Fig. 5

Effect of the cytoplasmic IDE on glucose production and transcriptomic analysis in IDE-deficient hepatocytes. The cytoplasmic isoform of IDE regulates glucose production in hepatocytes. (A) AML12-shRNA-IDE cells were transfected with a plasmid containing the cytoplasmic isoform of IDE (Met⁴²-IDE) and stimulated with glucagon (50 ng/mL) for 1 h. Afterwards, glucose production was quantified. Results are mean ± SEM. n = 3 per condition. *p value < 0.05 versus control by two-way ANOVA. Characterization of HepG2 cells with genetic depletion of IDE. HepG2-IDE-KO cells were generated using the CRISPR/Cas9 as described in the Methods section. (B) IDE protein levels. Upper panel, representative western blot image. Lower panel, quantification of IDE in control and HepG2-IDE-KO cells. Results are mean ± SEM. n = 4 per group. *p value < 0.05 versus control by Students’ T-test. (C) Assessment of the proteolytic activity of IDE in control and HepG2-IDE-KO cells. Results are mean ± SEM. n = 3 (control) and n = 5 (HepG2-IDE-KO). *p value < 0.05 versus control by Students´ T-test. Transcriptomic analyses in HepG2 cells. (D) Colored heatmap (left) shows gene expression (blue: low expression, red: high expression) across control and HepG2-IDE-KO samples. Right panel shows a functional profile of differentially expressed genes (NUC MET: nucleotides metabolism; CARB MET: carbohydrates metabolism; MIT FUNC: mitochondrial function; ME, OR, SIG: Membrane, organelles, signaling receptor; LIP MET: lipids metabolism). (E) Venn diagram. Number of up-regulated (left) and down-regulated (right) genes sharing one or more functional profiles. Genes with an adjusted p-value < 0.05 and a log2(Fold Change) > 1 were considered significantly differently expressed.