Figure 6.

Analyses of the role of βArr2 in CB₁R-induced, obesity-related muscle insulin resistance. (a) 2-Deoxyglucose was infused into anesthetized wild-type, βArr2-KO or βArr1-KO mice and its uptake measured in soleus muscle from lean mice or mice with diet-induced obesity 1 h following treatment with a single oral dose of 1 mg/kg (S)-MRI-1891 or vehicle. (b) 2-Deoxyglucose uptake measured as in panel (a), except that treatment with (S)-MRI-1891 was for 7 days at 0.1 mg/kg/day. (c) Insulin-induced Akt phosphorylation and its CB₁R-mediated inhibition were analyzed in mock-transfected and βArr2-siRNA-transfected C2C12 myotubes. Each treatment was tested in duplicate aliquots of cells, analyzed by Western blot using β-actin as loading control, and quantified by densitometry. The level of βArr2 knockdown is illustrated by the bar graph on the right. Note that the inhibition of insulin-induced akt-phosphorylation by the CB₁R agonist is inhibited by MRI-1891 and is absent in cells with βArr2 knockdown. (d) CB₁R-mediated inhibition of insulin-induced Akt phosphorylation is absent in C2C12 myotubes with Crip1a overexpression and (e) is enhanced in myotubes with Crip1a knockdown. (f) High-fat diet-induced obesity results in downregulation of Crip1a expression in soleus muscle from wild-type but not from βArr2-KO mice. *, significant difference (P < 0.05) within the indicated groups, as determined by 2-way ANOVA followed by Dunnett’s multiple comparisons test. #, significant difference (P < 0.05) between the groups, as determined by 2-way ANOVA followed by Sidaks’s multiple comparisons test. Columns and vertical bars represent mean ± SEM from 8–10 animals.