the generation of sex dimorphisms in the brain. Previous results showed that XX hypothalamic neurons grow faster, have longer axons, and exhibit higher expression of the neuritogenic gene neurogenin 3 (Ngn3) than XY before perinatal masculinization. Here we evaluated the participation of candidate X-linked genes in the development of these sex differences, focusing mainly on Kdm6a, a gene encoding for an H3K27 demethylase with functions controlling gene expression genome-wide. We established hypothalamic neuronal cultures from wild-type or transgenic Four Core Genotypes mice, a model that allows evaluating the effect of sex chromosomes independently of gonadal type. X-linked genes Kdm6a, Eif2s3x and Ddx3x showed higher expression in XX compared to XY neurons, regardless of gonadal sex. Moreover, Kdm6a expression pattern with higher mRNA levels in XX than XY did not change with age at E14, P0, and P60 in hypothalamus or under 17ß-estradiol treatment in culture. Kdm6a pharmacological blockade by GSK-J4 reduced axonal length only in female neurons and decreased the expression of neuritogenic genes Neurod1, Neurod2 and Cdk5r1 in both sexes equally, while a sex-specific effect was observed in Ngn3. Finally, Kdm6a downregulation using siRNA reduced axonal length and Ngn3 expression only in female neurons, abolishing the sex differences observed in control conditions. Altogether, these results point to Kdm6a as a key mediator of the higher axogenesis and Ngn3 expression observed in XX neurons before the critical period of brain masculinization.

Ductal Ngn3-expressing progenitors contribute to adult ß cell neogenesis in the pancreas.

Ductal cells have been proposed as a source of adult ß cell neogenesis, but this has remained controversial. By combining lineage tracing, 3D imaging, and single-cell RNA sequencing (scRNA-seq) approaches, we show that ductal cells contribute to the ß cell population over time. Lineage tracing using the Neurogenin3 (Ngn3)-CreERT line identified ductal cells expressing the endocrine master transcription factor Ngn3 that were positive for the δ cell marker somatostatin and occasionally co-expressed insulin. The number of hormone-expressing ductal cells was increased in Akita+/- diabetic mice, and ngn3 heterozygosity accelerated diabetes onset. scRNA-seq of Ngn3 lineage-traced islet cells indicated that duct-derived somatostatin-expressing cells, some of which retained expression of ductal markers, gave rise to ß cells. This study identified Ngn3-expressing ductal cells as a source of adult ß cell neogenesis in homeostasis and diabetes, suggesting that this mechanism, in addition to ß cell proliferation, maintains the adult islet ß cell population.

Protein Production and Purification of a Codon-Optimized Human NGN3 Transcription Factor from E. coli.

Neurogenin 3 (NGN3) transcription factor is vital for the development of endocrine cells of the intestine and pancreas. NGN3 is also critical for the neural precursor cell determination in the neuroectoderm. Additionally, it is one of the vital transcription factors for deriving human ß-cells from specialized somatic cells. In the current study, the production and purification of the human NGN3 protein from Escherichia coli (E. coli) is reported. First, the 642 bp protein-coding nucleotide sequence of the NGN3 gene was codon-optimized to enable enhanced protein expression in E. coli strain BL21(DE3). The codon-optimized NGN3 sequence was fused in-frame to three different fusion tags to enable cell penetration, nuclear translocation, and affinity purification. The gene insert with the fusion tags was subsequently cloned into an expression vector (pET28a( +)) for heterologous expression in BL21(DE3) cells. A suitable genetic construct and the ideal expression conditions were subsequently identified that produced a soluble form of the recombinant NGN3 fusion protein. This NGN3 fusion protein was purified to homogeneity (purity > 90%) under native conditions, and its secondary structure was retained post-purification. This purified protein, when applied to human cells, did not induce cytotoxicity. Further, the cellular uptake and nuclear translocation of the NGN3 fusion protein was demonstrated followed by its biological activity in PANC-1 cells. Prospectively, this recombinant protein can be utilized for various biological applications to investigate its functionality in cell reprogramming, biological processes, and diseases.

Ngn3-Positive Cells Arise from Pancreatic Duct Cells.

The production of pancreatic ß cells is the most challenging step for curing diabetes using next-generation treatments. Adult pancreatic endocrine cells are thought to be maintained by the self-duplication of differentiated cells, and pancreatic endocrine neogenesis can only be observed when the tissue is severely damaged. Experimentally, this can be performed using a method named partial duct ligation (PDL). As the success rate of PDL surgery is low because of difficulties in identifying the pancreatic duct, we previously proposed a method for fluorescently labeling the duct in live animals. Using this method, we performed PDL on neurogenin3 (Ngn3)-GFP transgenic mice to determine the origin of endocrine precursor cells and evaluate their potential to differentiate into multiple cell types. Ngn3-activated cells, which were marked with GFP, appeared after PDL operation. Because some GFP-positive cells were aligned proximally to the duct, we hypothesized that Ngn3-positive cells arise from the pancreatic duct. Therefore, we next developed an in vitro pancreatic duct culture system using Ngn3-GFP mice and examined whether Ngn3-positive cells emerge from this duct. We observed GFP expressions in ductal organoid cultures. GFP expressions were correlated with Ngn3 expressions and endocrine cell lineage markers. Interestingly, tuft cell markers were also correlated with GFP expressions. Our results demonstrate that in adult mice, Ngn3-positive endocrine precursor cells arise from the pancreatic ducts both in vivo and in vitro experiments indicating that the pancreatic duct could be a potential donor for therapeutic use.

Corrigendum: Role of TGF-ß/Smad Pathway in the Transcription of Pancreas-Specific Genes During Beta Cell Differentiation.

[This corrects the article DOI: 10.3389/fcell.2019.00351.].

Transient FOXO1 inhibition in pancreatic endoderm promotes the generation of NGN3+ endocrine precursors from human iPSCs.

In the multi-step differentiation protocol used to generate pancreatic endocrine cells from human pluripotent stem cells, the induction of NGN3+ endocrine precursors from the PDX1+/NKX6.1+ pancreatic endoderm is crucial for efficient endocrine cell production. Here, we demonstrate that transient, not prolonged FOXO1 inhibition results in enhanced NGN3+ endocrine precursors and hormone-producing cell production. FOXO1 inhibition does not directly induce NGN3 expression but stimulates PDX1+/NKX6.1+ cell proliferation. NOTCH activity, whose suppression is important for Ngn3 expression, is not suppressed but Wnt signaling is stimulated by FOXO1 inhibition. Reversely, Wnt inhibition suppresses the effects of FOXO1 inhibitor. These findings indicate that FOXO1 and Wnt are involved in regulating the proliferation of PDX1+/NKX6.1+ pancreatic endoderm that gives rise to NGN3+ endocrine precursors.

Reversible expansion of pancreatic islet progenitors derived from human induced pluripotent stem cells.

Transplantation of pancreatic islets is an effective therapy for severe type 1 diabetes. As donor shortage is a major problem for this therapy, attempts have been made to produce a large number of pancreatic islets from human pluripotent stem cells (hPSCs). However, as the differentiation of hPSCs to pancreatic islets requires multiple and lengthy processes using various expensive cytokines, the process is variable, low efficiency and costly. Therefore, it would be beneficial if islet progenitors could be expanded. Neurogenin3 (NGN3)-expressing pancreatic endocrine progenitor (EP) cells derived from hPSCs exhibited the ability to differentiate into pancreatic islets while their cell cycle was arrested. By using a lentivirus vector, we introduced several growth-promoting genes into NGN3-expressing EP cells. We found that SV40LT expression induced proliferation of the EP cells but reduced the expression of endocrine lineage-commitment factors, NGN3, NEUROD1 and NKX2.2, resulting in the suppression of islet differentiation. By using the Cre-loxP system, we removed SV40LT after the expansion, leading to re-expression of endocrine-lineage commitment genes and differentiation into functional pancreatic islets. Thus, our findings will pave a way to generate a large quantity of functional pancreatic islets through the expansion of EP cells from hPSCs.

Comparative analysis of enteroendocrine cells and their hormones between mouse intestinal organoids and native tissues.

Endocrine cells in the gastrointestinal tract secrete multiple hormones to maintain homeostasis in the body. In the present study, we generated intestinal organoids from the duodenum, jejunum, and ileum of Neurogenin 3 (Ngn3)-EGFP mice and examined how enteroendocrine cells (EECs) within organoid cultures resemble native epithelial cells in the gut. Transcriptome analysis of EGFP-positive cells from Ngn3-EGFP organoids showed gene expression pattern comparable to EECs in vivo. We also compared mRNAs of five major hormones, namely, ghrelin (Ghrl), cholecystokinin (Cck), Gip, secretin (Sct), and glucagon (Gcg) in organoids and small intestine along the longitudinal axis and found that expression patterns of these hormones in organoids were similar to those in native tissues. These findings suggest that an intestinal organoid culture system can be utilized as a suitable model to study enteroendocrine cell functions in vitro.

SIRT1 Activation Promotes ß-Cell Regeneration by Activating Endocrine Progenitor Cells via AMPK Signaling-Mediated Fatty Acid Oxidation.

Induction of ß-cell regeneration from endogenous cells represents a highly promising strategy in stem cell-based treatment for patients with diabetes. Recently, calorie restriction has been shown to affect the regulation of tissue and cell regeneration, including ß cells, via metabolic related mechanisms. Here, we examined the potential utility of sirtuin 1 (SIRT1), a calorie restriction mimetic, for stimulating ß-cell regeneration and the underlying mechanisms of such stimulation. The present results showed that SIRT1 activation with SRT1720 promoted ß-cell regeneration in streptozotocin (STZ)-induced ß-cell-deficient neonatal rats. This beneficial effect involved enhanced activation of neurogenin3 (NGN3)-positive endocrine progenitors from pancreatic ductal cells, rather than an expansion of residual ß cells. A dynamic expression profile of SIRT1 was observed in endocrine progenitors both during ß-cell regeneration in neonatal rats and in the second transition phase of mouse pancreas development. Consistently, SRT1720 treatment upregulated endocrine progenitor differentiation in cultured pancreatic rudiments. Upregulation of NGN3 by SIRT1 activation was through stimulating AMP-activated protein kinase (AMPK) signaling-mediated fatty acid oxidation (FAO) in human pancreatic progenitor cells; AMPK inhibition abolished these effects. The present findings demonstrate a promotional effect of SIRT1 activation on ß-cell restoration and endocrine progenitor differentiation that involves regulation of AMPK signaling-mediated FAO. Stem Cells 2019;37:1416-1428.

Role of TGF-ß/Smad Pathway in the Transcription of Pancreas-Specific Genes During Beta Cell Differentiation.

Autoimmune destruction of pancreatic beta cells causes absolute insulin deficiency and results in type 1 diabetes mellitus (T1DM). The substitution of healthy pancreatic beta cells for damaged cells would be the ideal treatment for T1DM; thus, the generation of pancreatic beta cells from adult stem cells represents an attractive avenue for research. In this study, a cocktail of factors was used to induce the differentiation of pancreatic beta cells from mesenchymal stem cells (MSCs). The differentiation program was divided into five stages, and the roles of the cocktail factors used during each stage were systematically elucidated. Activin A was found to phosphorylate Smad2 and Smad3 in stage III, thereby activating the TGF-ß/Smad pathway. Meanwhile, the endocrine-specific transcription factor, Ngn3, and the pancreas-specific miRNAs, miR-375 and miR-26a, were dramatically elevated in stage III. We next demonstrated that Smad4, an important transcription factor in the TGF-ß/Smad pathway, could bind to the promoter sequences of target genes and enhance their transcription to initiate the differentiation of beta cells. Use of SB-431542, an inhibitor of the TGF-ß/Smad pathway, demonstrated in vivo and in vitro that this pathway plays a critical role in the production of pancreatic beta cells and in modulating insulin secretion. Thus, the TGF-ß/Smad pathway is involved in the production of beta cells from adult stem cells by enhancing the transcription of Ngn3, miR-375, and miR-26a. These findings further underline the significant promise of cell transplant therapies for type 1 diabetes mellitus.

A Method for Identifying Mouse Pancreatic Ducts.

Proper identification of pancreatic ducts is a major challenge for researchers performing partial duct ligation (PDL), because pancreatic ducts, which are covered with acinar cells, are translucent and thin. Although damage to pancreatic ducts may activate quiescent ductal stem cells, which may allow further investigation into ductal stem cells for therapeutic use, there is a lack of effective techniques to visualize pancreatic ducts. In this study, we report a new method for identifying pancreatic ducts. First, we aimed to visualize pancreatic ducts using black, waterproof fountain pen ink. We injected the ink into pancreatic ducts through the bile duct. The flow of ink was observed in pancreatic ducts, revealing their precise architecture. Next, to visualize pancreatic ducts in live animals, we injected fluorescein-labeled bile acid, cholyl-lysyl-fluorescein into the mouse tail vein. The fluorescent probe clearly marked not only the bile duct but also pancreatic ducts when observed with a fluorescent microscope. To confirm whether the pancreatic duct labeling was successful, we performed PDL on Neurogenin3 (Ngn3)-GFP transgenic mice. As a result, acinar tissue is lost. PDL tail pancreas becomes translucent almost completely devoid of acinar cells. Furthermore, strong activation of Ngn3 expression was observed in the ligated part of the adult mouse pancreas at 7 days after PDL.

Mesenchymal cells are required for epithelial duct cell-to-beta cell maturation and function in an injured adult pancreas in the rat.

The islet, the endocrine portion of the pancreas - develops from an invagination of the pancreatic duct epithelial cells (PDECs) into the surrounding tissue. The contact of the PDECs with mesenchymal cells (MSCs) may be an essential drive for endocrine cell fate. During pancreatic development, cells that express Neurogenin-3 (Ngn3) biomarker are precursors of insulin- producing beta cells. These precursors have been reported in the neogenesis of islets from adult tissues following the surgical ligation of the main pancreatic duct (PDL). But the capacity of these precursors to induce the appropriate signals to complete the entire neogenesis program has been questioned. We studied the fate of co-culture of PDECs and MSCs from the ligated adult pancreas and established the exact location of adult stem- or progenitor-like cells that give rise to beta cells. PDECs were cultured in direct contact with or without MSCs in serum-containing culture media. The cytomorphology of the cells in co-cultures was determined and the immunocytochemical study of the cells was carried out using anti-Ngn3, anti-insulin and anti-cytokeratin-7 (CK7) antibodies. Both the PDEC/MSC- and PDEC/MSC+ cultures showed out- pocketing from duct epithelium by the end of the second week, which are distinct as cell clusters only in PDEC/MSC+ cells later in week four, exhibiting numerous branching ducts. Co-expression of Ngn3 with insulin was observed in both cultures from the second week. However, characterizations of these Ngn3+ cells in the PDEC/MSC+ culture revealed that these cells also co-expressed a CK7 biomarker. This study provides new evidence of the ductal epithelial nature of beta cells in injured adult pancreata; and that the mesenchymal stromal cells are required to sustain Ngn3 expression for beta cell maturation and function.

Phosphorylation of NEUROG3 Links Endocrine Differentiation to the Cell Cycle in Pancreatic Progenitors.

During pancreatic development, proliferating pancreatic progenitors activate the proendocrine transcription factor neurogenin 3 (NEUROG3), exit the cell cycle, and differentiate into islet cells. The mechanisms that direct robust NEUROG3 expression within a subset of progenitor cells control the size of the endocrine population. Here we demonstrate that NEUROG3 is phosphorylated within the nucleus on serine 183, which catalyzes its hyperphosphorylation and proteosomal degradation. During progression through the progenitor cell cycle, NEUROG3 phosphorylation is driven by the actions of cyclin-dependent kinases 2 and 4/6 at G1/S cell-cycle checkpoint. Using models of mouse and human pancreas development, we show that lengthening of the G1 phase of the pancreatic progenitor cell cycle is essential for proper induction of NEUROG3 and initiation of endocrine cell differentiation. In sum, these studies demonstrate that progenitor cell-cycle G1 lengthening, through its actions on stabilization of NEUROG3, is an essential variable in normal endocrine cell genesis.

Emerging roles of GLIS3 in neonatal diabetes, type 1 and type 2 diabetes.

GLI-similar 3 (GLIS3), a member of the Krüppel-like zinc finger protein subfamily, is predominantly expressed in the pancreas, thyroid and kidney. Glis3 mRNA can be initially detected in mouse pancreas at embryonic day 11.5 and is largely restricted to ß cells, pancreatic polypeptide-expressing cells, as well as ductal cells at later stage of pancreas development. Mutations in GLIS3 cause a neonatal diabetes syndrome, characterized by neonatal diabetes, congenital hypothyroidism and polycystic kidney. Importantly, genome-wide association studies showed that variations of GLIS3 are strongly associated with both type 1 diabetes (T1D) and type 2 diabetes (T2D) in multiple populations. GLIS3 cooperates with pancreatic and duodenal homeobox 1 (PDX1), v-maf musculoaponeurotic fibrosarcoma oncogene family, protein A (MAFA), as well as neurogenic differentiation 1 (NEUROD1) and potently controls insulin gene transcription. GLIS3 also plays a role in ß cell survival and likely in insulin secretion. Any perturbation of these functions may underlie all three forms of diabetes. GLIS3, synergistically with hepatocyte nuclear factor 6 (HNF6) and forkhead box A2 (FOXA2), controls fetal islet differentiation via transactivating neurogenin 3 (NGN3) and impairment of this function leads to neonatal diabetes. In addition, GLIS3 is also required for the compensatory ß cell proliferation and mass expansion in response to insulin resistance, which if disrupted may predispose to T2D. The increasing understanding of the mechanisms of GLIS3 in ß cell development, survival and function maintenance will provide new insights into disease pathogenesis and potential therapeutic target identification to combat diabetes.

Neurogenin 3 is regulated by neurotrophic tyrosine kinase receptor type 2 (TRKB) signaling in the adult human exocrine pancreas.

BACKGROUND: Reports of exocrine-to-endocrine reprogramming through expression or stabilization of the transcription factor neurogenin 3 (NGN3) have generated renewed interest in harnessing pancreatic plasticity for therapeutic applications. NGN3 is expressed by a population of endocrine progenitor cells that give rise exclusively to hormone-secreting cells within pancreatic islets and is necessary and sufficient for endocrine differentiation during development. In the adult human pancreas, NGN3 is expressed by dedifferentiating exocrine cells with a phenotype resembling endocrine progenitor cells and the capacity for endocrine differentiation in vitro. Neurotrophic tyrosine kinase receptor type 2 (TRKB), which regulates neuronal cell survival, differentiation and plasticity, was identified as highly overexpressed in the NGN3 positive cell transcriptome compared to NGN3 negative exocrine cells. This study was designed to determine if NGN3 is regulated by TRKB signaling in the adult human exocrine pancreas. METHODS: Transcriptome analysis, quantitative reverse transcriptase polymerase chain reaction (RTPCR) and immunochemistry were used to identify TRKB isoform expression in primary cultures of human islet-depleted exocrine tissue and human cadaveric pancreas biopsies. The effects of pharmacological modulation of TRKB signaling on the expression of NGN3 were assessed by Student's t-test and ANOVA. RESULTS: Approximately 30 % of cultured exocrine cells and 95 % of NGN3+ cells express TRKB on their cell surface. Transcriptome-based exon splicing analyses, isoform-specific quantitative RTPCR and immunochemical staining demonstrate that TRKB-T1, which lacks a tyrosine kinase domain, is the predominant isoform expressed in cultured exocrine tissue and is expressed in histologically normal cadaveric pancreas biopsies. Pharmacological inhibition of TRKB significantly decreased the percentage of NGN3+ cells, while a TRKB agonist significantly increased this percentage. Inhibition of protein kinase B (AKT) blocked the effect of the TRKB agonist, while inhibition of tyrosine kinase had no effect. Modulation of TRKB and AKT signaling did not significantly affect the level of NGN3 mRNA. CONCLUSIONS: In the adult human exocrine pancreas, TRKB-T1 positively regulates NGN3 independent of effects on NGN3 transcription. Targeting mechanisms controlling the NGN3+ cell population size and endocrine cell fate commitment represent a potential new approach to understand pancreas pathobiology and means whereby cell populations could be expanded for therapeutic purposes.