Volume 60, Issue 5, Pages 1017-1025 (May 2014) Cardiotrophin-1 eliminates hepatic steatosis in obese mice by mechanisms involving AMPK activation  David Castaño, Eduardo Larequi, Idoia Belza, Alma M. Astudillo, Eduardo Martínez-Ansó, Jesús Balsinde, Josepmaria Argemi, Tomás Aragon, María J. Moreno-Aliaga, Jordi Muntane, Jesús Prieto, Matilde Bustos  Journal of Hepatology  Volume 60, Issue 5, Pages 1017-1025 (May 2014) DOI: 10.1016/j.jhep.2013.12.012 Copyright © 2013 European Association for the Study of the Liver Terms and Conditions

Fig. 1 Expression of CT-1 in steatotic livers. Chronic treatment with rCT-1 decreases fat stores and reduces inflammatory cytokines in the liver from ob/ob mice. (A) Hepatic CT-1 mRNA and protein in ob/ob vs. wild-type (WT) mice and HFD-fed vs. control diet (CD)-fed animals. (B) CT-1 mRNA in livers from healthy livers (HL) and NAFLD. (C) Liver weight/body weight (LW/BW), serum transaminases ALT and AST. (D) Quantification of TG and DAG in livers from ob/ob mice after treatment with rCT-1 or saline (ad libitum controls and pair-fed [PF]) over 10days. Data are expressed as mean±SEM. (E) H&E staining of liver sections. (F) Hepatic expression of genes involved in inflammation (G) FA oxidation. Data are expressed as fold change relative to saline-treated ob/ob mice (dotted line). ∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001 vs. saline-treated group; #p<0.05, ##p<0.01, ###p<0.001 vs. rCT-1-treated group. Journal of Hepatology 2014 60, 1017-1025DOI: (10.1016/j.jhep.2013.12.012) Copyright © 2013 European Association for the Study of the Liver Terms and Conditions

Fig. 2 rCT-1 over 10days decreases lipogenesis, modifies lipid composition and increases insulin sensitivity in livers from ob/ob mice. (A) mRNA (fold change relative to saline controls; dotted line) and (B) protein levels of lipogenic genes. (C) Oleate (C18:1n-9) and desaturation index (C16:1n-10/C16:0) in liver. (D) FA composition of liver samples: saturated, monounsaturated and polyunsaturated FA. (E) SREBP1c (nSREBP1-c), CHREBP (nCHREBP) and laminin A/C in liver nuclear extracts. (F) Precursor SREBP-1c (pSREBP-1c) and CHREBP in liver cytosolic fraction. (G) Western blots and densitometry of p-AKT (Ser473) and p-IRS1 (Tyr896). (H) mRNA expression of LPL and FAT/CD36. Data are expressed as fold change relative to saline-treated ob/ob mice (dotted line). ∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001 vs. saline-treated group; #p<0.05, ##p<0.01, ###p<0.001 vs. rCT-1-treated group. Journal of Hepatology 2014 60, 1017-1025DOI: (10.1016/j.jhep.2013.12.012) Copyright © 2013 European Association for the Study of the Liver Terms and Conditions

Fig. 3 Chronic rCT-1 treatment phosphorylates SREBP-1c and AMPK, induces NAD+ biosynthesis and SIRT1 expression and activates PGC1α in livers from ob/ob mice. (A) Western blots and densitometric analysis for Ser372 phosphorylation of SREBP-1c and (B) Thr172 phosphorylation of AMPK and Ser431 phosphorylation of LKB1 in ob/ob mice after treatment with rCT-1 or saline (ad libitum controls and PF) over 10days. (C) NAMPT mRNA level expressed as fold change relative to saline-treated ob/ob mice (dotted line) and NAD+/NADH ratio. (D) SIRT1 protein levels and (E) Acetylated PGC1α in nuclear extracts from the 3 groups of ob/ob mice. ∗p<0.05, ∗∗∗p<0.001 vs. saline-treated group; #p<0.05, ##p<0.01 vs. rCT-1-treated group. Journal of Hepatology 2014 60, 1017-1025DOI: (10.1016/j.jhep.2013.12.012) Copyright © 2013 European Association for the Study of the Liver Terms and Conditions

Fig. 4 The effect of rCT-1 on liver lipid metabolism is blocked by inhibiting AMPK activation. (A) Liver samples from ob/ob mice 30min after injection of rCT-1 analyzed for p-AMPK (Thr172) and p-LKB1 (Ser428), AMP/ATP ratio, FA oxidation rate and mRNA of lipogenic genes.∗p<0.05, ∗∗p<0.001 vs. saline-treated group. (B) Cultured hepatocytes with or without rCT-1 under basal or lipogenic conditions (insulin+glucose [I+G]). FA oxidation assay was performed under basal conditions. Data are expressed as fold change relative to untreated hepatocytes under basal conditions (dotted line); ∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001 vs. untreated hepatocytes under basal conditions; ##p<0.01, ###p<0.001 vs. hepatocytes under lipogenic conditions. (C–E) Cultured hepatocytes were transduced with AdGFP or AdDNAMPKα1. (C) Western blots, (D) Sterol and FA synthesis, (E) FA oxidation rate. A-769662 is an AMPK activator, used as a positive control. Data are expressed as fold change relative to untreated transduced hepatocytes under basal conditions (dotted line); ∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001 vs. Ad-transduced hepatocytes under basal conditions; ##p<0.01, ###p<0.001 vs. Ad-transduced hepatocytes under lipogenic conditions. Journal of Hepatology 2014 60, 1017-1025DOI: (10.1016/j.jhep.2013.12.012) Copyright © 2013 European Association for the Study of the Liver Terms and Conditions