- Email: [email protected]
P0950 : Impact of kupffer cells on high fat induced insulin resistance and liver fetuin-A expression
POSTERS P0950 IMPACT OF KUPFFER CELLS ON HIGH FAT INDUCED INSULIN RESISTANCE AND LIVER FETUIN-A EXPRESSION N. Lanthier1 , V. Lebrun1 , M.-P. Berghmans1 , O. Molendi-Coste1 , I.A. Leclercq1 . 1 Laboratory of Hepato-Gastroenterology, Institut de Recherche Exp´erimentale et Clinique, Universit´e catholique de Louvain, Brussels, Belgium E-mail: [email protected] Background and Aims: Hepatokines (liver secreted proteins with possible distant action) are emerging potential players in insulin resistance in type 2 diabetic patients. Here, we explore the effect of a high fat diet on the expression of fetuin-A, one of those candidate liver proteins, and its relation with liver macrophage (Kupffer cell) activation. Methods: Male mice of 5 weeks of age were fed a normal diet (ND) or a high fat diet (HFD) for 3 days, known to initiate steatosis and insulin resistance. A preventive Kupffer cell (KC) depletion was obtained by intravenous injection of clodronate loaded liposomes and compared with PBS liposomes. The mRNA and protein expression of fetuin-A was evaluated by RT-PCR, Western-blot and immunofluorescence (IF) on different insulinsensitive tissues (liver, adipose tissue and muscle). Results: Short term HFD induced steatosis, KC activation and insulin resistance together with a significant increased expression of liver fetuin-A mRNA (1.5 fold, p < 0.01). However, liver fetuin-A protein expression remained unchanged under short term HFD. This increase in fetuin-A under high fat diet was not evidenced in the peripheral insulin sensitive tissues (skeletal muscle and adipose tissue) whether at the mRNA or at the protein level. Kupffer cell depletion in this setting did not reduce hepatic steatosis but significantly ameliorated insulin sensitivity proved by clamp studies. This amelioration in insulin sensitivity in KCdepleted mice was associated with a significant decrease in fetuin-A mRNA expression (0.7 fold, p < 0.01) compared to animals with KC. On immunofluorescence, fetuin-A was mostly expressed in centrilobular hepatocytes. Interestingly, while selectively depleting liver macrophages without affecting adipose tissue macrophage infiltration, intravenous clodronate injection was associated with a significant reduction in epididymal adipose tissue expansion compared to PBS injection (1.1% of body weight versus 1.6% of body weight, p < 0.001). Conclusions: This study demonstrates liver fetuin-A overexpression at the initiation of HFD feeding, concurrent with hepatic steatosis and insulin resistance. Targeting KC in this setting improved insulin sensitivity and was associated with a decreased adiposity and a reduced liver fetuin-A expression suggesting that fetuin-A acts as an hepatokine with pro-adiposity and pro-insulin resistance effects. P0951 MIR-21 INHIBITION AND FXR ACTIVATION SYNERGISTICALLY AMELIORATE DISEASE PATHOGENESIS IN A MOUSE MODEL OF NAFLD P.M. Rodrigues1 , M.B. Afonso1 , A.L. Sim˜ao1 , M. Caridade1 , C.C. Carvalho2 , A. Trindade2,3 , A. Duarte2,3 , P.M. Borralho1 , M.V. Machado4,5 , H. Cortez-Pinto4,5 , C.M.P. Rodrigues1 , R.E. Castro1 . 1 Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, 2 Reproduction and Development, Interdisciplinary Centre of Research in Animal Health (CIISA), Faculty of Veterinary Medicine, Universidade de Lisboa, Lisbon, 3 Gulbenkian Institute of Science, Oeiras, 4 Gastrenterology, Hospital Santa Maria, 5 IMM, Faculty of Medicine, Universidade de Lisboa, Lisbon, Portugal E-mail: [email protected] Background and Aims: Non-alcoholic fatty liver disease (NAFLD) comprises a spectrum of liver lesions, from simple steatosis to non-alcoholic steatohepatitis (NASH). Intrahepatic accumulation of fat represents a major triggering factor; still, disease pathogenesis S702
remains incomplete. Recently, microRNAs (miRNA/miRs) have been linked to NAFLD. In particular, mir-21 may contribute to disease progression by targeting peroxisome proliferator-activated receptor a (PPARa). Indeed, PPARa and farnesoid X receptor (FXR) constitute promising NAFLD therapeutic targets. We aimed to elucidate the role of miR-21 during NAFLD pathogenesis in mice, further evaluating the synergistic effect of miR-21 inhibition and FXR activation, using obeticholic acid (OCA). Methods: Wild type (WT; n = 24) and miR-21 knockout (KO; n = 24) mice were fed either a standard diet (SD; n = 12) or a fast food diet (FF; n = 12) for 25 weeks. 6 animals from each group had their diets supplemented with 60 mg/kg OCA (kindly provided by Intercept). Mice were weighed weekly while blood was collected and liver extracted and weighed at sacrifice. In parallel, human liver biopsies were obtained from morbid obese NAFLD patients at different disease stages (n = 28). Liver samples were processed for histological analysis and determination of miR-21 and PPARa expressions by qRT-PCR and immunoblotting, respectively. Serum was used for biochemical parameters analysis. Results: Our results show that, after 25 weeks, WT FF-fed mice develop NASH in parallel with an increase in both body weight and liver/body weight ratio, comparing with WT SD-fed mice (p < 0.05). Further, they exhibited increased miR-21 (p < 0.05) and decreased PPARa expressions (p < 0.05). Strikingly, miR-21 and PPARa also displayed an inverse and significant correlation in human patients, increasing from steatosis to less and more-severe NASH (p < 0.05). WT FF+OCA-fed animals displayed lower levels of miR-21, compared with WT FF-fed mice. KO FF-fed mice body weights and liver/body weight ratios were below WT FF-fed mice, as were serum levels of triglycerides and free fatty acids. Importantly, most of these beneficial effects were augmented in KO FF+OCA-fed mice (p < 0.05). Conclusions: In conclusion, our results indicate that miR-21 downregulation, likely leading to increased PPARa, together with FXR activation by OCA, ameliorate NAFLD pathogenesis, highlighting the therapeutic potential of novel synergistic therapies in NAFLD. (PTDC/BIM-MEC/0873/2012, SFRH/BD/88212/2012 and SFRH/BD/91119/2012, FCT, Portugal). P0952 TRANSIENT HEPATIC OVEREXPRESSION OF THE INSULINE-LIKE GROWTH FACTOR 2 (IGF2) INDUCES LIPID DROPLET FORMATION S.M. Kessler1 , S. Laggai1 , E. Van Wonterghem2 , R.E. Vandenbroucke2 , M. Ogris3 , C. Libert2 , A.K. Kiemer1 . 1 Pharmaceutical Biology, Saarland University, Saarbruecken, Germany; 2 Department for Molecular Biomedical Research, VIB, Ghent, Belgium; 3 Department of Pharmaceutical Chemistry, University of Vienna, Vienna, Austria E-mail: [email protected] Background and Aims: Although insulin-like growth factor 2 (IGF2) has been reported to be overexpressed in steatosis and steatohepatitis, a causal role of IGF2 in steatosis development remains elusive. Aim of our study was to decipher the role of IGF2 in steatosis development. Methods: Hydrodynamic gene delivery was used for transient IGF2 overexpression employing codon-optimized plasmid DNA. Lipids were quantified by vanilloid-sulfuric assay and stained by Scharlach Red. Lipid classes were determined by thin layer chromatography and free cholesterol by filipin staining. Lipid droplets (LD) were evaluated by fluorescence staining with LD540. mRNA and protein levels were quantified by real-time RT-PCR and Western blot, respectively. Results: Hydrodynamic gene delivery of the IGF2 plasmid resulted in a strong induction of hepatic IGF2 expression. The exogenously delivered IGF2 had no influence on endogenic IGF2 expression. The downstream kinase AKT was activated in IGF2 expressing
Journal of Hepatology 2015 vol. 62 | S263–S864
remains incomplete. Recently, microRNAs (miRNA/miRs) have been linked to NAFLD. In particular, mir-21 may contribute to disease progression by targeting peroxisome proliferator-activated receptor a (PPARa). Indeed, PPARa and farnesoid X receptor (FXR) constitute promising NAFLD therapeutic targets. We aimed to elucidate the role of miR-21 during NAFLD pathogenesis in mice, further evaluating the synergistic effect of miR-21 inhibition and FXR activation, using obeticholic acid (OCA). Methods: Wild type (WT; n = 24) and miR-21 knockout (KO; n = 24) mice were fed either a standard diet (SD; n = 12) or a fast food diet (FF; n = 12) for 25 weeks. 6 animals from each group had their diets supplemented with 60 mg/kg OCA (kindly provided by Intercept). Mice were weighed weekly while blood was collected and liver extracted and weighed at sacrifice. In parallel, human liver biopsies were obtained from morbid obese NAFLD patients at different disease stages (n = 28). Liver samples were processed for histological analysis and determination of miR-21 and PPARa expressions by qRT-PCR and immunoblotting, respectively. Serum was used for biochemical parameters analysis. Results: Our results show that, after 25 weeks, WT FF-fed mice develop NASH in parallel with an increase in both body weight and liver/body weight ratio, comparing with WT SD-fed mice (p < 0.05). Further, they exhibited increased miR-21 (p < 0.05) and decreased PPARa expressions (p < 0.05). Strikingly, miR-21 and PPARa also displayed an inverse and significant correlation in human patients, increasing from steatosis to less and more-severe NASH (p < 0.05). WT FF+OCA-fed animals displayed lower levels of miR-21, compared with WT FF-fed mice. KO FF-fed mice body weights and liver/body weight ratios were below WT FF-fed mice, as were serum levels of triglycerides and free fatty acids. Importantly, most of these beneficial effects were augmented in KO FF+OCA-fed mice (p < 0.05). Conclusions: In conclusion, our results indicate that miR-21 downregulation, likely leading to increased PPARa, together with FXR activation by OCA, ameliorate NAFLD pathogenesis, highlighting the therapeutic potential of novel synergistic therapies in NAFLD. (PTDC/BIM-MEC/0873/2012, SFRH/BD/88212/2012 and SFRH/BD/91119/2012, FCT, Portugal). P0952 TRANSIENT HEPATIC OVEREXPRESSION OF THE INSULINE-LIKE GROWTH FACTOR 2 (IGF2) INDUCES LIPID DROPLET FORMATION S.M. Kessler1 , S. Laggai1 , E. Van Wonterghem2 , R.E. Vandenbroucke2 , M. Ogris3 , C. Libert2 , A.K. Kiemer1 . 1 Pharmaceutical Biology, Saarland University, Saarbruecken, Germany; 2 Department for Molecular Biomedical Research, VIB, Ghent, Belgium; 3 Department of Pharmaceutical Chemistry, University of Vienna, Vienna, Austria E-mail: [email protected] Background and Aims: Although insulin-like growth factor 2 (IGF2) has been reported to be overexpressed in steatosis and steatohepatitis, a causal role of IGF2 in steatosis development remains elusive. Aim of our study was to decipher the role of IGF2 in steatosis development. Methods: Hydrodynamic gene delivery was used for transient IGF2 overexpression employing codon-optimized plasmid DNA. Lipids were quantified by vanilloid-sulfuric assay and stained by Scharlach Red. Lipid classes were determined by thin layer chromatography and free cholesterol by filipin staining. Lipid droplets (LD) were evaluated by fluorescence staining with LD540. mRNA and protein levels were quantified by real-time RT-PCR and Western blot, respectively. Results: Hydrodynamic gene delivery of the IGF2 plasmid resulted in a strong induction of hepatic IGF2 expression. The exogenously delivered IGF2 had no influence on endogenic IGF2 expression. The downstream kinase AKT was activated in IGF2 expressing
Journal of Hepatology 2015 vol. 62 | S263–S864