Investigating the Impact of FGFR5 Expression on β-cell Metabolism, Maturity and Intracellular Signaling
Year: 2023
Type: dissertation
Abstract: Pancreatic β-cells exist in an incredibly dynamic microenvironment that impacts cell metabolism, maturity, and function. Our research group previously showed that pancreatic β-cells plated on laminin matrix express reduced levels of FGFR1, a kinase receptor linked to β-cell metabolism. Due to recent evidence that adult β-cells also express FGFR5, a co-receptor for FGFR1, we hypothesize that laminin would impact FGFR5 expression with consequent effects on β-cell metabolism. Using a genetically encoded sensor for NADPH/NADP+ redox state (Apollo-NADP+), we show overexpression of FGFR5 enhances glucose-stimulated NADPH metabolism in β-cell lines as well as mouse and human β-cells. This enhanced response was accompanied by increased expression of transcripts for glycolytic enzymes (Glucokinase/Gck, Pyruvate kinase M2/Pkm2) and the functional maturity marker Urocortin 3 (Ucn3). Culturing β-cells on laminin matrix also stimulated upregulation of endogenous FGFR5 expression, and similarly enhanced β-cell glucose-stimulated NADPH-metabolism as well as Gck and Pkm2 transcript expression. Collectively these data reveal that β-cells respond to laminin by increasing FGFR5 expression to enhance β-cell glucose metabolism. We next examined changes to intracellular signaling pathways activated by FGFR5 expression. Using live cell imaging and qPCR we showed that the FGFR5-induced changes to metabolism are due to activation of the MEK/ERK signaling pathway. We then examined if the metabolic changes induced by MEK/ERK activation, specifically increased glucose consumption, had subsequent effects on nutrient sensing pathways. Using Western immunoblot and a variety of live cell imaging techniques, we showed FGFR5-induced metabolism increased activation of the nutrient sensing mTOR pathway. Lastly, we used data mining to uncover gene correlations to FGFR5 based on a human online β-cell database. Findings from the data mining analysis were validated using qPCR and identified the gene Rcc2 (Regulator of chromosome condensation 2) as being upregulated by FGFR5 in β-cell lines. We then expanded our data mining approach to better understand changes associated with late-stage β-cell maturation by centering our analysis on UCN3, a β-cell maturity marker. Our UCN3 focused analysis allowed us to construct a “late-stage maturation map” for β-cells which identified correlation to notable integrin subunits and solute carrier genes.
Source: TSpace (University of Toronto)
Author: Pal, Vidhant
Institution: University of Toronto
Language: English
Subfield: Surgery
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W7132935735