Involvement of the inorganic phosphate transporter Pit1 in insulin signaling and metabolism
Duration of Inovarion intervention
In this context, the objective of the project was to study the role of the PiT1 protein on weight balance and metabolism in genetically modified animals. The objective was also to decipher the modalities of PiT1’s dialogue with molecular actors involved in metabolism.
Uncertainties, difficulties and technological complexities
The work undertaken presented the following technological and conceptual uncertainties, difficulties and constraints:
- no PiT1 molecular partners identified
- no known signal paths for PiT1
- no tools available for PiT1 detection
Description of the work carried out
The interaction of Pit1 with the major players in the insulin pathway was analyzed by immunoprecipitation directed against the insulin receptor and Insulin receptor substrate 1 (IRS1) in the presence of insulin. These experiments revealed an association of the IRS1/USP7 complex, a deubiquitination enzyme, which is enhanced in MEFs invalidated for Pit1. The PiT1/USP7 interaction was next investigated. As no good murine anti-PiT1 antibody was available, a strategy using a plasmid coding for the murine PiT1 protein tagged with the V5 detection peptide was chosen. After insulin stimulation, PiT1/USP7 interaction was examined in MEFs, primary cultured hepatocytes or HepG2 cells transfected with Pit1-V5 plasmid. All these experiments confirmed a direct PiT1/USP7 interaction. Thus, PiT1 deletion stabilizes the IRS1/USP7 interaction, which will result in the maintenance of insulin signalling in response to glucose, thus improving glucose tolerance.
All the results obtained led to the writing of an original article entitled « Disruption of the Phosphate Transporter Pit1 in Hepatocytes Improves Glucose Metabolism and Insulin Signaling by Modulating the USP7/IRS1 Interaction » by Forand A, Koumakis E, Rousseau A, Sassier Y, Journe C, Merlin JF, Leroy C, Boitez V, Codogno P, Friedlander G, Cohen I published in Cell Reports in 2016 (doi: 10.1016/j.celrep.2016.08.012; PMID: 27568561).