Figure 7.

Hepatocyte-specific deletion of GNE promotes insulin signaling and glucose uptake in the skeletal muscle by causing increased hepatic production of FGF21. A, B. 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. Glucose disposal rate (GDR, A) and insulin-stimulated glucose disposal rate (IS-GDR, B) during hyperinsulinemic–euglycemic clamp (N = 10–11/group). C. During a GTT [³H]-2-deoxyglucose uptake was evaluated in the extensor digitorum longus (EDL) and soleus muscles (N = 10–11/group). D, E. Insulin receptor (IR) expression and signaling downstream of IR were evaluated following the clamp by immunoblot (E). Quantitation of immunoblots is shown in E (N = 10–11/group). F, G. Plasma FGF21 concentrations in mice without fasting or after 6 h or 16 h fast (F, N = 8–14/group). FGF21 mRNA expression levels were evaluated by qRT-PCR in liver, white adipose tissue (WAT), brown adipose tissue (BAT), skeletal muscle (Skel Muscl) and pancreas (G, N = 6–12/group). H, I. Male GNE^fl/fl, FGF21^fl/fl or GNE^fl/fl;FGF21^fl/fl mice were injected with control AAV8-GFP or AAV8-Cre at 5 weeks of age, and fed control diet for 2 week. Plasma FGF21 levels (H, N = 9–13/group) and plasma glucose levels after 6 h fast (I, N = 11–12/group). J. During a GTT [³H]-2-deoxyglucose uptake was evaluated in soleus and EDL skeletal muscle (N = 7/group). K, L. Two weeks post AAV8 injection, mice were injected with vehicle (PBS) or insulin (0.5 U/kg, 5 min), and Akt-ser473 phosphorylation in the soleus were assessed by immunoblot (K). Quantitation by densitometry for phosphorylated Akt (L, N = 3–6/group). M. Glucagon tolerance tests (16 μg/kg glucagon) after 6 h fast at 2 weeks post AAV8 injections (N = 11–12/group). N. Glucose tolerance tests (1 g/kg glucose) after 6 h fast at 2 weeks post AAV8 injection (N = 11–12/group). O. Insulin tolerance tests (0.5 U/kg insulin) after 6 h fast at 2 weeks post AAV8 injection (N = 11–12/group). Data are represented as mean ± SEM. Significance was determined by unpaired two tailed Student's t test (A, B, ProIR, IRα and IRβ in E, G), unpaired two tailed Student's t test with Welch's correction (C, pIRβ, pAkt, pGSK in E), one-way ANOVA with Tukey's multiple comparison (J), Brown-Forsythe and Welch ANOVA with Dunnett's T3 multiple comparisons test (H, I), two-way ANOVA with Tukey's multiple comparison test (F, L), or two-way repeated measures ANOVA with Bonferroni multiple comparison test to compare blood glucose among groups (M–O).