Non-alcoholic fatty liver (NAFLD) occurs as simple hepato-steatosis and its evolution into severe non-alcoholic steatohepatitis (NASH) is strictly connected to obesity, but the pathogenesis is still obscure. Recently, a multiple hit theory suggested that from gut and adipose tissue different adipokines and inflammatory markers influenced the liver where steatosis, collagen deposition and inflammation might occur. Sirtuin 1 (Sirt1) is a NAD-dependent enzyme involved in longevity and in the regulation of multiple cellular functions and metabolism. Here we hypothesized that Sirt 1 down-regulation might exacerbate liver damage and mitochondrial lesions in hepatocytes in dietary-induced obesity in mice. To best analyse these events, we studied male C57BL/6 mice (WT) and Sirt 1 deficient heterozygous mice (HET) (by an established colony in CSIC generated from founders from Dr David Sinclair laboratory, Harvard University, Boston, USA). Animals housed in calorimetric rooms on a 12h: 12h light/dark cycle were placed for 16 wks, from 12 to 28 wks of age, on a standard rodent diet (8% fat -13.0 kJ/g) or on a high fat diet (HFAT- 35% lard-18.8kJ/g Tekland Research Diet–TD03584). Body weight was monitored at the beginning, at 3wks and at 16 wks before euthanasia. Livers were fixed in 4% paraformaldehyde overnight for histopathology, immunohistochemistry or in Karnowsky mixture (4% paraformaldehyde and 1% glutaraldehyde) then post-fixed in 1% osmium tetroxide for transmission electron microscopy analysis (TEM). By H&E, Masson trichrome and Sirius red staining at bright and polarized microscopy significant increase in steatosis and fibrosis in HET mice received HFAT diet vs WT fed HFAT diet were detected (Fig.1 and Fig.2). Interstitial collagen type I, type III, and basal membrane associated collagen type IV fibres were detected in pericentral vascular wall, intermediate lobular area and in thickened sinusoidal wall at light microscopy and at TEM (Fig.3). In contrast, WT or HET animals fed standard diet (as control groups) displayed negligible liver changes. Remarkably, HET mice fed HFAT diet showed abnormal giant mitochondria and activated Kupffer cells, strongly positive to Perls Prussian Blue iron staining, that were lacking in other groups. Finally, in HET obese mice myofibroblasts immersed in an abundant extracellular matrix and fibrous debris were observed by TEM (Fig.4). To highlight the crucial role of oxidative damage and caspase 1 complex in the progression to NASH, we analysed 4HNE and pro-caspase 1 expressions. Remarkably, strong 4HNE and moderate caspase 1 signals were present in pericentral and midzonal hepatocytes in HET HFAT diet mice but weak in WT HFAT diet group and absent in controls. All these data suggest that Sirt 1 is critical to limit fibrogenesis and inflammation associated to irreversible NASH in HFAT diet induced obesity in mice.

Acknowledgements

New Pet Food Italia Srl generously supports this study. Authors thank Mr G. Bozzoni and Mrs S. Castrezzati for expert TEM sample preparation and trimming.

Figures:

Figure 1. Sirt 1+/- mice liver receiving high fat-diet. Centrolobular collagen deposition by Sirius Red staining at bright field at 400x.

Figure 2. Polarized dark field microscopy of the same field. Note in green–Collagen type III, in yellow-orange-Collagen type I, V = centrolobular vein at 400x.

Figure 3. Sirt1+/- mice liver receiving high fat-diet. TEM micrograph. Disse space (D) engulfed of fibrillary deposits, note endothelial cell filled with lipid debris (E). Bar=2 μm

Figure 4. Sirt1+/- mice liver. TEM micrograph: Interstitial fibroblast immersed in an abundant extracellular matrix (white arrow) between hepatocytes (H), L-lipid droplets. Bar=10μm.

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EMC Abstracts - https://emc-proceedings.com/abstract/sirtuin-1-downregulation-exacerbates-fibrosis-and-inflammation-in-obese-mice-liver/