Lipid, fetuin-A and macrophage zonation in high fat diet foz-foz mice with non-alcoholic steatohepatitis
(2017) XXIXth Belgian Week of Gastroenterology — Location: Antwerpen (9.February.2017)
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- Introduction Non-alcoholic fatty liver disease (NAFLD) is characterized by steatosis (accumulation of triglycerides in the liver) and insulin resistance. A subgroup of patients can develop a more serious condition called non-alcoholic steatohepatitis (NASH) with increased risk of fibrosis development. Innate immunity, cell injury, lipid metabolism and severity of insulin resistance constitute potential mechanisms underlying disease progression. Fetuin-A , an emerging player in insulin resistance in type 2 diabetic patients, is described as a liver-derived protein increased in human NAFLD. Aim Here, we explore the effect of a high fat diet on the expression of fetuin-A and its relation with the development of steatosis, cell injury and liver macrophage (Kupffer cell) activation in a mouse model of obesity and NASH. Methods : Male foz/foz mice were fed a normal diet (ND) or a high fat diet (HFD) for 12 (long term HFD or LHFD) or 30 weeks (very long term HFD or VLHFD) (n=4/group) to induce early or definite fibrosing NASH, respectively. Liver tissue homogenates were prepared for Western blot protein studies and total RNA was extracted for gene expression analysis. Liver paraffin-embedded sections were used for hematoxylin and eosin staining, Sirius red staining and double immunofluorescence detection of F4/80 and fetuin-A. Results : Compared to foz/foz mice fed a ND, HFD-fed foz/foz mice developed obesity, insulin resistance and either steatosis (LHFD) or steatohepatitis with steatosis, hepatocyte ballooning, inflammation and fibrosis (VLHFD). In ND fed mice, fetuin-A staining was positive in the cytoplasm of zone 3 centrilobular hepatocytes while F4/80+ Kupffer cells were located in the sinusoids of the intermediate lobular zone 2. In LHFD fed mice, lipid deposition occurred in the hepatocytes of the zone 3 centrilobular areas. Fetuin-A protein was also located in the cytoplasm of these zone 3 centrilobular hepatocytes. F4/80+ macrophages distributed mainly in the sinusoids of the intermediate lobular zones 2, as seen in ND fed mice. However, liver m-RNA expression showed a 2-fold increased level of F4/80+ macrophage mRNA compared to ND (p<0.05), suggesting activation. In VLHD, we observed a loss of zonation of liver steatosis with the presence of fat loaded hepatocytes in all liver lobular zones. Fetuin-A was highest in periportal fat-ladden hepatocytes and next to inflammatory infiltrates. There was a 4-fold F4/80 mRNA increased level upon VLHFD compared to ND (p<0.05). Three types of F4/80+ cells were recognized on the morphology: elongated cells located in liver sinusoids compatible with liver resident Kupffer cells, cells forming lipogranuloma together with fat loaded hepatocytes and small inflammatory cells located in inflammatory foci compatible with recruited macrophages. Interestingly, F4/80+ cells from lipogranuloma were positive for fetuin-A protein staining. Liver fetuin-A mRNA levels remained unchanged either in LHFD or VLHFD compared to ND. Similarly, liver fetuin-A protein level was also stable under HFD. Conclusion : We demonstrate that VLHFD foz-foz mice develop NASH together with zonal changes of steatosis, liver macrophage activation and fetuin-A expression in fatty hepatocytes and macrophages. A shift of steatosis and fetuin-A from the centrilobular region in ND and LHFD to the periportal zone was observed in VLHFD, together with macrophage activation, recruitment and fetuin-A co-localization in macrophages forming the lipogranuloma. The stable liver fetuin-A protein level could be compatible with a redistribution of this protein and/or the profile of a secretory factor. Taken together, we could imagine that lipid deposition and macrophage infiltration may be important factors in the liver tissue remodeling observed during NASH development. Further work is planned to delineate whether fetuin-A presence in macrophages is linked with a production and/or a simple storage in those cells in this model as well as the role of this protein in NASH progression and insulin resistance pathogenesis.
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Lanthier, N., Horsmans, Y., & Leclercq, I. (2017). Lipid, fetuin-A and macrophage zonation in high fat diet foz-foz mice with non-alcoholic steatohepatitis. Acta Gastro-Enterologica Belgica, 80(1), A07. https://hdl.handle.net/2078.5/178144 (Original work published 2017)