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. 2021 Nov 8;11(1):21827.
doi: 10.1038/s41598-021-00501-5.

Albino mice with the point mutation at the tyrosinase locus show high cholesterol diet-induced NASH susceptibility

Kaushalya Kulathunga #  1   2 , Arata Wakimoto #  1 , Yukiko Hiraishi  1 , Manoj Kumar Yadav  1 , Kyle Gentleman  3 , Eiji Warabi  1 , Tomoki Sakasai  1 , Yoshihiro Miwa  1   4 , Seiya Mizuno  5 , Satoru Takahashi  6   7   8 , Michito Hamada  9   10
Affiliations

Albino mice with the point mutation at the tyrosinase locus show high cholesterol diet-induced NASH susceptibility

Kaushalya Kulathunga et al. Sci Rep. .

Abstract

Non-alcoholic fatty liver disease (NAFLD) constitutes a metabolic disorder with high worldwide prevalence and increasing incidence. The inflammatory progressive state, non-alcoholic steatohepatitis (NASH), leads to liver fibrosis and carcinogenesis. Here, we evaluated whether tyrosinase mutation underlies NASH pathophysiology. Tyrosinase point-mutated B6 (Cg)-Tyrc-2J/J mice (B6 albino) and C57BL/6J black mice (B6 black) were fed with high cholesterol diet (HCD) for 10 weeks. Normal diet-fed mice served as controls. HCD-fed B6 albino exhibited high NASH susceptibility compared to B6 black, a phenotype not previously reported. Liver injury occurred in approximately 50% of B6 albino from one post HCD feeding, with elevated serum alanine aminotransferase and aspartate aminotransferase levels. NASH was induced following 2 weeks in severe-phenotypic B6 albino (sB6), but B6 black exhibited no symptoms, even after 10 weeks. HCD-fed sB6 albino showed significantly higher mortality rate. Histological analysis of the liver revealed significant inflammatory cell and lipid infiltration and severe fibrosis. Serum lipoprotein analysis revealed significantly higher chylomicron and very low-density lipoprotein levels in sB6 albino. Moreover, significantly higher small intestinal lipid absorption and lower fecal lipid excretion occurred together with elevated intestinal NPC1L1 expression. As the tyrosinase point mutation represents the only genetic difference between B6 albino and B6 black, our work will facilitate the identification of susceptible genetic factors for NASH development and expand the understanding of NASH pathophysiology.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Severe B6 (sB6) albino mice develop NASH with 2 weeks of HCD feeding. (a) Survival curve of HCD fed B6 albino (dotted line, n = 27) and B6 black (thick line, n = 17) under HCD feeding. *p < 0.05 B6 albino compared with B6 black (log-rank test). (b) Serum ALT, AST levels of HCD-fed B6 black, mild B6 (mB6) albino and sB6 albino for 10 weeks. (c) Body weight of HCD-fed B6 albino and B6 black for 10 weeks. All the data are presented as the mean ± s.e.m. *p < 0.05 sB6 albino compared with B6 black (Welch’s t-test). (d) Serum ALT, AST levels of HCD-fed B6 black, mild B6 (mB6) albino and severe B6 (sB6) albino mice for 14 days. (e) Body weight of 2 weeks HCD-fed sB6 albino and B6 black. B6 black (n = 3), sB6 albino (n = 6) mice. (f) Serum total cholesterol (T-CHO), triglyceride (TG) and glucose levels of 2 weeks HCD-fed B6 black (n = 3) and sB6 albino (n = 6) mice. (g) Representative macroscopic appearance of the liver. (h) Relative weight of the liver (B6 black n = 14, sB6 albino n = 25), spleen (n = 14, n = 25), and gWAT (n = 9, n = 19) after 2 weeks of HCD feeding. (i) Representative microscopic view of liver sections observed using HE, MT, ORO, anti- mouse Mac2 staining, and under polarized light (Scale bar = 50 μm). (j) Quantified area of liver section steatosis and fibrosis (B6 black n = 3, sB6 albino n = 6). The data are from one representative experiment of at least two independent experiments and are presented as the means ± s.e.m. *p < 0.05 compared with B6 black (Welch’s t-test).
Figure 2
Figure 2
Rapid onset of NASH phenotype in sB6 albino mice upon HCD induction. (a) Representative microscopic images of liver tissues before HCD (a), 1-day (b), and 3-days (c) after HCD feeding. Liver sections were stained with HE, MT, and ORO. Samples were analyzed under polarized light to observe cholesterol crystal accumulation. (Scale bar = 50 μm). Black arrowhead: lipid droplet, white arrowhead: immune cell, green arrowhead: fibrotic region, yellow arrowhead: cholesterol crystal. Data are from one representative experiment of at least two independent experiments.
Figure 3
Figure 3
Severe B6 albino mice show impaired small intestinal cholesterol homeostasis. (a) Serum lipoprotein species before and 1-day after HCD feeding. T-CHO, CM-CHO, VLDL-CHO, LDL-CHO, and HDL-CHO are shown (B6 black n = 5, sB6 albino n = 5). (b) Serum LPL enzyme activity in sB6 albino (n = 3) and B6 black (n = 3) mice 1-day after cholesterol feeding. (c) Representative ORO staining of fecal matter from B6 black and B6 albino 1-day after HCD feeding (Scale bar = 1 mm). (d) Quantification of the lipid amount in feces normalized to B6 black amounts. Data from four independent experiments were pooled (B6 black n = 15, sB6 albino n = 18). (e) Representative view of ORO staining of SI 1.5 h after oral gavage with 2% cholesterol in 200 μl corn oil (Scale bar = 25 μm). (f) Quantification of ORO-positive area in intestinal tissue (B6 black n = 3, sB6 albino n = 3). (g) Relative expression of intestinal cholesterol transporter genes in HCD-fed B6 black and sB6 albino mice (B6 black n = 8, sB6 albino n = 11). Data were normalized using 36b4 mRNA levels. (h) Representative SI NPC1L1 staining (Scale bar = 100 μm). Data are from one representative experiment of at least two independent experiments and are presented as the means ± s.e.m. *p < 0.05 compared with B6 black (Welch’s t-test).
Figure 4
Figure 4
Mice with Tyrosinase c.291G > T mutation show the same phenotype to B6 albino with HCD feeding. (a) Tyrosinase transcript. CRISPR albino with C.291G > T mutation in Tyrosinase of C57BL/6J mouse using CRISPR/Cas9 system. (b) Transition graph of weight with HCD feeding in B6 black group and CRISPR albino group. (c) Serum ALT and AST values of B6 black group (black bar), mild CRISPR albino group (beige bar) and severe CRISPR albino group (white bar) with HCD feeding. (d) Liver histology of each group on day 14 of HCD feeding. Steatosis, inflammatory cell accumulation and fibrosis only observed in severe CRISPR albino livers. All the data are presented as the mean ± s.e.m. *p < 0.05 severe CRISPR albino (n = 7) compared with B6 black (n = 5) (Welch’s t-test).
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