Hepatic Steatosis in the Mouse Model of Wilson Disease Coincides with a Muted Inflammatory Response

Abstract

Wilson disease (WND) is caused by inactivation of the copper transporter ATP7B and copper accumulation in tissues. WND presentations vary from liver steatosis to inflammation, fibrosis, and liver failure. Diets influence the liver phenotype in WND, but findings are inconsistent. To better understand the impact of excess calories on liver phenotype in WND, the study compared C57BL/6J Atp7b^-/- and C57BL/6J mice fed for 12 weeks with Western diet or normal chow. Serum and liver metabolites, body fat content, liver histology, hepatic proteome, and copper content were analyzed. Wild-type and Atp7b^-/- livers showed striking similarities in their responses to Western diet, most notably down-regulation of cholesterol biosynthesis, altered nuclear receptor signaling, and changes in cytoskeleton. Western diet increased body fat content and induced liver steatosis in males and females regardless of genotype; however, the effects were less pronounced in Atp7b^-/- mice compared with those in the wild type mice. Although hepatic copper remained elevated in Atp7b^-/- mice, liver inflammation was reduced. The diet diminished signaling by Rho GTPases, integrin, IL8, and reversed changes in cell cycle machinery and cytoskeleton. Overall, high calories decreased inflammatory response in favor of steatosis without improving markers of cell viability. Similar changes of cellular pathways during steatosis development in wild-type and Atp7b^-/- mice explain histologic overlap between WND and non-alcoholic fatty liver disease despite opposite copper changes in these disorders.

Figure 1

Effect of WD on the weight and fat mass accumulation in C57BL/6J (wt) and Atp7b^−/− mice (ko). A: The weight changes are shown for wt (grey) and ko (blue) mice on NC (light shade) and WD (dark shade). B: The overall weight gain. C: Body fat mass. D: Representative images of the abdominal cavity of male mice from different feeding groups. GraphPad version 8 software was used for statistics. ∗P < 0.05 by two-way analysis of variance. ko, knockout; NC, normal chow; WD, Western diet; wt, wild-type.

Figure 2

Wt and ko mice on WD have similar levels of serum metabolites but different fat accumulation in the liver. A–C: WD increases serum cholesterol (A) and glucose (B), whereas triglycerides (TG) remain unchanged (C) in both genotypes. D: TG levels in the liver. The levels of serum metabolites changes are shown for wt (grey) and ko (blue) mice on NC (light shade) and WD (dark shade). E: Oil Red O staining is noticeably reduced in Atp7b^−/− on WD compared with wt; males (m), females (f). ∗∗P < 0.01, ∗∗∗P < 0.001 by two-way analysis of variance. Scale bar = 100 μm. ko, knockout; NC, normal chow; WD, Western diet; wt, wild-type.

Figure 3

Quantitative analysis of WD-induced changes in liver proteome in wt and ko male mice. A: Principal component analysis of different feeding groups (wt_NC data are in green, wt_WD is red, ko_NC is orange, and ko_WD is blue). B: The heatmap showing protein profiles within the four feeding groups. From left to right: ko WD, ko NC, wt WD, wt NC. (Proteome Discoverer software version 2.4). C and D: A simplified cartoon illustrating main pathways involved in hepatic fat processing (C) and abundances of these proteins in male livers as determined by tandem mass tag–labeling mass spectrometry (D). Wt (grey) and ko (blue) mice on NC (light shade) and on WD (dark shade). E: The mRNA levels for HMG-CoA reductase and SCD1 as determined by quantitative PCR. F: Western blot and quantitation for FATP2 (left) and ABCG8 (right). The changes in liver proteome are shown for wt (grey) and ko (blue) mice on NC (light shade) and WD (dark shade). GraphPad version 8 software was used for statistics. n = 3 per group (B). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 by two-way analysis of variance. ABCG5/8, ATP-binding cassette sub-family G member 5/8; CD36, cluster of differentiation 36; DAG, diacylglycerol; FA, fatty acid; FasN, fatty acid synthase; FATP2 and FATP5, fatty acid transport protein 2 and 5; FC, free cholesterol; ko, knockout; NC, normal chow; SCD1, stearoyl-CoA desaturase-1; TG, triglycerides; WD, Western diet; wt, wild-type.

Figure 4

Similarities and differences between the wt and ko liver response to WD identified by IPA. A and B: The most significantly changed (P value <10⁻⁵) metabolic and signaling pathways in the wt_WD liver compared with wt_NC (A) and in the ko_WD liver compared with ko_NC (B). In each panel, the blue indicates down-regulation/inhibition; orange, activation/up-regulation; and white or grey, dysregulation in no particular direction. C: Major predicted upstream regulators and biological processes affected by WD in the wt liver. ko, knockout; NC, normal chow; WD, Western diet; wt, wild-type.

Figure 5

WD modulates changes in the metabolic and signaling pathways in the ko liver. A: The heatmap (left) and the corresponding P values (right) compare changes in canonical pathways for the wt and ko livers in response to WD, as well as changes in the ko_NC liver compared with wt_NC. Darker purple color in a heatmap illustrates more significant changes (lower P value); the colors in the table indicate whether the pathway is inhibited (blue), activated (orange), or dysregulated in no particular direction (black). B: Major predicted upstream regulators and biological processes affected by WD in the ko liver. ko, knockout; NC, normal chow; WD, Western diet; wt, wild-type.

Figure 6

Changes in the tissue copper content in response to WD. A: Representative rhodanine staining of liver tissues (female mice). There is no rhodamine signal in the wt groups regardless of the diet, but a noticeable decrease of rhodanine staining in ko_WD animals compared with ko_NC animals. B: Copper levels, at 10 weeks, are significantly elevated in ko animals regardless of diet. Copper levels in wt mice do not differ between the feeding groups or the sex. Copper levels in ko mice are lower compared with NC at that age. At 16 weeks, copper changes occur in both genotypes due to WD. In wt males, hepatic copper decreases in response to WD, whereas it increases in wt females. Atp7b^−/− males on WD show significantly reduced copper levels compared with ko_NC males. C: Spleen copper levels are significantly reduced in ko females (P = 0.0004), but not in ko males on WD compared with ko_NC (P = 0.9448). D: There are no changes of copper content in adipose tissue. The changes the tissue copper content are shown for wt (grey) and ko (blue) mice on NC (light shade) and WD (dark shade). GraphPad version 8 software was used for statistics. . ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001 by two-way analysis of variance. Scale bars: 1 mm (A, top row); 200 μm (A, middle row); 100 μm (A, bottom row). ko, knockout; NC, normal chow; WD, Western diet; wt, wild-type.

Figure 7

WD mutes the signaling pathways and ameliorates changes in protein expression in Atp7b^−/− mice. A: The heatmap and Z-score values illustrate the status of signaling pathways in ko_NC and ko_WD when their proteomes are compared with normal wt mice. Orange indicates activation; blue, inhibition. B: Examples of pathways and associated proteins changed in response to WD. Each genotype is compared with wt_NC. Numbers in bold indicate the number of altered proteins within the respective pathway. Created with Ingenuity Pathway Analysis software version 01-20-04. C: The volcano plot for ko_NC (40 down- and 132 up-regulated proteins) and ko_WD mice (32 down- and 33 up-regulated proteins) when compared with wt_NC. Pink and green panels indicate changes larger than |log2| = 1.3. Bottom panels show an enlarged area the corresponding top panel with up-regulated proteins (highlighted in blue) illustrating how several of them change in response to WD. Created with Proteome Discoverer software version 2.4. Cyt P450, cytochrome P450 family; GST, glutathione S-transferase; INFα, interferon alpha regulatory protein; ko, knockout; MT1 and 2, metallothionein 1 and 2; , N/A, no change; NC, normal chow; STMN1, stathmin; WD, Western diet; wt, wild-type.

Figure 8

WD improves liver histology in Atp7b^−/− mice and decreases inflammatory markers. A: Hematoxylin and eosin (H&E) staining of female mice show improvement of liver histology in ko animals on WD. B: Inflammatory scoring performed by a pathologist (0, no inflammation; 1, minimal; 2, mild; 3, moderate; 4, severe, which did not occur). C–E: Quantitative PCR data showing reduction in CD24a (C), INFα (D), and iNOS levels (E). The changes are shown for wt (grey) and ko (blue) mice on NC (light shade) and WD (dark shade). GraphPad version 8 software was used for statistics. . ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 by two-way analysis of variance. Scale bars: 1 mm (A, top row); 200 μm (A, middle row); 100 μm (A, bottom row). ko, knockout; NC, normal chow; WD, Western diet; wt, wild-type.

Supplemental Figure S1

Weight measurements for both sexes and abdominal fat accumulation in female mice: A and B: Weight gain during the experiment for males (A) and females (B). C: Images of abdominal cavity for female mice show higher fat content in Western diet (WD) groups. From left to right: wild-type (wt) normal chow (NC), wild-type Western diet (WD), knockout (ko) normal chow, and knockout Western diet.

Supplemental Figure S2

Analysis of serum metabolites and the Oil-red-O staining of tissue sections for males and females. A: Serum cholesterol for males (wt: NC 126.4 ± 10.53 mg/dL versus WD 182.5 ± 16.82 mg/dL; ko: NC 123 ± 31.63 mg/dL versus WD 167 ± 9.59 mg/dL) and females (wt: NC 116 ± 13.69 mg/dL versus WD 190 ± 103.49 mg/dL; ko: NC 87.5 ± 9.85 mg/dL versus WD 150.6 ± 15.58 mg/dL). B: Serum glucose in males (wt: NC 181 ± 22.24 mg/dL versus WD 215 ± 27.69 mg/dL and ko: NC 123.4 ± 27.32 mg/dL versus WD 197.6 ± 23.77 mg/dL) and females (wt: NC 155 ± 24.86 mg/dL versus WD 189.4 ± 33.95 mg/dL and ko: NC 105.25 ± 42.09 mg/dL versus WD 189.4 ± 33.95 mg/dL). C: Oil Red O staining of liver sections from an additional two male and female mice. D: Serum levels of TG are unchanged between the four different feeding groups in male (wt: NC 105.4 ± 37.02 mg/dL versus WD 87.25 ± 27.66 mg/dL, ko: NC 111 ± 33.13 mg/dL versus WD 101.6 ± 40.36 mg/dL) and female (wt: NC 94.2 ± 18.14 mg/dL versus WD 89.6 ± 39.53 mg/dL; ko: NC 117.5 ± 25.51 mg/dL versus WD 91.4 ± 22.01 mg/dL). E: Triglyceride (TG) levels in the liver. Pairwise comparison was made within each group. ∗P < 0.05, ∗∗P < 0.01 by two-tailed t-test. Scale bars = 100 μm. f, female; m, male; ko, knockout; NC, normal chow; WD, Western diet; wt, wild-type.

Supplemental Figure S3

Box plot analysis of distribution of protein abundances in individual samples. ko, knockout; NC, normal chow; WD, Western diet; wt, wild-type.

Supplemental Figure S5

Representative hematoxylin and eosin (H&E) staining of liver sections. A: Effect of WD on liver histology of 16-week–old males. B: H&E staining of liver section from male (m) and female (f) mice at 10 weeks of age. At this age, only mild steatosis is seen in the wt livers in response to WD; steatosis is not apparent in the ko samples. From left to right: wt NC: wild-type normal chow, wild-type Western diet, knockout normal chow, and knockout Western diet. Scale bars: 1 mm (A, top row); 200 μm (A, middle row); 100 μm (A, bottom row, and B). ko, knockout; NC, normal chow; WD, Western diet; wt, wild-type.

Supplemental Figure S6

Immunofluorescence staining of liver sections to compare inflammation. F4/80 staining (A) and quantitation of stained macrophages (B) show no significant difference between the genotypes as well as feeding groups. Scale bars = 20 μm. ko, knockout; NC, normal chow; WD, Western diet; wt, wild-type.