The DNA damage checkpoint protein ATM promotes hepatocellular apoptosis and fibrosis in a mouse model of non-alcoholic fatty liver disease

Steatoapoptosis is a hallmark of non-alcoholic fatty liver disease (NAFLD) and is an important factor in liver disease progression. We hypothesized that increased reactive oxygen species resulting from excess dietary fat contribute to liver disease by causing DNA damage and apoptotic cell death, and tested this by investigating the effects of feeding mice high fat or standard diets for 8 weeks. High fat diet feeding resulted in increased hepatic H2O2, superoxide production, and expression of oxidative stress response genes, confirming that the high fat diet induced hepatic oxidative stress. High fat diet feeding also increased hepatic steatosis, hepatitis and DNA damage as exemplified by an increase in the percentage of 8-hydroxyguanosine (8-OHG) positive hepatocytes in high fat diet fed mice. Consistent with reports that the DNA damage checkpoint kinase Ataxia Telangiectasia Mutated (ATM) is activated by oxidative stress, ATM phosphorylation was induced in the livers of wild type mice following high fat diet feeding. We therefore examined the effects of high fat diet feeding in Atm-deficient mice. The prevalence of apoptosis and expression of the pro-apoptotic factor PUMA were significantly reduced in Atm-deficient mice fed the high fat diet when compared with wild type controls. Furthermore, high fat diet fed Atm−/− mice had significantly less hepatic fibrosis than Atm+/+ or Atm+/− mice fed the same diet. Together, these data demonstrate a prominent role for the ATM pathway in the response to hepatic fat accumulation and link ATM activation to fatty liver-induced steatoapoptosis and fibrosis, key features of NAFLD progression

<i>Ccr2^−/−</i> and <i>Cd44^−/−</i> mice display altered inflammatory cell recruitment during LD feeding.

<p>(A–I) Composition of Liver Leukocytes in mice fed SD or LD (*P<0.05 compared to SD controls; **P<0.05 compared to all other groups). Cells are identified as in figure legend 2.</p

LD fed hepatitis susceptible B6 mice display uniform increases in hepatic leukocyte populations.

<p>(A–H) Composition of liver leukocytes in B6, BALB/c, and AKR mice fed SD or LD (*P<0.05 compared to SD controls). Leukocyte populations are defined as follows: NK cells, CD49b⁺ CD3⁻; NK-T cells, CD3⁺ CD49b⁺; B-cells, CD19⁺ B220⁺; dendritic cells, CD11c⁺, macrophages, CD11b⁺ Ly6C⁻, neutrophils, CD11b⁺ Ly6C⁺.</p

Hepatic leukocytes from B6 mice bind to hyaluronic acid in a CD44 dependent manner.

<p>(A) HA binding of CD45⁺ cells from a representative SD fed B6, <i>Ccr2^−/−</i> and <i>Cd44^−/−</i>mouse. (B) HA binding of CD45⁺ cells from a representative four week LD fed B6, <i>Ccr2^−/−</i> and <i>Cd44^−/−</i>mouse. (C) Cumulative HA binding results over time (n = 3–6 per group). (D) In CD45⁺ leukocytes from LD fed B6 mice, HA binding occurs on a subpopulation of CD44⁺ cells.</p

Monocytes in the liver of LD fed mice display an inflammatory phenotype.

<p>Co-expression of CCR2 and Gr-1 (A), as well as CCR2 and iNOS (B) in livers of CCR2 reporter mice. Flow cytometric analysis of hepatic CCR2⁺, CD11b⁺, Ly6C⁻ cells (solid line) confirms increase expression of Gr-1 (C) and iNOS (D) on CCR2⁺, CD11b⁺ cells compared to gray shaded regions which represent CCR2⁺, CD11b⁻⁻ populations stained for the same markers.</p

<i>Ccr2^−/−</i> and <i>Cd44^−/−</i> mice are resistant to hepatitis during LD feeding despite lipid accumulation and elevated serum enzymes.

<p>(A) Infiltration of CD45⁺ inflammatory cells into the livers of B6, <i>Ccr2^−/−</i>, and <i>Cd44^−/−</i> at one and four weeks of LD or SD feeding (*P<0.05 compared to SD controls, **P<0.05 compared to all groups, n = 3–6 per group). (B and C) Respective low power images from <i>Ccr2^−/−</i> and <i>Cd44^−/−</i> mice fed the LD for four weeks. In C, arrows point to inflammatory infiltrates restricted to large liver vessels. (D) Histological scoring of mice confirms moderate to severe inflammation in B6 LD mice and mild to moderate inflammation in <i>Cd44^−/−</i> mice (**P<0.05 compared to all groups, *P<0.05 compared to SD fed or <i>Ccr2−/−</i> LD fed mice; scores represent means of n = 6–12 mice per group). (E) Lithogenic diet feeding did not induce obesity in any genotype. (F) Lithogenic diet feeding induced significant hepatomegaly in all strains (*P<0.05 compared to all other mice, n = 4–6 per group). (G–H) Oil-red-o staining of livers from <i>Ccr2^−/−</i>(G), and <i>Cd44^−/−</i> (H) mice demonstrate an accumulation of excess lipid irrespective of genotype. (I) Steatosis scoring demonstrates a significant increase in steatosis in all genotypes in response to LD feeding for 4 weeks compared to SD fed mice (**P<0.05 compared to all groups n = 6–10 mice per group). (J–L) Serum levels of hepatic enzymes are elevated in LD fed mice irrespective of genotype. (J and K, *P<0.05 against all LD groups; L, *P<0.05 compared to LD fed B6 and <i>Ccr2^−/−</i> mice; n = 4–5 per group). B and C, bar represents 100µm; G–H, bar represents 20µm.</p

C57BL/6 mice are highly susceptible to diet induced hepatitis.

<p>(A) Liver leukocytes (CD45⁺ cells) were quantified in C57BL/6 (B6), BALB/c, and AKR mice fed either a standard diet (SD) or 4 weeks of lithogenic diet (LD) (*P<0.05 compared to SD controls, **P<0.05 compared to all groups; n = 3–5 per group). (B and E) H&E staining of liver from LD fed B6 mice under low and high power magnification respectively. Arrows in B point to inflammatory foci and E shows a representative inflammatory focus. (C and F) H&E staining of liver from LD fed AKR mice under low and high power magnification respectively. Oil-red-O staining showing lipid accumulation in BALB/c (G), B6 (H) and AKR mice (I) fed the LD but not in those fed the SD (J–L). Bars in B and C = 100µm; bars in D–L = 20µm.</p

Livers from LD fed B6 mice display activated stellate cells and fibrotic lesions.

<p>(A) Liver tissue sections were stained with α-smooth muscle actin antibody (αSMA) and cell nuclei were stained with DAPI. α-smooth muscle actin signal (green fluorescence) was normalized to cell number (blue fluorescence) (n = 4 mice per group, 5 tissue sections per mouse; *<i>P</i><0.05 compared to <i>Ccr2^−/−</i> mice; **P<0.05 compared to all groups). A representative section from LD fed B6 mice (B), <i>Ccr2^−/−</i> mice (C) and <i>Cd44^−/−</i> mice (D). (E–H) Low and (J–L) high power magnification of pico-sirius stained SD fed B6 mice (E), LD fed B6 mice (F,J), <i>Ccr2^−/−</i> mice (G,K) and <i>Cd44^−/−</i> mice (H,L). (I) Fibrosis score was determined by sirius-red positive staining (**P<0.05 compared to all groups, *P<0.05 compared to B6 SD and <i>Ccr2^−/−</i> LD). In E–H and J–L the bar corresponds to 25µm.</p

Hepatitis susceptible mouse strains display increased liver leukocyte CD44 expression in response to LD feeding.

<p>Hepatic leukocyte preparations were gated on forward and side-scatter and CD44 levels of B6 (A), BALB/c (B) and AKR (C) mice were examined by flow cytometry. Solid lines represent mice on LD and shaded lines represent mice on SD.</p