Although pancreatic islet transplantation has been largely successful there are a number of caveats for this treatment method, mainly concerning the limited availability of donor islets. There has therefore been an advance in the field of regenerative medicine for the generation of functional β-cells for transplantation for the treatment of diabetes. Pancreatic development is tightly regulated by a cascade of transcription factors and regulatory signalling pathways. Extensive research has been carried out into mapping the transcription factors onto specific stages of pancreatic development. Understanding these regulatory networks has been critical in the establishment of in-vitro differentiation protocols. There is a myriad of data supporting pancreatic plasticity and the ability of pancreatic exocrine cells to trans-differentiate towards an endocrine lineage. Previously in our laboratory, using the human pancreatic ductal cell line PANC-1, a small compound molecule (N-cyclopropyl-5-(2-thienyl)-3-isoxazole-carboxamide) referred to in this study as isoxazole, was shown to reprogramme PANC-1 cells towards an endocrine phenotype. The aim of this current project was to generate a metabolic profile of isoxazole treated PANC-1 cells and optimise the in-vitro cell culture environment to enhance trans-differentiation, with a hypothesis that metabolism may be driving some aspects of endocrine differentiation. Following a 48 hr treatment of PANC-1 cells with isoxazole, cells showed distinct changes in cell morphology with the formation of islet-like clusters and 'pseudo-ductal' structures. Morphometric data analysis using the CellProfiler software showed that isoxazole treatment induced enhanced cellular elongation, whilst decreasing cell symmetry and compactness, phenotypic of a migratory cell. Isoxazole up-regulated key genes involved in pancreatic development like Ngn3 and NeuroD1 and also mature β-cell markers such as insulin, SLC30A8 and the GLUT2 glucose transporter SLC2A2 measured by qRT-PCR. In addition to changes in gene expression, a metabolic profile of PANC-1 cells following isoxazole treatment was also generated using LC and GC mass spectrometry. This profile highlighted significantly altered metabolites from isoxazole treated cells and used cell culture medium, suggesting active utilisation of these metabolites during the trans-differentiation process. Many of the metabolites altered by isoxazole treatment were involved in the tricarboxylic acid (TCA) cycle. Interestingly, two metabolites (arginine and aspartate) were significantly lowered in the cell culture medium following isoxazole treatment when compared to the DMSO vehicle control. Supplementation of the cell culture medium with arginine and aspartate led to the enhanced expression of β-cell associated genes and transcription factors such as Ngn3, NeuroD1, insulin, and SLC30A8. Changes were also observed at a protein level, with an up-regulation in the levels of SUSD2, a novel endocrine progenitor cell surface marker, following isoxazole treatment. Collectively, this data suggests a key role for amino acid metabolism in endocrine differentiation, which could be used as a potential tool for enhancing the generation of pancreatic β-cells for transplantation.
|Date of Award
|1 Aug 2017
- The University of Manchester
|Mark Dunne (Supervisor) & Karen Cosgrove (Supervisor)
- pancreatic beta cell development, isoxazole, endocrine differentiation, cell culture, insulin, gene expression, amino acid metabolism, arginine, aspartate, asparagine