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. 2019 Aug 29;9(1):12514.
doi: 10.1038/s41598-019-48973-w.

High-fat and high-cholesterol diet decreases phosphorylated inositol-requiring kinase-1 and inhibits autophagy process in rat liver

Hisao Naito  1 , Yuki Yoshikawa-Bando  2 , Yuan Yuan  3 , Sayuki Hashimoto  4 , Kazuya Kitamori  4 , Hiroshi Yatsuya  1 , Tamie Nakajima  5
Affiliations

High-fat and high-cholesterol diet decreases phosphorylated inositol-requiring kinase-1 and inhibits autophagy process in rat liver

Hisao Naito et al. Sci Rep. .

Abstract

Precise molecular pathways involved in the progression of non-alcoholic steatohepatitis (NASH) remain to be elucidated. As Mallory-Denk bodies were occasionally observed in the enlarged hepatocytes in NASH model rat (SHRSP5/Dmcr) fed high-fat and high-cholesterol (HFC) diet, we aimed to clarify the roles of autophagy and endoplasmic reticulum (ER) stress in NASH progression. Male SHRSP5/Dmcr were randomly divided into 4 groups. Two groups were fed a control diet; the other two groups were fed a HFC diet for 2 and 8 weeks, respectively. The HFC diet increased the autophagy-related proteins levels and microtubule-associated protein 1 light chain 3-II/I ratio after 2 and 8 weeks, respectively. However, regarding ER stress-related proteins, the HFC diet decreased the levels of phosphorylated (p-) inositol-requiring kinase-1 (p-IRE-1) and p-protein kinase RNA-like ER kinase after 2 weeks. Additionally, the HFC diet increased anti-ubiquitin-positive cells and the level of the autophagy substrate p62, suggesting that the HFC diet induced dysfunction in ubiquitin-dependent protein degradation pathways. In conclusion, the HFC diet arrested the autophagy process in the liver; this was particularly associated with decreases in p-IRE-1 expression.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
(A) Representative images of p62 immunostaining of samples from SHRSP5/Dmcr rats fed control and HFC diets for 2 and 8 weeks. Arrows indicate the accumulation in the cytoplasm of enlarged hepatocytes. Scale bar 100 μm. (B) Representative images of ubiquitin immunostaining of samples from rats fed control and HFC diets for 8 weeks. Scale bar 100 μm.
Figure 2
Figure 2
(A) Representative Western blot images of p62 and autophagy-related proteins (Atg) 3, 7 and 12, and microtubule-associated protein 1 light chain 3 (LC3), beclin-1, class III phosphatidylinositol 3-kinase (VPS34) and UV radiation resistance-associated gene protein (UVRAG). Samples were obtained from SHRSP5/Dmcr rats fed control and HFC diets for 2 and 8 weeks. (B) Data are presented as means (bar) and dot plots for individual data. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Wide-length blots/gel image are presented in Supplementary information. Data was analysed by two-way analysis of variance between diet and feeding duration (week). P values of diet, and week, D × W (diet × week interaction effect) were expressed within each graph. *Significant difference between HFC diet and control-diet groups, P < 0.05.
Figure 3
Figure 3
Electron microscopic image. Giant lysosomes (arrow) and accumulation of autophagosomes (top-right), related to autologous cells with heterochromatin that adhered to the nuclear membrane, are seen in the livers of rats fed the HFC diet for 8 weeks. Scale bar 2 μm (control-diet group) and 5 μm (HFC-diet group).
Figure 4
Figure 4
(A) Representative Western blot images of the endoplasmic reticulum (ER) stress-related proteins binding immunoglobulin protein/glucose-regulated protein-78 (GRP78), phosphorylated (p-) inositol-requiring enzyme 1 (p-IRE-1)/IRE-1 ratio, p-protein kinase RNA-like ER kinase (p-PERK)/PERK ratio and activating transcription factor-6 (ATF6). (B) Data are presented as means (bar) and dot plots of individual data. GAPDH was used as a loading control. Wide-length blots/gel image are presented in Supplementary information. Data was analysed by two-way analysis of variance between diet and feeding duration (week). P values of diet, week, D × W (diet × week interaction effect) were expressed within in each graph. *Significant difference between the HFC diet and control-diet groups, P < 0.05.
See this image and copyright information in PMC

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