Figure 1: Differential roles of IR and IGF1R in regulating proliferation and glycolysis.

(a) Schematic showing IR/IGF1R double knockout preadipocytes reconstituted with normal IR, IGF1R, and chimeric receptors IR/IGF1R and IGF1R/IR. (b) Relative mRNA levels of endogenous IR and IGF1R from wild-type and DKO cells, as well as overexpressed recombinant receptors. Data were shown as mean±s.e.m. copy number per ng total RNA, n=3. Fold overexpression of each recombinant receptor to endogenous receptor are highlighted. (c) Immunoblotting of phosphorylated and total receptors in lysates from three independent lines of cells expressing normal IR and chimeric receptor IR/IGF1R stimulated with 10 nM insulin or cells expressing normal IGF1R and chimeric receptor IGF1R/IR stimulated with 10 nM IGF-1 for 5 min. (d) Proliferation rates of wild-type, DKO and receptor-reconstituted cells grown in DMEM+10% FBS. Cell doubling times per day are shown as mean±s.e.m. (**P<0.01; ***P<0.001. One-way ANOVA followed by Newman–Keuls post-hoc analysis, n=6). (e) Proliferation rates of wild-type, DKO cells and combined data on cells expressing receptors with IR-ICD and IGF1R-ICD grown in DMEM+10% FBS. Data are shown as mean±s.e.m. (*P<0.05; **P<0.01. One-way ANOVA followed by Newman–Keuls post-hoc analysis, n=12). (f) Glycolysis induced by insulin or IGF-1 (100 nM) stimulation for 6 h. Cells were serum starved overnight, stimulated with ligands and ECAR values as an indicator for glycolysis were measured using a Seahorse X24 Bioanalyzer. Fold change of glycolysis rates in response to stimulation were calculated for each cell line and presented as mean±s.e.m. (Student t-test, n=5). (g) Maximal glycolytic capacity induced by insulin or IGF-1 (100 nM) stimulation for 6 h. Fold change of the maximal glycolytic capacity as measured by ECAR upon ligand stimulation was calculated for each cell line and shown as mean±s.e.m. (*P<0.05, Student t-test, n=5).