Figure 2.

Hepatocyte-specific deletion of GNE decreases insulin receptor (IR) sialylation and protein levels, and impairs IR signaling in the liver. A–C. Male GNE^fl/fl mice were injected with control AAV8-GFP or AAV8-Cre at 5 weeks of age, and fed control diet for 2–4 weeks. Hyperinsulinemic–euglycemic clamps were performed, and glucose infusion rate (GIR, A), hepatic glucose production (HGP, B) and HGP suppression rates (C) were measured (N = 10,11/group). D, E. After the clamp, livers were isolated, and expression of IR precursor (Pro-IR), IRα and IRβ, phosphorylation of IRβ-Tyr1146, Akt-Ser473 and GSK3α/β-Ser21/9 were assessed by immunoblot (D). E. Quantitation by densitometry (N = 8–11/group). F. Insulin receptor (IR) mRNA expression in the liver by qRT-PCR (N = 12/group). G–H. Two weeks post AAV8 injection, mice were injected with vehicle (PBS) or insulin (2 U/kg, 5 min), and liver Akt-ser473 phosphorylation and insulin receptor expression were assessed by immunoblot (G). H. Quantitation by densitometry for phosphorylated Akt (N = 6/group). I. Insulin Receptors (IR) were immunoprecipitated from liver lysate, and IR sialylation and galactosylation were assessed by SNA-lectin, MAL-II lectin or ECL-lectin blotting. Findings for 3 samples per group are shown. J. Separate sets of immunoprecipitated IRβ were treated with neuraminidase (NA), and its electromobility and terminal galactosylation were assessed by IRα/IRβ immunoblot and ECL-lectin blot. Findings for 2 samples per group are shown. Data are represented as mean ± SEM. Significance was determined by unpaired two tailed Student's t test (A, Pro-IR and IRβ in E, F), unpaired two tailed Student's t test with Welch's correction (C, IRα, pIRβ, pAkt and pGSK3 in E) and two-way ANOVA with Tukey's multiple comparison analysis (B, H).