Fig. 1: Kinase- and domain-dependent roles of IR in regulating metabolism and growth.
a Schematic representation of DKO preadipocytes reconstituted with IR, K1030R mutated IR (K1030R), mutated IR lacking 79 amino acids from the C-terminus (∆CT), and mutated IR lacking most of the intracellular domain, but with the 36 amino acid juxtamembrane domain (JMO). b Relative mRNA levels of recombinant receptors of IR, K1030R, ∆CT, and JMO as determined by qPCR using cDNA standards for quantitation. The dotted line is the level of IR mRNA in WT cells. Data are means ± SEM copy number per ng total RNA (n = 4). c Immunoblotting of IR-beta subunit (IRβ) in lysates of the membrane (Mem), cytosolic (Cyt), and nucleus fraction from DKO, IR, K1030R, ∆CT, and JMO cells. d Immunoblotting of phosphorylated and total receptor protein levels, IRS-1Y608 phosphorylation, and AktS473 phosphorylation in lysates from DKO, IR, K1030R, ∆CT, and JMO cells stimulated with 100 nM insulin for 15 min. e Triglyceride accumulation in DKO, IR, K1030R, ∆CT, and JMO cells by Oil red O staining day 7 after induction of differentiation (n = 3). Data are means ± SEM. P-values vs. DKO, one-way ANOVA. f Proliferation rates of DKO, IR, K1030R, ∆CT, and JMO cells (n = 4) per day are shown as means ± SEM. P-values vs. DKO, one-way ANOVA. g mRNA levels of Pfkl in DKO, IR, K1030R, ∆CT, and JMO cells. Cells were FBS starved for 5 h with DMEM containing 0.1% BSA, then stimulated with or without 100 nM insulin for 6 h. mRNA levels of genes were analyzed by qPCR. Data are means ± SEM (n = 4). Gene expression levels of DKO cells at the basal were set at 1. P-values are basal vs insulin, two-way ANOVA. TBP expression was used to normalize gene expression. h Glycolytic rate (maximal glycolytic capacity) induced by insulin (100 nM) stimulation for 6 h. Fold change of the maximal glycolytic capacity as measured by ECAR on insulin stimulation was calculated for each cell line. Data are means ± SEM (n = 5–6). P-values are basal vs insulin stimulation.
