Authors
J. HAAS (1), E. BAUGÉ (1), A. VERRIJKEN (2), S. QUEMENER (1), N. HENNUYER (1), F. LALLOYER (1), A. DEPRINCE (1), C. GHEERAERT (1), R. PAUMELLE-LESTRELIN (1), S. CARON-HOUDE (1), L. VAN GAAL (3), P. LEFEBVRE (1), D. DOMBROWICZ (1), S. FRANCQUE (4), B. STAELS (1) / [1] Institut Pasteur, Lille, France, INSERM U1011, [2] Antwerp University Hospital, Edegem, Belgium, Department of Endocrinology, Diabetology and Metabolism, [3] ANTWERP UNIVERSITY HOSPITAL, Edegem, Belgium, Dept. of Gastroenterology and Hepatology, [4] Antwerp University Hospital, Edegem, Belgium, Dept. of Gastroenterology and Hepatology
Introduction
Nonalcoholic fatty liver disease (NAFLD) is rapidly becoming a major global health problem with its prevalence rising in concert with the epidemic of obesity. Critically, the molecular processes leading to the development of NAFLD and its more severe subtype, non-alcoholic steatohepatitis (NASH), remain poorly understood.
Aim
To identify molecular mechanisms implicated in development and resolution of NASH in humans.
Methods
A cohort of 170 patients covering the spectrum of NAFLD were biopsied at baseline and one year following dietary intervention or bariatric surgery totaling 253 biopsies. The biopsies were scored histologically according to the NAFLD Activity Score (NAS) criteria and each patient received a full clinical workup before and after the intervention. Expression microarray was used to assess gene expression changes in these biopsies and identify genes whose expression was correlated with histological features of NAFLD. In animal studies, acute overexpression of human or murine ApoF was achieved using hydrodynamic injection via tail vain, and adenovirus-mediated shRNA expression was employed for transient knockdown of endogenous Apof. All experiments were performed using wildtype C57BL6/J mice fed a normal chow diet.
Results
In the human biopsies, we identified Apolipoprotein F (ApoF) as the strongest transcript inversely correlated with steatosis score in males (Spearman rho=-0.675, nominal p=7.6x10-8). Upon further investigation, we found ApoF also inversely correlated with lobular inflammation scores (Spearman rho=-0.612, nominal p=2.3x10-6), and hence, significantly reduced in males with NASH. These findings were confirmed using publicly available data from previously published NAFLD cohorts. Following bariatric surgery-mediated resolution of NASH, APOF transcript levels were increased nearly 2-fold (nominal p=3.5x10-4). To determine whether ApoF may play a causative role in NAFLD development, we assessed intracellular lipid metabolism using radiolabeled 14C-oleate. We found that transfecting Apof in Hepa1c1c7 cells led to reduced incorporation of the label into phosphatidylcholine (PC) while incorporation into triglycerides (TG) was unaffected. Due to the requirement of PC for efficient VLDL secretion, we suspected that changes in ApoF may affect plasma lipid levels. Indeed, overexpression of either human or murine ApoF in mice led to reduced plasma TG levels, associated with a reduction in hepatic VLDL production. Conversely, acute knockdown of ApoF led to increased plasma cholesterol levels 7 days post injection combined with increased liver weight.
Conclusions
We have identified ApoF as a transcript whose expression is strongly diminished with NAFLD severity and improves following NASH reversion in humans. Through a combination of animal and cell-based studies we demonstrate that reduced ApoF affects phosphatidylcholine homeostasis and leads to increased plasma cholesterol levels and likely elevated hepatic TGs. These findings highlight the importance of hepatic PC metabolism in the development of NASH and suggest ApoF is a novel player in this setting.
