Casitas b-lymphoma (CBL) is an adaptor protein that rapidly becomes phosphorylated following insulin receptor activation in adipose and muscle cells. While CBL null mice exhibits improved insulin sensitivity and enhanced glucose clearance, the molecular mechanisms have not been fully elucidated. Here we depleted CBL or the associated protein SORBS1 also known as Cbl Associated Protein (CAP) in C2C12 myocytes via lentiviral delivery of specific shRNAs to investigate the molecular mechansisms. Methods: We determined AMPK activation by western blotting, mitochondrial mass by flow Cytometry, 2-deoxyuglucose uptake, mitochondrial respiration with SeaHorse analyser. We determined mitochondrial OXPHOS complex activities by spectrophotometric methods and mitochondrial respiratory complex assembly by Blue Native gel electrophoresis and western blot. Cellular proteome and metabolome was analysed by MS and NMR. ANOVA Statistical analysis of biochemical assays was performed using GraphPad Prism6. Bioinformatic analysis of proteome and metabolome data was carried out in R/Bioconductor package. Results: CBL depletion increased AMP- regulated kinase activation in C2C12 cells, Consistently, CBL KD myocytes showed significantly increased basal and insulin-stimulated glucose uptake, increased expression of transcriptional coactivators PGC1α and PGC1β, and higher mitochondrial mass compared to control cells. Cellular respiration rates in CBL and CAP KD cells showed greater proton leak than control cells. Respiratory chain complex I (CI) activity and assembly into supercomplexes I+III+IV were significantly reduced. Furthermore, cellular proteome and metabolome analyses revealed alterations affecting proteins involved in CI biogenesis, fatty acid degradation and glycolysis. Conclusions: Our findings demonstrate that the CBL/CAP pathway couples insulin signalling to efficient mitochondrial respiratory function and metabolic adaptation in muscle cells.
Physiology 2019 (Aberdeen, UK) (2019) Proc Physiol Soc 43, PC151
Poster Communications: Cbl/CAP are essential for mitochondria respiration complex assembly and bioenergetics efficiency in muscle cells.
C. Aye1, D. E. Hammond1, S. Rodriguez-Cuenca2, M. K. Doherty3, P. D. Whitfield3, M. Phelan1, C. Yang1, R. Perez-Perez4, X. Li1, A. Diaz-Ramos5, A. Vidal-Puig2, A. Zorzano5, C. Ugalde4, S. Mora1
1. Physiology, University of Liverpool, Liverpool, United Kingdom. 2. University of Cambridge, Cambridge, United Kingdom. 3. University of Highlands and Islands, Inverness, United Kingdom. 4. Hospital 12 Octubre, Madrid, Spain. 5. IRB, Barcelona, Spain.
View other abstracts by:
Where applicable, experiments conform with Society ethical requirements.