Volume 18, Issue 1, Pages 99-105 (July 2013) A Noncanonical, GSK3-Independent Pathway Controls Postprandial Hepatic Glycogen Deposition  Min Wan, Karla F. Leavens, Roger W. Hunter, Shlomit Koren, Alexander von Wilamowitz-Moellendorff, Mingjian Lu, Santhosh Satapati, Qingwei Chu, Kei Sakamoto, Shawn C. Burgess, Morris J. Birnbaum  Cell Metabolism  Volume 18, Issue 1, Pages 99-105 (July 2013) DOI: 10.1016/j.cmet.2013.06.001 Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 Deletion of Akt2 in the Liver Results in Hepatic Insulin Resistance (A) Chow-fed 2-month-old male control (Akt2(lox/lox)) and liver-specific Akt2 KO (Akt2(lox/lox); AFP > CRE) mice were subjected to a euglycemic-hyperinsulinemic clamp. GIR, glucose infusion rate; HGP, hepatic glucose production; Rd, rate of glucose disposal. Data are expressed as mean ± SEM; n = 4 for each genotype; ∗p < 0.05; ∗∗p < 0.01 by Student’s t test. (B) Glucose infusion rates during the euglycemic-hyperinsulinemic clamp. 2- to 3-month-old male control (Akt2(lox/lox)) and liver-specific Akt2 KO (Akt2(lox/lox); AFP > CRE) mice were subjected to a euglycemic clamp with infusion of insulin at 2.5 mU/kg/min. See also Figure S1. (C) G6pc and Pck1 mRNA levels in the liver at the termination of the clamp. (D) Hepatic glycogen content after the termination of the clamp. Data are expressed as mean ± SEM; n = 5–7 for each condition; ∗p < 0.05; ∗∗p < 0.01 by two-way ANOVA followed by the Bonferroni post hoc test. Cell Metabolism 2013 18, 99-105DOI: (10.1016/j.cmet.2013.06.001) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 Postprandial Hepatic Glycogen Accumulation Is Blunted in the Akt2(lox/lox); AFP > CRE Mice (A) Livers from control and Akt2 KO mice were perfused ex vivo, and glucose production, glycogenolysis, and gluconeogenesis was measured by isotopomer analysis of incorporated deuterium. Data are expressed as mean ± SEM; n = 4–6 for each condition; ∗p < 0.05; ∗∗p < 0.01 by two-way ANOVA followed by the Bonferroni post hoc test. (B–D) Male control (Akt2(lox/lox)) and liver-specific Akt2 KO (Akt2(lox/lox); AFP > CRE) mice (2 months old) were fasted overnight, refed, and, at 1 and 4 hr, assayed for (B) blood glucose and serum insulin, (C) gene expression, and (D) hepatic glycogen (left, 1 hr refed; right, 4 hr refed). Data are expressed as mean ± SEM, n = 5–7; ∗p < 0.05; ∗∗p < 0.01 by two-way ANOVA followed by the Bonferroni post hoc test. See also Figures S2D and S2E. Cell Metabolism 2013 18, 99-105DOI: (10.1016/j.cmet.2013.06.001) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 GSK3 Phosphorylation Is Not Required for Insulin-Dependent Accumulation of Glycogen in Liver (A and B) Immunoblot for phosphorylated Akt and GSK3α and GSK3β in livers from fasting mice at 1 hr (A) or 4 hr (B) after refeeding (upper panels). Quantification of Akt and GSK3 phosphorylation is shown in lower panels. (C) Hepatic glycogen content at the termination of the clamp. Male control or GSK3αS21A/S21A;βS9A/S9A mice (3 months old) were infused with PBS or insulin at 10 mU/kg/min while glucose was clamped as in Figure 2. Data are expressed as mean ± SEM; n = 6–7 for each condition; ∗p < 0.05 by two-way ANOVA followed by the Bonferroni post hoc test. See also Figure S3. (D–F) GSK3αS21A/S21A;βS9A/S9A mice and their controls were fasted overnight followed by 1 hr refed. (D) Blood glucose levels, (E) serum insulin levels, and (F) hepatic glycogen content were assayed at fasting and refeeding. n = 5 for each condition. Data are expressed as mean ± SEM; ∗∗p < 0.01 by two-way ANOVA followed by the Bonferroni post hoc test. See also Figures S2A–S2C. Cell Metabolism 2013 18, 99-105DOI: (10.1016/j.cmet.2013.06.001) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 Glycogen Synthase Activity Is Blunted after Refeeding in the Akt2(lox/lox); AFP > CRE Mice (A) Immunoblot for phosphorylated glycogen synthase (GS) and glycogen phosphorylase (GP) in livers from fasting mice and 1 hr after refeeding. (B) Hepatic GS activity after 1 hr refeeding. Data are expressed as mean ± SEM; ∗∗p < 0.01 by two-way ANOVA followed by the Bonferroni post hoc test. See also Figure S4. Cell Metabolism 2013 18, 99-105DOI: (10.1016/j.cmet.2013.06.001) Copyright © 2013 Elsevier Inc. Terms and Conditions