(A and B) i.p. Glucose tolerance tests (IGTT). (C and D) i.p. pyruvate tolerance tests (PTT). In B and D, an anti-GCGR Ab (10 mg/kg i.p) was administered approximately 24 hours prior to glucose or pyruvate injections, respectively. (E) i.p. glucagon challenge test (GCT). (F–I) In vitro studies with primary mouse hepatocytes. The ability of glucagon to stimulate intracellular cAMP accumulation (F), glucose production (G), and the expression of the G6Pase (H) and Pepck (I) genes was studied using primary hepatocytes prepared from hep-barr2–KO mice and their control littermates. Gene expression data were normalized relative to the expression of β-actin (quantitative reverse-transcriptase PCR [qRT-PCR] analysis). Hepatocyte data are shown as mean ± SEM of at least 3 independent experiments. (J and K) Hepatic glycogen and triglyceride content. All studies were carried out with male mice consuming RC (mouse age, 11–20 weeks). Data represent mean ± SEM (n = 7–10 mice per group) (A–K). *P < 0.05; **P < 0.01, versus control (Student’s t test). (L and M) GCGR internalization assays. Primary hepatocytes prepared from hep-barr2–KO (L) or hep-barr1–KO (M) mice and their control littermates were stimulated with glucagon (100 nM) for 30 minutes at 37 °C. Cell surface GCGRs were labeled with I¹²⁵-glucagon. Data represent the mean ± SEM of 3 independent experiments, each carried out in triplicate. **P < 0.01, versus cells not exposed to glucagon (Student’s t test).