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. 2023;15(5):1027-1049.
doi: 10.1016/j.jcmgh.2023.01.016. Epub 2023 Feb 7.

Loss of SQSTM1/p62 Induces Obesity and Exacerbates Alcohol-Induced Liver Injury in Aged Mice

Hui Qian  1 , Xiaojuan Chao  1 , Shaogui Wang  1 , Yuan Li  1 , Xiaoxiao Jiang  1 , Zhaoli Sun  2 , Thomas Rülicke  3 , Kurt Zatloukal  4 , Hong-Min Ni  1 , Wen-Xing Ding  5
Affiliations

Loss of SQSTM1/p62 Induces Obesity and Exacerbates Alcohol-Induced Liver Injury in Aged Mice

Hui Qian et al. Cell Mol Gastroenterol Hepatol. 2023.

Abstract

Background: Alcohol-associated liver disease (ALD) is a worldwide health problem, of which the effective treatment is still lacking. Both detrimental and protective roles of adipose tissue have been implicated in ALD. Although alcohol increases adipose tissue lipolysis to promote alcohol-induced liver injury, alcohol also activates brown adipose tissue (BAT) thermogenesis as an adaptive response in protecting against alcohol-induced liver injury. Moreover, aging and obesity are also risk factors for ALD. In the present study, we investigated the effects of autophagy receptor protein SQSTM1/p62 on adipose tissue and obesity in alcohol-induced liver injury in both young and aged mice.

Methods: Young and aged whole-body SQSTM1/p62 knockout (KO) and their age-matched wild-type (WT) mice were subjected to chronic plus binge (Gao-binge) alcohol feeding. Blood, adipose and liver tissues were collected for biochemical and histologic analysis.

Results: Aged but not young SQSTM1/p62 KO mice had significantly increased body weight and fat mass compared with the matched WT mice. Gao-binge alcohol feeding induced white adipose atrophy and decreased levels of SQSTM1/p62 levels in adipose tissue in aged WT mice. SQSTM1/p62 KO aged mice were resistant to Gao-binge alcohol-induced white adipose atrophy. Alcohol feeding increased the expression of thermogenic genes in WT mouse BAT, which was significantly blunted in SQSTM1/p62 KO aged mice. Alcohol-fed aged SQSTM1/p62 KO mice showed significantly higher levels of serum alanine aminotransferase, hepatic triglyceride, and inflammation compared with young and aged WT mice fed with alcohol. Alcohol-fed SQSTM1/p62 KO mice also increased secretion of proinflammatory and angiogenic adipokines that may promote alcohol-induced liver injury.

Conclusions: Loss of SQSTM1/p62 in aged mice leads to obesity and impairs alcohol-induced BAT adaptation, resulting in exacerbated alcohol-induced liver injury in mice.

Keywords: ALD; Adipokine; Adipose Tissue; Autophagy; Steatosis.

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Figures

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Graphical abstract
Figure 1
Figure 1
Increased level of hepatic p62 and its aggregation in patients with alcoholic hepatitis (AH). (A) Western blot analysis of total liver lysates of healthy donors (normal liver) and AH patients, respectively. LE, long exposure; SE, short exposure. (B) Densitometry analysis of p62 and LC3B expression. Results are presented as mean ± standard error (n = 5–8). ∗P < .05 with Student t test. (C and D) Representative images of H&E and immunohistochemical staining of p62 in liver tissues from AH patients and healthy donors. Arrowheads: hepatocyte ballooning; solid black arrows: lipid droplets; scale bar: 50 μm.
Figure 2
Figure 2
Increased hepatic p62 aggregation in aged mice after Gao-binge alcohol feeding. Young (3-month-old or 3M) and aged (14-month-old or 14M) male mice were subjected to Gao-binge alcohol model. (A) Western blot analysis of total liver lysates from WT young mice. LE, long exposure; SE, short exposure. (B) Densitometry analysis of the Western blot result from (A). (C) Western blot analysis of total liver lysates from WT aged mice. (D) Densitometry analysis of the Western blot result from (C). (E) Representative images of liver immunofluorescence staining of p62 in WT young (3M) and aged mice (14M) with or without alcohol treatment. Ctrl, control; EtOH, ethanol; scale bar: 20 μm. (F) Western blot analysis of p62 in the hepatic detergent insoluble fractions from young and aged WT mice fed with control or Gao-binge alcohol. (G) Densitometry analysis of the Western blot results from (F). Results are presented as mean ± standard error (n = 4–9).
Figure 3
Figure 3
The levels of detergent insoluble ubiquitinated proteins in young and aged WT and p62 KOmice with or without Gao-binge alcohol feeding. (A and B) Young (3M) and (C and D) aged (14M) male mice were subjected to Gao-binge alcohol model. Hepatic detergent insoluble fractions were prepared and subjected to Western blot analysis of p62 and ubiquitin (Ub). (B and D) Densitometry analysis of the Western blot results from (A and C), respectively. Western blot analysis of p62 and Ub from hepatic detergent insoluble fractions young and aged mice fed with the control diet (E) or alcohol (G). (F and H) Densitometry analysis of the Western blot results from (E and G), respectively. Results are presented as mean ± standard error (n = 3–4). ∗P < .05; one-way analysis of variance followed by Tukey post hoc test.
Figure 4
Figure 4
p62 knockout mice develop mature-onset obesity during aging. (A) Representative images of the mice and gross anatomy of livers from young and aged mice with the indicated genotype and treatment. Scale bar: 1 cm. (B) Measurement of mice body weight (in grams). (C) Measurement of liver weight (in grams). (D) Liver to body weight (BW) ratio. (E) Average of food intake (in mL) per day per mouse. (F) Normalized food intake by body weight of the mice in each group. (G) Serum ethanol concentration of young and aged mice with the indicated genotype and treatment. Results are presented as mean ± standard error (n ≥ 4). ∗P < .05; #P < .05 between young and aged group; one-way analysis of variance followed by Tukey post hoc test.
Figure 5
Figure 5
p62 KO exacerbates alcohol-induced liver injury and steatosis in aged mice. Levels of serum ALT (A), hepatic triglycerides (B), and hepatic cholesterol (C) in young and aged mice with the indicated genotype and treatment. Results are presented as mean ± standard error (n ≥ 6). ∗P < .05, one-way analysis of variance with Tukey post hoc test. (D and E) Representative images of H&E and Oil Red O staining of liver sections from young mice and aged mice with the indicated genotype and treatment. Scale bar: 50 μm.
Figure 6
Figure 6
Gao-binge alcohol induces white adipose tissues atrophy in WT but not p62 KO aged mice. (A) Representative images of gross anatomy of eWAT (epididymal white adipose tissue) section from mice with the indicated genotype and treatment. Scale bars: 1 cm. (B) Measurement of eWAT (in grams) and (C) eWAT to body weight (BW) ratio. (D) Representative images of H&E of eWAT section from mice with the indicated genotype and treatment. Scale bar: 50 μm. (E) Quantification of the number of eWAT adipocytes with different area (pixel) based on per 100× H&E staining. Each dot represents the number of eWAT adipocytes per image, and at least n ≥ 3 mice were quantified in each group. (F) Quantification of serum free fatty acids (FFA) concentration and (G) serum free glycerol concentration from mice with the indicated genotype and treatment. Data are presented as mean ± standard error (n ≥ 4). ∗P < .05, one-way analysis of variance followed by Tukey post hoc test.
Figure 7
Figure 7
Alcohol decreases p62 expression and alters adipose lipolysis in eWAT. (A and B) Western blot analysis of total eWAT lysates from young and aged mice with the indicated genotype and treatment; Ponceau S staining of total proteins is also included. SE, short exposure; LE, long exposure. (C and D) Densitometry analysis of the Western blot results from (A) and (B), respectively. Note that phosphorylated HSL is not included in the quantification because the change is obvious on the blot.
Figure 8
Figure 8
Alcohol promotes white adipose tissue inflammation in p62 KO aged mice. (A) Representative images of immunostaining of F4/80 positive macrophages/Kupffer cells (top) and MPO staining (bottom) of infiltrated neutrophils in eWAT from aged (14M) mice with the indicated genotype and treatment. Dark arrow: crown-like structures (CLS). (B) Quantification of MPO and F4/80 positive cells from (A). (C) Quantitative polymerase chain reaction analysis of the indicated eWAT RNAs from aged (14M) mice; results were normalized to Actb and expressed as fold change compared with WT young group. Data shown are means ± standard error (n = 3–5). ∗P < .05, one-way analysis followed by Tukey post hoc test.
Figure 9
Figure 9
Impaired BAT activation in aged p62 KO mice fed with Gao-binge alcohol. (A) Representative images of gross anatomy of BAT (brown adipose tissue) sections from young and aged mice with the indicated genotype and treatment. Scale bars: 1 cm. (B) Measurement of BAT weight (in grams) and BAT to body weight ratio. (C) Representative images of H&E staining of BAT sections from young and mice with the indicated genotype and treatment. Scale bar: 50 μm. (D) Quantitative polymerase chain reaction analysis of the indicated mRNAs in BAT sections from young and aged mice with the indicated genotype and treatment. Data are presented as mean ± standard error (n = 3–5). ∗P < .05, ∗∗P < .001, #P < .05 between young and aged group. One-way analysis of variance followed by Tukey post hoc test.
Figure 10
Figure 10
p62 is required for alcohol-induced BAT activation in mice. (A and B) Western blot analysis of BAT lysates from young and aged mice with the indicated genotype and treatment. The result of quantification on Western blot is shown below each corresponding band and is presented as fold change, mean ± standard error. (C) Densitometry analysis of OXPHOUS from (B). SE, short exposure; LE, long exposure.
Figure 11
Figure 11
Hepatic de novo lipogenesis (DNL) genes and fatty acid beta-oxidation may not be critical in Gao-binge alcohol-induced steatosis in aged mice. (A) Western blot analysis of total liver lysates from aged mice with the indicated genotype and treatment. (B) Densitometry analysis of FASN and ACCα from (A). (C) mRNA expression analysis of Fasn and Accα in liver from aged mice with the indicated genotype and treatment. (D) Measurement of serum β-Hydroxybutyrate (D), mRNA expression of Fgf21 (E), and serum FGF21 (F) in aged mice with the indicated genotype and treatment. Results are presented as mean ± standard error (n = 3–4). ∗P < .05, one-way analysis of variance with Tukey post hoc test.
Figure 12
Figure 12
Effects of alcohol on adipokine secretion in mice. Young (3M) and aged (14M) male p62 KO mice and their age-matched WT littermates were subjected to Gao-binge alcohol model. (A) Representative results of adipokine array assay on the serum from young and aged mice with the indicated genotype and treatment. Serum from different groups were assessed for adipokine expression using an antibody adipokine array, where each group consists of the serum collected from 8 mice. (B and C) Densitometry analysis of the adipokine array result from (A).
Figure 13
Figure 13
Alcohol promotes liver inflammation in p62 KO aged mice. (A) Representative images of immunostaining of F4/80 resident macrophages/Kupffer cells (top), CD68 circulating macrophages (middle), and MPO (bottom) staining of infiltrated neutrophils in livers from young and aged mice with the indicated genotype and treatment. (B) Quantification of F4/80, CD68, and MPO positive cells from (A); data are presented as mean ± standard error (n = 3–4). (C) Quantitative polymerase chain reaction analysis of the indicated inflammation-related mRNAs extracted from livers of aged mice with the indicated genotype and treatment. Data are presented as mean ± standard error (n = 3–4). ∗P < .05, one-way analysis of variance followed by Tukey post hoc test.
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