Intracellular ATP Signaling Contributes to FAM3A-Induced PDX1 Upregulation in Pancreatic Beta Cells

FAM3A is a recently identified mitochondrial protein that stimulates pancreatic-duodenal homeobox 1 (PDX1) and insulin expressions by promoting ATP release in islet β cells. In this study, the role of intracellular ATP in FAM3A-induced PDX1 expression in pancreatic β cells was further examined. Acute FAM3A inhibition using siRNA transfection in mouse pancreatic islets significantly reduced PDX1 expression, impaired insulin secretion, and caused glucose intolerance in normal mice. In vitro, FAM3A overexpression elevated both intracellular and extracellular ATP contents and promoted PDX1 expression and insulin secretion. FAM3A-induced increase in cellular calcium (Ca2+) levels, PDX1 expression, and insulin secretion, while these were significantly repressed by inhibitors of P2 receptors or the L-type Ca2+ channels. FAM3A-induced PDX1 expression was abolished by a calmodulin inhibitor. Likewise, FAM3A-induced β-cell proliferation was also inhibited by a P2 receptor inhibitor and an L-type Ca2+ channels inhibitor. Both intracellular and extracellular ATP contributed to FAM3A-induced PDX1 expression, insulin secretion, and proliferation of pancreatic β cells.

Pancreatic Differentiation of Stem Cells Reveals Pathogenesis of a Syndrome of Ketosis-Prone Diabetes

Genetic analysis of an adult patient with an unusual course of Ketosis-Prone Diabetes (KPD) and lacking islet autoantibodies demonstrated a nucleotide variant in the<i> </i>5’-UTR of <i>PDX1</i>, a beta-cell development gene. When differentiated to the pancreatic lineage, his induced pluripotent stem cells stalled at the definitive endoderm stage. Metabolomic analysis of the cells revealed that this was associated with leucine hypersensitivity during transition from the definitive endoderm to the pancreatic progenitor stage, and RNA-sequencing showed defects in leucine-sensitive mTOR pathways contribute to the differentiation deficiency. CRISPR-Cas9 manipulation of the <i>PDX1</i> variant demonstrated that it is necessary and sufficient to confer leucine sensitivity and the differentiation block, likely due to disruption of binding of the transcriptional regulator NFY to the <i>PDX1</i> 5’-UTR, leading to decreased PDX1 expression at the early pancreatic progenitor stage. Thus, the combination of an underlying defect in leucine catabolism characteristic of KPD with a functionally relevant heterozygous variant in a critical beta-cell gene that confers increased leucine sensitivity and inhibits endocrine cell differentiation resulted in the phenotype of late-onset beta-cell failure in this patient. We define the molecular pathogenesis of a diabetes syndrome and demonstrate the power of multi-omics analysis of patient-specific stem cells for clinical discovery.

Pancreatic Differentiation of Stem Cells Reveals Pathogenesis of a Syndrome of Ketosis-Prone Diabetes

Genetic analysis of an adult patient with an unusual course of Ketosis-Prone Diabetes (KPD) and lacking islet autoantibodies demonstrated a nucleotide variant in the<i> </i>5’-UTR of <i>PDX1</i>, a beta-cell development gene. When differentiated to the pancreatic lineage, his induced pluripotent stem cells stalled at the definitive endoderm stage. Metabolomic analysis of the cells revealed that this was associated with leucine hypersensitivity during transition from the definitive endoderm to the pancreatic progenitor stage, and RNA-sequencing showed defects in leucine-sensitive mTOR pathways contribute to the differentiation deficiency. CRISPR-Cas9 manipulation of the <i>PDX1</i> variant demonstrated that it is necessary and sufficient to confer leucine sensitivity and the differentiation block, likely due to disruption of binding of the transcriptional regulator NFY to the <i>PDX1</i> 5’-UTR, leading to decreased PDX1 expression at the early pancreatic progenitor stage. Thus, the combination of an underlying defect in leucine catabolism characteristic of KPD with a functionally relevant heterozygous variant in a critical beta-cell gene that confers increased leucine sensitivity and inhibits endocrine cell differentiation resulted in the phenotype of late-onset beta-cell failure in this patient. We define the molecular pathogenesis of a diabetes syndrome and demonstrate the power of multi-omics analysis of patient-specific stem cells for clinical discovery.

Non-functional pancreatic neuroendocrine tumours: ATRX/DAXX and alternative lengthening of telomeres (ALT) are prognostically independent from ARX/PDX1 expression and tumour size

ObjectiveRecent studies have found aristaless-related homeobox gene (ARX)/pancreatic and duodenal homeobox 1 (PDX1), alpha-thalassemia/mental retardation X-linked (ATRX)/death domain-associated protein (DAXX) and alternative lengthening of telomeres (ALT) to be promising prognostic biomarkers for non-functional pancreatic neuroendocrine tumours (NF-PanNETs). However, they have not been comprehensively evaluated, especially among small NF-PanNETs (≤2.0 cm). Moreover, their status in neuroendocrine tumours (NETs) from other sites remains unknown.DesignAn international cohort of 1322 NETs was evaluated by immunolabelling for ARX/PDX1 and ATRX/DAXX, and telomere-specific fluorescence in situ hybridisation for ALT. This cohort included 561 primary NF-PanNETs, 107 NF-PanNET metastases and 654 primary, non-pancreatic non-functional NETs and NET metastases. The results were correlated with numerous clinicopathological features including relapse-free survival (RFS).ResultsATRX/DAXX loss and ALT were associated with several adverse prognostic findings and distant metastasis/recurrence (p<0.001). The 5-year RFS rates for patients with ATRX/DAXX-negative and ALT-positive NF-PanNETs were 40% and 42% as compared with 85% and 86% for wild-type NF-PanNETs (p<0.001 and p<0.001). Shorter 5-year RFS rates for ≤2.0 cm NF-PanNETs patients were also seen with ATRX/DAXX loss (65% vs 92%, p=0.003) and ALT (60% vs 93%, p<0.001). By multivariate analysis, ATRX/DAXX and ALT status were independent prognostic factors for RFS. Conversely, classifying NF-PanNETs by ARX/PDX1 expression did not independently correlate with RFS. Except for 4% of pulmonary carcinoids, ATRX/DAXX loss and ALT were only identified in primary (25% and 29%) and NF-PanNET metastases (62% and 71%).ConclusionsATRX/DAXX and ALT should be considered in the prognostic evaluation of NF-PanNETs including ≤2.0 cm tumours, and are highly specific for pancreatic origin among NET metastases of unknown primary.

Epigenetic landscape of pancreatic neuroendocrine tumours reveals distinct cells of origin and means of tumour progression

Recent data suggest that Pancreatic Neuroendocrine Tumours (PanNETs) originate from α- or β-cells of the islets of Langerhans. The majority of PanNETs are non-functional and do not express cell-type specific hormones. In the current study we examine whether tumour DNA methylation (DNAme) profiling combined with genomic data is able to identify cell of origin and to reveal pathways involved in PanNET progression. We analyse genome-wide DNAme data of 125 PanNETs and sorted α- and β-cells. To confirm cell identity, we investigate ARX and PDX1 expression. Based on epigenetic similarities, PanNETs cluster in α-like, β-like and intermediate tumours. The epigenetic similarity to α-cells progressively decreases in the intermediate tumours, which present unclear differentiation. Specific transcription factor methylation and expression vary in the respective α/β-tumour groups. Depending on DNAme similarity to α/β-cells, PanNETs have different mutational spectra, stage of the disease and prognosis, indicating potential means of PanNET progression.

Tectorigenin enhances PDX1 expression and protects pancreatic β-cells by activating ERK and reducing ER stress

Pancreas/duodenum homeobox protein 1 (PDX1) is an important transcription factor that regulates islet β-cell proliferation, differentiation, and function. Reduced expression of PDX1 is thought to contribute to β-cell loss and dysfunction in diabetes. Thus, promoting PDX1 expression can be an effective strategy to preserve β-cell mass and function. Previously, we established a PDX1 promoter-dependent luciferase system to screen agents that can promote PDX1 expression. Natural compound tectorigenin (TG) was identified as a promising candidate that could enhance the activity of the promoter for the PDX1 gene. In this study, we first demonstrated that TG could promote the expression of PDX1 in β-cells via activating extracellular signal-related kinase (ERK), as indicated by increased phosphorylation of ERK; this effect was observed under either normal or glucotoxic/lipotoxic conditions. We then found that TG could suppress induced apoptosis and improved the viability of β-cells under glucotoxicity and lipotoxicity by activation of ERK and reduction of reactive oxygen species and endoplasmic reticulum (ER) stress. These effects held true in vivo as well: prophylactic or therapeutic use of TG could obviously inhibit ER stress and decrease islet β-cell apoptosis in the pancreas of mice given a high-fat/high-sucrose diet (HFHSD), thus dramatically maintaining or restoring β-cell mass and islet size, respectively. Accordingly, both prophylactic and therapeutic use of TG improved HFHSD-impaired glucose metabolism in mice, as evidenced by ameliorating hyperglycemia and glucose intolerance. Taken together, TG, as an agent promoting PDX1 expression exhibits strong protective effects on islet β-cells both in vitro and in vivo.