Pancreatic Transcription Factors Research Articles

Pancreatic Differentiation of Oral Minor Salivary Gland Stem Cells.

Stem cells from various sources including major salivary glands have been used to establish pancreatic differentiation in an attempt to provide new treatment options for patients with diabetes mellitus. In contrast, the potential of using the more easily accessible intraoral minor salivary glands has not been evaluated so far. Salivary stem cells were isolated from normal labial minor salivary glands that were removed during the excision of a mucocele and were attempted to differentiate into pancreatic cell lines using a culture medium enriched with activin A, retinoic acid and GLP-1.Real time RT-PCR was used to evaluate the expression of the genes of pancreatic transcription factors MafA, Ptf1a, Hb9 and Arx. Complementary, 22 labial minor salivary gland paraffin-embedded specimens were examined using immunohistochemistry for the presence of the relevant gene products of the pancreatic transcription factors Arx, MafA, Ptf1a and Pdx1. The differentiated salivary stem cells(cells of passage 3) expressed the genes of the pancreatic transcription factors MafA, Ptf1a, Hb9 and Arx even on the first day of the experiment while immunohistochemistry also confirmed the presence of the protein products of Arx, MafA, Ptf1a as well as Pdx1[> 50% of the specimens for Arx(5/8) and MafA(7/8), < 50% for Ptf1a(5/11) and Pdx1(5/11)] in ducts, mesenchymal connective tissue and acinar cells. Labial minor salivary glands may share gene and protein characteristics with pancreas suggesting a possible usefulness for pancreatic regeneration or substitution in cases of deficiency.

Developmentally dynamic changes in DNA methylation in the human pancreas

Development of the human pancreas requires the precise temporal control of gene expression via epigenetic mechanisms and the binding of key transcription factors. We quantified genome-wide patterns of DNA methylation in human fetal pancreatic samples from donors aged 6 to 21 post-conception weeks. We found dramatic changes in DNA methylation across pancreas development, with > 21% of sites characterized as developmental differentially methylated positions (dDMPs) including many annotated to genes associated with monogenic diabetes. An analysis of DNA methylation in postnatal pancreas tissue showed that the dramatic temporal changes in DNA methylation occurring in the developing pancreas are largely limited to the prenatal period. Significant differences in DNA methylation were observed between males and females at a number of autosomal sites, with a small proportion of sites showing sex-specific DNA methylation trajectories across pancreas development. Pancreas dDMPs were not distributed equally across the genome and were depleted in regulatory domains characterized by open chromatin and the binding of known pancreatic development transcription factors. Finally, we compared our pancreas dDMPs to previous findings from the human brain, identifying evidence for tissue-specific developmental changes in DNA methylation. This study represents the first systematic exploration of DNA methylation patterns during human fetal pancreas development and confirms the prenatal period as a time of major epigenomic plasticity.

Chromosome 20p11.2 deletions cause congenital hyperinsulinism via the loss of FOXA2 or its regulatory elements.

Persistent congenital hyperinsulinism (HI) is a rare genetically heterogeneous condition characterised by dysregulated insulin secretion leading to life-threatening hypoglycaemia. For up to 50% of affected individuals screening of the known HI genes does not identify a disease-causing variant. Large deletions have previously been used to identify novel regulatory regions causing HI. Here, we used genome sequencing to search for novel large (>1 Mb) deletions in 180 probands with HI of unknown cause and replicated our findings in a large cohort of 883 genetically unsolved individuals with HI using off-target copy number variant calling from targeted gene panels. We identified overlapping heterozygous deletions in five individuals (range 3-8 Mb) spanning chromosome 20p11.2. The pancreatic beta-cell transcription factor gene, FOXA2, a known cause of HI was deleted in two of the five individuals. In the remaining three, we found a minimal deleted region of 2.4 Mb adjacent to FOXA2 that encompasses multiple non-coding regulatory elements that are in conformational contact with FOXA2. Our data suggests that the deletions in these three children may cause disease through the dysregulation of FOXA2 expression. These findings provide new insights into the regulation of FOXA2 in the beta-cell and confirm an aetiological role for chromosome 20p11.2 deletions in syndromic HI.

Heparanase 2 (Hpa2)- a new player essential for pancreatic acinar cell differentiation

Heparanase 2 (Hpa2, HPSE2) is a close homolog of heparanase. Hpa2, however, lacks intrinsic heparan sulfate (HS)-degrading activity, the hallmark of heparanase enzymatic activity. Mutations of HPSE2 were identified in patients diagnosed with urofacial syndrome (UFS), a rare genetic disorder that exhibits abnormal facial expression and bladder voiding dysfunction, leading to renal damage and eventually renal failure. In order to reveal the role of HPSE2 in tissue homeostasis, we established a conditional Hpa2-KO mouse. Interestingly, the lack of Hpa2 was associated with a marked decrease in the expression of key pancreatic transcription factors such as PTF1, GATA6, and Mist1. This was associated with a two-fold decrease in pancreas weight, increased pancreatic inflammation, and profound morphological alterations of the pancreas. These include massive accumulation of fat cells, possibly a result of acinar-to-adipocyte transdifferentiation (AAT), as well as acinar-to-ductal metaplasia (ADM), both considered to be pro-tumorigenic. Furthermore, exposing Hpa2-KO but not wild-type mice to a carcinogen (AOM) and pancreatic inflammation (cerulein) resulted in the formation of pancreatic intraepithelial neoplasia (PanIN), lesions that are considered to be precursors of invasive ductal adenocarcinoma of the pancreas (PDAC). These results strongly support the notion that Hpa2 functions as a tumor suppressor. Moreover, Hpa2 is shown here for the first time to play a critical role in the exocrine aspect of the pancreas.

Expression and clinicopathological characteristics of PDX1, PTF1A, and SALL4 in large and small ducts of ectopic pancreas located in gastro-duodenum and jejunum

An ectopic pancreas is defined as pancreatic tissue outside its normal location, anatomically separated from the pancreas.The transcription factor pancreas/duodenum homeobox protein 1 (PDX1) is involved in maintaining the pancreas and functions in early pancreatic development, beta cell differentiation, and endocrine non beta cells. Pancreatic transcription factor 1 subunit alpha (PTF1A) affects exocrine cell formation and regulation of acinar cell identity, and is expressed in exocrine cells as a transcription factor. The depletion of SALL4 disrupts self-renewal and induces differentiation.To clarify which of PDX1, PTF1A, or SALL4 determines the difference in Heinrich's classification, we examined the localization and number of positive cells. We analyzed the differential expression of PDX1, PTF1A, and SALL4 in large and small ducts in ectopic pancreas by immunohistochemistry. Results showed that the number of PTF1A-positive cells in large ducts was more widespread in type I than in type II in the gastro-duodenum, and more SALL4-positive cells were noticed in large ducts than in small ducts in the gastro-duodenum of type II. Our results revealed that PTF1A might promote exocrine differentiation in developing the pancreatic tissues, and that those with widespread expression differentiate into exocrine cells.

Abstract A070: Comparison of pancreatic cancer mouse models identifies potential drivers of tumor initiation and progression

Abstract The five-year survival rate of pancreatic ductal adenocarcinoma (PDAC) is currently 11% [CP1] and by 2030 PDAC is expected to become the second leading cause of cancer related death. Understanding PDAC initiation and maintenance is important for the advancement of treatment. Mouse models allow us to study the mechanisms of PDAC initiation and progression. The most common genetically engineered mouse model (GEMM) of PDAC utilizes a knock-in of Cre recombinase [FB2] into the pancreatic transcription factor 1a (Ptf1a)locus to drive pancreas specific oncogenic KRAS expression. Alternative GEMMs express Cre recombinase from the Mist1/Bhlha15 locus (Mist1-CreER) or from elastase enzyme promoter-driven constructs (Ela-CreER). How these models differ in their ability to promote neoplasia is unknown. We hypothesized that differences in GEMMs may provide insight into mechanisms of PDAC initiation and progression. Here, we compare the tamoxifen-inducible Ptf1a-CreER, [FB3] Mist1-CreER and Ela-CreER models in their response to oncogenic KRAS expression. KRASG12D was induced with tamoxifen gavage 6-8 weeks postpartum and caerulein was used to induce pancreatitis. Histological analyses (H&amp;E and IHC for amylase, cytokeratin 19, Fos and Jun) were performed on fixed tissue to assess associated phenotypic changes. Tissues from Ptf1a-CreER, Mist1-CreER and Ela-CreER mice were harvested 3 weeks after tamoxifen administration and processed for RNA sequencing. Phenotypically, in the presence of oncogenic KRAS and after pancreatitis induction, Ptf1a-CreER pancreata exhibited extensive tissue remodeling - acinar to ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN) - with a dense fibroinflammatory response in contrast to both Mist1-CreER and Ela-CreER pancreata. Mist1-CreER and Ela-CreER pancreata had much more variable penetrance with tissues often showing little to no ADM and PanINs despite oncogenic KRAS expression and prior pancreatitis. At the gene expression level, Mist1-CreER pancreata showed fewer differentially expressed genes when compared to wild type tissue than Ptf1a-CreER under the same conditions. In total, less than 70 differentially expressed genes were common between the two models. Members of the AP-1 transcriptional complex (fos and c-jun) previously implicated in neoplastic progression were specifically increased only in Ptf1a-CreER mice.[FB4] Taken together, our histological, and transcriptional data indicates mice lacking one copy of Ptf1a (Ptf1a-CreER) undergo more dramatic histological changes after pancreatitis injury and oncogenic KRAS induction when compared to the GEMMs with both copies of Ptf1a intact. Additionally, mice in the Ptf1a-CreER group show a more marked molecular response to oncogenic KRAS induction, suggesting the absence of a single copy of the acinar fate transcription factor Ptf1a disrupted pancreatic homeostasis and poised it for transformation. Further comparison of PDAC GEMMs will yield clearer insight into mechanisms of PDAC initiation and progression. Citation Format: Joyce K. Thompson, Fatemeh Mousavi, Justine Lau, Emily Wu, Christopher Pin, Filip Bednar. Comparison of pancreatic cancer mouse models identifies potential drivers of tumor initiation and progression [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A070.

Flavin Adenine Dinucleotide (FAD) and Pyridoxal 5'-Phosphate (PLP) Bind to Sox9 and Alter the Expression of Key Pancreatic Progenitor Transcription Factors.

Cofactor flavin adenine dinucleotide (FAD), a compound with flavin moiety and a derivative of riboflavin (vitamin B2), is shown to bind to Sox9 (a key transcription factor in early pancreatic development) and, subsequently, induce a large increase in markers of pancreatic development, including Ngn3 and PTF1a. Pyridoxal 5'-phosphate (PLP), the active form of vitamin B6, also binds to Sox9 and results in a similar increase in pancreatic development markers. Sox9 is known to be specifically important for pancreatic progenitors. Previously, there was no known link between FAD, PLP, or other co-factors and Sox9 for function. Thus, our findings show the mechanism by which FAD and PLP interact with Sox9 and result in the altered expression of pancreatic progenitor transcription factors involved in the pancreas development.

Integration of genomic analysis and transcript expression of ABCC8 and KCNJ11 in focal form of congenital hyperinsulinism.

The focal form of CHI is caused by an autosomal recessive pathogenic variant affecting the paternal homologue of genes ABCC8 or KCNJ11 and a second somatic event specifically occurring in the affected islet of Langerhans. The approach of this study was to integrate the genetic changes occurring in pancreatic focal lesions of CHI at the genomic and transcriptional level. Patients receiving therapeutic surgery and with proven ABCC8 or KCNJ11 pathogenic variants were selected and analyzed for loss of heterozygosity (LOH), changes in copy number and uniparental disomy (UPD) on the short am of chromosome 11 by molecular microarray analysis and methylation-specific MLPA. Gene expression was analyzed by RT-PCR and Massive Analysis of cDNA Ends (MACE). Both genes, ABCC8 and KCNJ11, are located in proximity to the Beckwith-Wiedemann (BWS) imprinting control region on chromosome 11p15. Somatic paternal uniparental isodisomy (UPD) at chromosome 11p was identified as second genetic event in focal lesions resulting in LOH and monoallelic expression of the mutated ABCC8/KCNJ11 alleles. Of five patients with samples available for microarray analysis, the breakpoints of UPD on chromosome 11p were different. Samples of two patients were analyzed further for changes in gene expression. Profound downregulation of growth suppressing genes CDKN1 and H19 was detected in focal lesions whereas growth promoting gene ASCL2 and pancreatic transcription factors of the endocrine cell lineage were upregulated. Paternal UPD on the short arm of chromosome 11 appears to be the major second genetic event specifically within focal lesions of CHI but no common breakpoint for UDP can be delineated. We show for the first time upregulation of growth promoting ASCL2 (achaete-scute homolog 2) suggestive of a driving factor in postnatal focal expansion in addition to downregulation of growth suppressing genes CDKN1C and H19.

The role of DNA demethylation in liver to pancreas transdifferentiation

BackgroundInsulin producing cells generated by liver cell transdifferentiation, could serve as an attractive source for regenerative medicine. The present study assesses the relationship between DNA methylation pTFs induced liver to pancreas transdifferentiation.ResultsThe transdifferentiation process is associated with DNA demethylation, mainly at gene regulatory sites, and with increased expression of these genes. Active inhibition of DNA methylation promotes the pancreatic transcription factor-induced transdifferentiation process, supporting a causal role for DNA demethylation in this process.ConclusionsTransdifferentiation is associated with global DNA hypomethylation, and with increased expression of specific demethylated genes. A combination of epigenetic modulators may be used to increase chromatin accessibility of the pancreatic transcription factors, thus promoting the efficiency of the developmental process.

N6-methyladenosine modification-mediated mRNA metabolism is essential for human pancreatic lineage specification and islet organogenesis

Pancreatic differentiation from human pluripotent stem cells (hPSCs) provides promising avenues for investigating development and treating diseases. N6-methyladenosine (m6A) is the most prevalent internal messenger RNA (mRNA) modification and plays pivotal roles in regulation of mRNA metabolism, while its functions remain elusive. Here, we profile the dynamic landscapes of m6A transcriptome-wide during pancreatic differentiation. Next, we generate knockout hPSC lines of the major m6A demethylase ALKBH5, and find that ALKBH5 plays significant regulatory roles in pancreatic organogenesis. Mechanistic studies reveal that ALKBH5 deficiency reduces the mRNA stability of key pancreatic transcription factors in an m6A and YTHDF2-dependent manner. We further identify that ALKBH5 cofactor α-ketoglutarate can be applied to enhance differentiation. Collectively, our findings identify ALKBH5 as an essential regulator of pancreatic differentiation and highlight that m6A modification-mediated mRNA metabolism presents an important layer of regulation during cell-fate specification and holds great potentials for translational applications.

Pancreatic Transdifferentiation Using β-Cell Transcription Factors for Type 1 Diabetes Treatment.

Type 1 diabetes is a chronic illness in which the native beta (β)-cell population responsible for insulin release has been the subject of autoimmune destruction. This condition requires patients to frequently measure their blood glucose concentration and administer multiple daily exogenous insulin injections accordingly. Current treatments fail to effectively treat the disease without significant side effects, and this has led to the exploration of different approaches for its treatment. Gene therapy and the use of viral vectors has been explored extensively and has been successful in treating a range of diseases. The use of viral vectors to deliver β-cell transcription factors has been researched in the context of type 1 diabetes to induce the pancreatic transdifferentiation of cells to replace the β-cell population destroyed in patients. Studies have used various combinations of pancreatic and β-cell transcription factors in order to induce pancreatic transdifferentiation and have achieved varying levels of success. This review will outline why pancreatic transcription factors have been utilised and how their application can allow the development of insulin-producing cells from non β-cells and potentially act as a cure for type 1 diabetes.

1273-P: The Interplay between Polygenic and Rare HNF1A Variant Risk in Type 2 Diabetes

Rare variants in HNF1A, a pancreatic beta cell transcription factor, have been shown to be associated with T2D and monogenic diabetes. Here, we examine the effect of functionally validated rare (MAF&amp;lt;1%) nonsynonymous variants (RNVs) in a functional domain of HNF1A on T2D risk, and whether their effect is influenced by an overall genetic susceptibility to T2D. Using exome sequencing data of 3,443 African ancestry (AA) and 157,182 European ancestry (EA) individuals in the UK Biobank and 5,018 AA, 5,6EA and 7,1Hispanic-Latino Ancestry (HA) individuals in the BioMe Biobank, we identified 132 AA, 287 EA, and 64 HA RNV carriers (NRNVs= 30) of HNF1A variants that impair protein function ≤ 60%. T2D Polygenic Risk Scores (PRSs) were built using either trans-ethnic or EA GWAS summary statistics and standardized per ancestry. We tested the association between RNV carrier status and the PRS with T2D risk, and meta-analyzed the ancestry-specific results. Carrying any of the HNF1A RNV mutations was associated with a 1.5 increase in the odds of T2D in the EA, albeit non-significantly (P=0.06) , but not in the AA (OR=1.1, P=0.6) or HA (OR=1 , P=1) . We then restricted analyses to RNVs within a functional domain, which results in more pronounced effects in the AA (OR=1.5, P=0.07) and EA (OR=1.9, P=0.002) but not in the HA (OR=0.8, P=0.5) . Per SD increase in the PRS, there is a significant 1.7, 1.6 and 2.1 higher odds of T2D in AA, EA and HA individuals, respectively. Even though we observed no interaction between the PRS and HNF1A RNVs (P &amp;gt;; 0.05) , the association between RNVs and T2D was more pronounced among EA individuals with a high PRS. Functional domain carriers (NEA = 50) in the top PRS quintile had a 3.6 (P=0.0008) higher odds of T2D as compared to non-carriers in the same quintile. Our study shows the value of large biobanks in studying rare variants, the importance of variant selection, and the need to include non-European ancestry populations. We demonstrate the utility of combining rare and common variant risks to better assess T2D risk. Disclosure L.Stalbow: None. M.H.Preuss: None. N.Chami: None. R.A.J.Smit: None. R.J.Loos: None.

Epigallocatechin-3-gallate alleviates type 2 diabetes mellitus via β-cell function improvement and insulin resistance reduction.

Objective(s):Epigallocatechin-3-gallate (EGCG) has a good therapeutic effect on type 2 diabetes mellitus (T2DM). This work was designed to explore EGCG’s effectiveness in insulin resistance (IR) and pancreas islet β-cell function in a rat model of T2DM. Materials and Methods:Eight-week-old male Sprague Dawley rats were randomly divided into 6 groups, including the Control (normal diet), Diabetes (high-sucrose high-fat [HSHF] diet combined with tail vein injection of streptozotocin [STZ] for T2DM induction) and Treatment Diabetic rats which were treated with metformin [500 mg/kg/d] or EGCG [25, 50 or 100 mg/kg/d] intragastric administration for 10 weeks. With the exception of control animals, the other groups were fed the HSHF diet. EGCG’s effects on IR and insulin secretion were assessed by measuring body weights, and fasting blood glucose (FBG), postprandial blood glucose (PBG) and insulin levels. The morphological and molecular changes of pancreas islet β-cells were examined by hematoxylin-eosin (H&E) staining, transmission electron microscopy (TEM) and immunofluorescence.Results:Rats fed the HSHF diet combined with STZ treatment had increased body weights and blood glucose amounts, accompanied by IR and impaired β-cell function, induced T2DM, and EGCG dose-dependently restored the above indicators. Additionally, EGCG upregulated the pancreatic transcription factors pancreatic duodenal homeobox protein-1 (PDX-1) and musculoaponeurotic fibrosarcoma oncogene homolog A (MafA).Conclusion:These results suggest that EGCG reduces blood glucose amounts, and improve IR and islet β-cell disorder in T2DM.

Gymnemic Acid Ameliorates Pancreatic β-Cell Dysfunction by Modulating Pdx1 Expression: A Possible Strategy for β-Cell Regeneration.

Endogenous pancreatic β-cell regeneration is a promising therapeutic approach for enhancing β-cell function and neogenesis in diabetes. Various findings have reported that regeneration might occur via stimulating β-cell proliferation, neogenesis, or conversion from other pancreatic cells to β-like cells. Although the current scenario illustrates numerous therapeutic strategies and approaches that concern endogenous β-cell regeneration, all of them have not been successful to a greater extent because of cost effectiveness, availability of suitable donors and rejection in case of transplantation, or lack of scientific evidence for many phytochemicals derived from plants that have been employed in traditional medicine. Therefore, the present study aims to investigate the effect of gymnemic acid (GA) on β-cell regeneration in streptozotocin-induced type 1 diabetic rats and high glucose exposed RIN5-F cells. The study involves histopathological and immunohistochemical analysis to examine the islet's architecture. Quantitative polymerase chain reaction (qPCR) and/or immunoblot were employed to quantify the β-cell regeneration markers and cell cycle proliferative markers. The immunoexpression of E-cadherin, β-catenin, and phosphoinositide 3-kinases/protein kinase B were significantly increased in GA-treated diabetic rats. On the other hand, treatment with GA upregulated the pancreatic regenerative transcription factor viz. pancreatic duodenal homeobox 1, Neurogenin 3, MafA, NeuroD1, and β-cells proliferative markers such as CDK4, and Cyclin D1, with a simultaneous downregulation of the forkhead box O, glycogen synthase kinase-3, and p21cip1 in diabetic treated rats. Adding to this, we noticed increased nuclear localization of Pdx1 in GA treated high glucose exposed RIN5-F cells. Our results suggested that GA acts as a potential therapeutic candidate for endogenous β-cell regeneration in treating type 1 diabetes.

Differential Responses of Neural Retina Progenitor Populations to Chronic Hyperglycemia.

Diabetic retinopathy is a frequent complication of longstanding diabetes, which comprises a complex interplay of microvascular abnormalities and neurodegeneration. Zebrafish harboring a homozygous mutation in the pancreatic transcription factor pdx1 display a diabetic phenotype with survival into adulthood, and are therefore uniquely suitable among zebrafish models for studying pathologies associated with persistent diabetic conditions. We have previously shown that, starting at three months of age, pdx1 mutants exhibit not only vascular but also neuro-retinal pathologies manifesting as photoreceptor dysfunction and loss, similar to human diabetic retinopathy. Here, we further characterize injury and regenerative responses and examine the effects on progenitor cell populations. Consistent with a negative impact of hyperglycemia on neurogenesis, stem cells of the ciliary marginal zone show an exacerbation of aging-related proliferative decline. In contrast to the robust Müller glial cell proliferation seen following acute retinal injury, the pdx1 mutant shows replenishment of both rod and cone photoreceptors from slow-cycling, neurod-expressing progenitors which first accumulate in the inner nuclear layer. Overall, we demonstrate a diabetic retinopathy model which shows pathological features of the human disease evolving alongside an ongoing restorative process that replaces lost photoreceptors, at the same time suggesting an unappreciated phenotypic continuum between multipotent and photoreceptor-committed progenitors.

Transcriptional changes and the role of ONECUT1 in hPSC pancreatic differentiation

Cell type specification during pancreatic development is tightly controlled by a transcriptional and epigenetic network. The precise role of most transcription factors, however, has been only described in mice. To convey such concepts to human pancreatic development, alternative model systems such as pancreatic in vitro differentiation of human pluripotent stem cells can be employed. Here, we analyzed stage-specific RNA-, ChIP-, and ATAC-sequencing data to dissect transcriptional and regulatory mechanisms during pancreatic development. Transcriptome and open chromatin maps of pancreatic differentiation from human pluripotent stem cells provide a stage-specific pattern of known pancreatic transcription factors and indicate ONECUT1 as a crucial fate regulator in pancreas progenitors. Moreover, our data suggest that ONECUT1 is also involved in preparing pancreatic progenitors for later endocrine specification. The dissection of the transcriptional and regulatory circuitry revealed an important role for ONECUT1 within such network and will serve as resource to study human development and disease.

Effects of Metformin and Sitagliptin on islet cell function in insulin-resistant prediabetic KKay mice

Objective: To investigate the effects of metformin and sitagliptin on the function of islet β cells in insulin resistance pre-diabetic KKay mice. Methods: Thirty 6-week-old KKAy mice were randomly divided into two groups: normal diet fed group (NC group, n=10) and high-fat diet fed group (n=20). At 8 weeks, KKAy mice were randomly divided into two groups: metformin intervention group (met group, n=10) and sitagliptin intervention group (SP group, n=10), which were treated by gavage for 8 weeks. Glucose tolerance was measured by oral glucose tolerance test (OGTT), serum insulin level and plasma lipid level were measured by tail blood sampling, and HOMA-β and HOMA-IR were calculated. The mice were killed after blood collection, and the pancreas of KKAy mice was taken. The β cell volume of each group was compared by immunofluorescence staining of insulin and glucagon, respectively. The proliferation and apoptosis of β cell were analyzed by Ki67/INS. The expressions of pancreatic transcription factors PDX-1 and MafA were detected by Western blot. Results: ① The OGTT results indicated that blood glucose of KKAy mice at fast, 30, 60 and 120 min after oral administration of glucose in the Met and SP groups were decreased significantly compared with the NC group, and the area under the blood glucose time curve (AUC) was significantly reduced (P＜0.01, P＜0.01). There was no significant difference between the Met group and the SP group in blood glucose level at 30 and 60 min after oral administration of glucose. Compared with the SP group, the blood glucose of Met group at 120 min was decreased significantly (P＜0.05). There was no significant difference in AUC between the two groups. ② The results of the insulin tolerance test (ITT) indicated that, compared with NC, the fasting blood glucose and the blood glucose at 30, 60 and 90 min after insulin injection in KKAy mice in the Met and SP groups were decreased significantly, and the area under the ITT blood glucose curve (AUC) was increased significantly (P＜0.01), while there was no significant difference between the Met and SP groups. ③ In the NC group, the brightness of the areas of the islet β cells was low and the edges were scattered. After treated with metformin, the areas and brightness of the β cells were increased. After treatment with sitagliptin, the area and brightness of the β cells were increased significantly. In the NC group, the α cells were disordered in the islet distribution and the brightness was large. After the administration of metformin, the α cell area and the brightness were decreased, and distributed to the edge of the islet to a certain extent. After the administration of sitagliptin, there was a significant decrease in the area of the α cells, with a significant decrease in the brightness and distribution at the edge of the islet. ④ Compared with the NC group, the expression levels of pancreatic MafA in the Met group and SP group were increased significantly, which were 1.63 times and 1.58 times, respectively (P＜0.01, P＜0.01). There was no significant difference in the expression of pancreatic PDX-1 between the groups. Conclusion: In pre-diabetes mellitus KKAy mice with insulin resistance, metformin can maintain the function and morphology of pancreatic islets, and sitagliptin may promote the proliferation of islet βcells, improve the expression level of insulin transcription factor MafA, and prevent the occurrence and development of diabetes.

Activation of Pancreatic Stellate Cells Is Beneficial for Exocrine but Not Endocrine Cell Differentiation in the Developing Human Pancreas.

Pancreatic stellate cells (PaSCs) are non-endocrine, mesenchymal-like cells that reside within the peri-pancreatic tissue of the rodent and human pancreas. PaSCs regulate extracellular matrix (ECM) turnover in maintaining the integrity of pancreatic tissue architecture. Although there is evidence indicating that PaSCs are involved in islet cell survival and function, its role in islet cell differentiation during human pancreatic development remains unclear. The present study examines the expression pattern and functional role of PaSCs in islet cell differentiation of the developing human pancreas from late 1st to 2nd trimester of pregnancy. The presence of PaSCs in human pancreata (8–22 weeks of fetal age) was characterized by ultrastructural, immunohistological, quantitative RT-PCR and western blotting approaches. Using human fetal PaSCs derived from pancreata at 14–16 weeks, freshly isolated human fetal islet-epithelial cell clusters (hIECCs) were co-cultured with active or inactive PaSCs in vitro. Ultrastructural and immunofluorescence analysis demonstrated a population of PaSCs near ducts and newly formed islets that appeared to make complex cell-cell dendritic-like contacts. A small subset of PaSCs co-localized with pancreatic progenitor-associated transcription factors (PDX1, SOX9, and NKX6-1). PaSCs were highly proliferative, with significantly higher mRNA and protein levels of PaSC markers (desmin, αSMA) during the 1st trimester of pregnancy compared to the 2nd trimester. Isolated human fetal PaSCs were identified by expression of stellate cell markers and ECM. Suppression of PaSC activation, using all-trans retinoic acid (ATRA), resulted in reduced PaSC proliferation and ECM proteins. Co-culture of hIECCs, directly on PaSCs or indirectly using Millicell® Inserts or using PaSC-conditioned medium, resulted in a reduction the number of insulin+ cells but a significant increase in the number of amylase+ cells. Suppression of PaSC activation or Notch activity during the co-culture resulted in an increase in beta-cell differentiation. This study determined that PaSCs, abundant during the 1st trimester of pancreatic development but decreased in the 2nd trimester, are located near ductal and islet structures. Direct and indirect co-cultures of hIECCs with PaSCs suggest that activation of PaSCs has opposing effects on beta-cell and exocrine cell differentiation during human fetal pancreas development, and that these effects may be dependent on Notch signaling.

Starvation Stress in the Sbds -Null Zebrafish: Implications for Human Shwachman-Diamond Syndrome

Shwachman-Diamond Syndrome (SDS) is an autosomal recessive disorder characterized by exocrine pancreatic insufficiency, bone marrow dysfunction, and an increase risk of acute myeloid leukemia. Patients with SDS exhibit abnormalities in other organs, including bone, liver and immunologic and endocrine systems. Almost all cases of SDS result from biallelic mutations in the SBDS gene1. SBDS is involved in ribosomal biogenesis and stabilization of mitotic spindle microtubules2,3. Recently, it has also been shown a reduction cell proliferation and mTOR and STAT3 expression, both with an important role in cellular growth and tumorigenesis, in SDS derived leukocytes.4Genetic ablation of Sbds results in early embryonic lethality (ED 7.5) in mice5. Zebrafish provide an attractive, alternative model organism to study SDS. Genome analysis of zebrafish (Danio rerio) revealed the presence of a single sbds gene, encoding a protein 90% identical to the human orthologue.We created sbds zebrafish knockout strains that phenocopies the human Shwachman-Diamond Syndrome. Survival rate of mutants was normal at 15 days post fertilization (dpf) (100%) but decline significantly at 21 dpf (4%). Western blot analysis showed a progressive decrease of Sbds during the first 8 dpf, with total absence at 10 dpf. These results suggest that larval viability is due to maternally deposited sbds transcript and corresponding protein, which was confirmed by RT-qPCR and immunoblot at earliest developmental stages. Zebrafish sbds mutants showed neutropenia at 5 and 15 days post fertilization (dpf) but normal macrophage number and hemoglobinization. Pancreas, liver, digestive tract, and eye showed histologic evidence of atrophy at 21 dpf. Moreover, we observed an important reduction in the intestinal epithelium at 15 dpf.To further characterize effects of Sbds loss, we tested expression of genes involved in lipid metabolism (acc, ppar α , ppar γ and srbp1) and pancreatic transcription factors (ptf1a and prox1) by qPCR. Our results showed that they were not affected at 10 dpf but upregulated at 18 dpf. After staining 15 dpf larvae with oil red we observed, in the mutants compared to their sibling wild type or heterozygous, significantly reduced accumulation of lipids. In addition, we found an increase of expression of genes involved in autophagy (atg5, atg7 and mTOR), and upregulation of casp9 and tp53 expression.With these results we thought that reduced viability was due to a problem in nutrient absorption and therefore starvation. We carried out two starvation experiments, a diet restriction from 10 to 15 dpf, and from 15 to 21 dpf. Unlike sbds mutants, mortality rates were not affected comparing to control groups in any of these experiments. However, genes related to lipid metabolism and autophagy were upregulated and histology showed a reduction in intestinal epithelium as well. Moreover, oil red staining showed lower lipid accumulation in starved fish as we observed in sbds mutants.With these results, we conclude sbds -null zebrafish showed signs of malnutrition and an effect in fatty acid-metabolism gene expression as seen in starved fish. Our results make it a highly relevant model to investigate new mechanistic hypotheses and develop new small molecule therapies for Shwachman-Diamond syndrome.1. Boocock GR, Morrison JA, Popovic M, et al. Mutations in SBDS are associated with Shwachman-Diamond syndrome. Nat Genet . 2003;33(1):97-101.2. Austin KM, Gupta ML, Jr., Coats SA, et al. Mitotic spindle destabilization and genomic instability in Shwachman-Diamond syndrome. J Clin Invest . 2008;118(4):1511-1518.3. Finch AJ, Hilcenko C, Basse N, et al. Uncoupling of GTP hydrolysis from eIF6 release on the ribosome causes Shwachman-Diamond syndrome. Genes Dev . 2011;25(9):917-929.4. Bezzerri V, Vella A, Calcaterra E, et al. New insights into the Shwachman-Diamond Syndrome-related haematological disorder: hyper-activation of mTOR and STAT3 in leukocytes. 2016;6:33165.5. Zhang S, Shi M, Hui CC, Rommens JM. Loss of the mouse ortholog of the Shwachman-Diamond syndrome gene (Sbds) results in early embryonic lethality. Mol Cell Biol . 2006;26(17):6656-6663. DisclosuresNo relevant conflicts of interest to declare.

Dose‐dependent Improvement in Cardiovascular Complications and Hyperglycemia Reversal Post‐mesenchymal Stem Cells Delivery in Diabetic Infarcted Rats

Study objective To study the effect of intravenous delivery of single and double doses of mesenchymal stem cells (MSCs) on cardiovascular complications and hyperglycemia in diabetic infarcted rats. Hypothesis Diabetes has been one of the leading causes of deaths, and majority of these deaths are concomitant with myocardial infarction. Currently available clinical management strategies have failed to provide desired support to respective failing organs and reduce continuous increasing mortality rate. Hence, in this study improvement in cardiovascular complications and diabetic conditions were assessed in diabetic infarcted rats post-MSCs infusion. Methods Diabetic infarcted animal models were created by streptozotocin (STZ, 40mg/Kg body weight) followed by intraperitoneal injection of isoproterenol in Wister rats (150 to 250 g). These animals received intravenous infusion of single and two consecutive doses of human umbilical cord blood (hUCB) derived MSCs. After seven days biochemical, glucose and insulin levels were quantified along with the pancreatic and cardiac markers transcript expression which were compared with the sham and control groups. Results Double dose infusion of hUCB-MSCs in diabetic infarcted rats showed greater improvement compared to single dose in cardiovascular complications which were defined by Tropinin-1, creatinine kinase, beta-hydroxy butyrate and acetoacetate levels, cardiac transcript expression levels and histological analysis. While improvement in diabetic condition was observed by blood glucose and insulin levels and pancreatic transcription factors expression levels compared to sham and control group. Conclusion This study suggests that two consecutive doses of hUCB-MSCs intravenous infusion exert comparatively better effects on the myocardial infarction and hyperglycemia in pre-clinical settings.