1. Prevention of Steatosis by Hepatic JNK1
Guadalupe Sabio, Julie Cavanagh-Kyros, Hwi Jin Ko, Dae Young Jung, Susan Gray, John Y. Jun, Tamera Barrett, Alfonso Mora, Jason K. Kim, Roger J. Davis 
Cell Metabolism 
Volume 10, Issue 6, Pages (December 2009)
DOI: /j.cmet
Copyright © 2009 Elsevier Inc. Terms and Conditions

2. Figure 1
Mice with Hepatocyte-Specific Deficiency of JNK1 Are Glucose Intolerant
(A) The liver and quadriceps muscle of Alb-cre Jnk1+/+ (LWT) mice, Alb-cre Jnk1LoxP/LoxP (LKO) mice, and Jnk1−/− (KO) mice were examined by immunoblot analysis by probing with antibodies to JNK1 and GAPDH.
(B) LWT and LKO mice were fed a chow diet (ND) or a high fat diet (HFD) for 16 wks. JNK activity in epididymal (white) fat and liver was measured in a protein kinase (KA) assay using cJun and ATP[γ-32P] as substrates. The cell extracts used for the protein kinase assay were also examined by immunoblot analysis by probing with an antibody to GAPDH.
(C) The phosphorylation of IRS1 on Ser-307 in the liver was examined using ND- and HFD- fed LWT and LKO mice by immunoblot analysis by probing with antibodies to IRS1 and phosphoSer-307 IRS1.
(D) Chow- fed LWT and LKO mice were fasted overnight and administered glucose (2g/kg) by intraperitoneal injection. The activation of AKT in the liver was examined by immunoblot analysis by probing with antibodies to AKT and phosphoAKT.
(E) Glucose tolerance tests of chow-fed LWT and LKO mice were performed by measurement of blood glucose concentration in animals following intraperitoneal injection of glucose (1g/kg). The data presented represent the mean ± SD (n = 10 ∼15). Statistically significant differences are indicated (∗p < 0.05; ∗∗p < 0.01).
(F) Chow- fed LWT and LKO mice were fasted overnight or fed ad libitum and the blood glucose concentration was measured (mean ± SD; n = 10 ∼15). Statistically significant differences are indicated (∗∗∗p < 0.001).
Cell Metabolism  , DOI: ( /j.cmet )
Copyright © 2009 Elsevier Inc. Terms and Conditions

3. Figure 2 JNK1 Deficiency in Hepatocytes Increases Insulin Clearance
(A) Chow-fed LWT and LKO mice were fasted overnight. In (A), the concentration of blood insulin was measured (mean ± SD; n = 10). No statistically significant difference between LKO and LWT mice was detected.
(B) The effect of administration of glucose (2 g/kg body mass) by intraperitoneal injection on blood insulin concentration was examined (mean ± SD; n = ∼13–15). Statistically significant differences between LKO and LWT are indicated (∗p < 0.01).
(C) The concentration of insulin C-peptide in the blood was measured (mean ± SD; n = ∼10–15). Statistically significant differences between LKO and LWT are indicated (∗∗∗∗p < ).
(D) The effect of administration of glucose (2 g/kg body mass) on insulin C-peptide concentration in the blood was examined (mean ± SD; n = 15). No statistically significant difference between LKO and LWT was detected.
(E) Mice were injected with human insulin (1.5 U/kg body mass). The concentration of human insulin in the blood was measured (mean ± SD; n = 15). Statistically significant differences between LKO and LWT are indicated (∗p < 0.05).
(F) The expression of the insulin receptor, Ceacam-1, and Gapdh mRNA in the liver was measured by quantitative RT-PCR (Taqman) assays. The expression of insulin receptor (InsR) and Caecam-1 mRNA was normalized to the amount of 18S RNA in each sample (mean ± SD; n = 7). Statistically significant differences between LKO and LWT are indicated (∗p < 0.05; ∗∗∗p < 0.001).
Cell Metabolism  , DOI: ( /j.cmet )
Copyright © 2009 Elsevier Inc. Terms and Conditions

4. Figure 3
Mice with JNK1-Deficient Hepatocytes Exhibit Hepatic Insulin Resistance
(A–F) Insulin sensitivity was measured using a hyperinsulinemic-euglycemic clamp in conscious chow-fed LKO and LWT mice. In (A), basal hepatic glucose production (HGP) is shown. In (B), insulin-stimulated rate of HGP is shown. In (C), hepatic insulin action, expressed as insulin-mediated percent suppression of basal HGP is portrayed. In (D), insulin-stimulated whole body glucose turnover is shown. In (E), whole-body fat mass measured using 1H-MRS is shown. In (F), whole-body lean mass is shown. The data presented are the mean ± SE for ∼6–8 experiments. Statistically significant differences between LKO mice and LWT mice are indicated (∗p < 0.05).
(G and H) Chow-fed LKO and LWT mice were administered insulin (0.3U/kg body mass) by intravenous injection (5 min). The activation of AKT in the liver was examined by immunoblot analysis by probing with antibodies to AKT and phospho-AKT. The relative amount of phospho-AKT is presented as the mean ± SD (n = 3). Statistically significant differences between LKO and LWT mice are indicated (∗p < 0.05).
(I) Chow-fed LKO and LWT mice were administered insulin (0.3U/kg body mass) by intravenous injection (5 min). The phosphorylation of IRS1 on Ser307 in the liver was examined by immunoblot analysis by probing with antibodies to IRS1 and phospho-Ser307 IRS1.
Cell Metabolism  , DOI: ( /j.cmet )
Copyright © 2009 Elsevier Inc. Terms and Conditions

5. Figure 4 JNK1 Deficiency in Hepatocytes Causes Steatosis
(A) Chow-fed LWT and LKO mice were fasted overnight. Representative sections of the liver stained with oil red O are presented.
(B) The amount of hepatic triglyceride was measured in mice fasted overnight (mean ± SD; n = 10). Statistically significant differences between LKO and LWT are indicated (∗p < 0.05).
(C) The amount of hepatic lipogenesis was measured in mice fasted 6 hr (mean ± SD; n = ∼8–9). Statistically significant differences between LKO and LWT are indicated (∗p < 0.05).
(D) The expression of genes that encode lipogenic transcription factors and coactivators in the liver of chow-fed LKO and LWT mice that were fasted overnight (C/ebpα, C/ebpβ, Pgc1β, Pparγ, and Srebp1) was measured by quantitative RT-PCR assays of the amount of mRNA and was normalized to the amount of 18S RNA in each sample (mean ± SD; n = 7). Statistically significant differences between LKO and LWT are indicated (∗p < 0.05; ∗∗p < 0.01).
(E) The expression of genes that encode enzymes that promote lipogenesis in the liver of chow-fed LKO and LWT mice that were fasted overnight (Fas, fatty acid synthase; Acsl1/4, acetyl-CoA synthetase long chain family member 1/4; Acacα/β, acetyl-CoA carboxylase α/β; Acot3, Acetyl-CoA thioesterase; Dgat1, Diacylglycerol O-acyltransferase homolog 1; Glyk, Glycerol kinase; Mttp, microsomal triglyceride transfer protein) was measured by quantitative RT-PCR assays of the amount of mRNA and was normalized to the amount of 18S RNA in each sample (mean ± SD; n = 7). Statistically significant differences between LKO and LWT are indicated (∗p < 0.05; ∗∗p < 0.01).
Cell Metabolism  , DOI: ( /j.cmet )
Copyright © 2009 Elsevier Inc. Terms and Conditions