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ER stress and autophagy dysfunction contribute to fatty liver in diabetic mice.

Zhang Q, Li Y, Liang T, Lu X, Zhang C, Liu X, Jiang X, Martin RC, Cheng M, Cai L - Int. J. Biol. Sci. (2015)

Bottom Line: Likewise, autophagy functioned well in the early stage but suppressed in the later stage.The inactivation of unfolded protein response and suppression of autophagy were positively related to the development of steatohepatitis, which linked to metabolic abnormalities in the compromised hepatic tissues in diabetic condition.We conclude that the adaption of ER stress and impairment of autophagy play an important role to exacerbate lipid metabolic disorder contributing to steatohepatitis in diabetes.

View Article: PubMed Central - PubMed

Affiliation: 1. Department of Infectious Diseases, Affiliated Hospital of Guiyang Medical College, Guiyang, Guizhou, China, 550004 ; 4. Kosair Children's Hospital Research Institute, the Department of Pediatrics of the University of Louisville, Louisville, KY 40202, USA.

ABSTRACT
Diabetes mellitus and nonalcoholic fatty liver disease (NAFLD) are often identified in patients simultaneously. Recent evidence suggests that endoplasmic reticulum (ER) stress and autophagy dysfunction play an important role in hepatocytes injury and hepatic lipid metabolism, however the mechanistic interaction between diabetes and NAFLD is largely unknown. In this study, we used a diabetic mouse model to study the interplay between ER stress and autophagy during the pathogenic transformation of NAFLD. The coexist of inflammatory hepatic injury and hepatic accumulation of triglycerides (TGs) stored in lipid droplets indicated development of steatohepatitis in the diabetic mice. The alterations of components for ER stress signaling including ATF6, GRP78, CHOP and caspase12 indicated increased ER stress in liver tissues in early stage but blunted in the later stage during the development of diabetes. Likewise, autophagy functioned well in the early stage but suppressed in the later stage. The inactivation of unfolded protein response and suppression of autophagy were positively related to the development of steatohepatitis, which linked to metabolic abnormalities in the compromised hepatic tissues in diabetic condition. We conclude that the adaption of ER stress and impairment of autophagy play an important role to exacerbate lipid metabolic disorder contributing to steatohepatitis in diabetes.

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(A) The images of immunofluorescent staining for detection of LC3BII expression. (B-D) The expressions of LC3BII, P62, T-p70 and P-P70 were detected by Western blotting assay. Data are presented as mean ± SD. m: mouse age in months. OVE: OVE26. P-p70: phospho-p70 S6 Kinase; T-p70: total p70 S6 kinase. * P<0.05 compared to FVB controls. & P <0.05 compared to OVE26 mice aged 3 month.
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Figure 5: (A) The images of immunofluorescent staining for detection of LC3BII expression. (B-D) The expressions of LC3BII, P62, T-p70 and P-P70 were detected by Western blotting assay. Data are presented as mean ± SD. m: mouse age in months. OVE: OVE26. P-p70: phospho-p70 S6 Kinase; T-p70: total p70 S6 kinase. * P<0.05 compared to FVB controls. & P <0.05 compared to OVE26 mice aged 3 month.

Mentions: The clearance routes for the deleterious unfolded/misfolded proteins are not only endoplasmic reticulum-associated degradation (ERAD) but also ER stress-activated autophagy. If not timely removed, the unfolded/misfolded proteins can be toxic to cells to trigger cell death. Therefore, autophagy could play a very important role in keeping cellular homeostasis in the compromised liver caused by metabolic abnormalities of diabetes. The autophagy signaling components including LC3BII, P62, phospho-p70 S6 Kinase (P-p70) and total p70 S6 kinase (T-p70) were further evaluated. LC3BII, an important autophagy effector associate with lipid droplets movement, was reported being concentrated in autophagosome membranes during the autophagic process 22. Therefore, we firstly investigated the cytoplasmic distribution of LC3BII in the hepatocytes by immunofluorescent staining. As shown in the Fig 4A, positive staining of LC3BII represented by red fluorescence extensively diffused in the hepatocytes of OVE26 mice, whereas no fluorescence signaling of LC3BII was detected in the same age FVB controls (Fig 5A). To study the dynamic changes, the protein levels of LC3BII were further quantified by Western blot. The hepatic LC3BII protein levels in the all OVE26 mice were higher than that in the same age FVB controls, which consisted to the result of fluorescence staining. Interestingly, the hepatic LC3BII level in the OVE26 mice was significantly increased at month 5 compared to that at month 3, but the increase was blunted at month 8 (Fig 5B), implying a compromised autophagy function. Since changes in LC3BII levels could be caused by either autophagosome formation or degradation in lysosomes, and this issue needed to be clarified. P62 is an important component in the autophagy signaling. It has been shown that the aggregating p62 and ubiquitinylated proteins serve as a nucleating scaffold for autophagosome biogenesis 23. In addition, p62 can bind directly to LC3 proteins via a specific sequence motif, and acts as autophagy receptors for ubiquitinated proteins 24. P62 is also required for the aggregation of ubiquitinylated proteins and delivers ubiquitinylated cargos to the proteasome 25. Therefore, p62 was further evaluated by Western blot in the OVE26 mice and FVB controls. Our results showed decreased P62 expressions in the OVE26 mice at month 5 and month 8 compared to the same aged FVB controls (Fig 5C), which provided further evidence of autophagy dysfunction because of loss of autophagosome biogenesis. mTOR/p70S6K signaling pathway plays an important role in regulation of autophagy 26. It has been shown that an mTOR independent p70S6K inhibitor prevents LC3-I conversion to LC3-II, a critical process in autophagosome formation, in the situation of massive autophagy 27. Therefore, we next examined the mTOR substrates p70S6K levels in the hepatic tissues of OVE26 mice. The expressions of phospho-p70S6K and total p70S6K proteins were analyzed by Western blot and the ratio of phospho-p70S6K and total p70S6K was determined. The results showed that phospho-p70S6K protein level was up-regulated significantly at month 5 but the increase was blunted at month 8 in the liver tissues of OVE26 mice (Fig 5D), implying less autophagosomes formation in the liver at later stage of diabetes. All these results suggested suppression of hepatic autophagy in the later stage of OVE26 mice, and the dysfunction of autophagy was associated with the lipid metabolic abnormalities by diabetes.


ER stress and autophagy dysfunction contribute to fatty liver in diabetic mice.

Zhang Q, Li Y, Liang T, Lu X, Zhang C, Liu X, Jiang X, Martin RC, Cheng M, Cai L - Int. J. Biol. Sci. (2015)

(A) The images of immunofluorescent staining for detection of LC3BII expression. (B-D) The expressions of LC3BII, P62, T-p70 and P-P70 were detected by Western blotting assay. Data are presented as mean ± SD. m: mouse age in months. OVE: OVE26. P-p70: phospho-p70 S6 Kinase; T-p70: total p70 S6 kinase. * P<0.05 compared to FVB controls. & P <0.05 compared to OVE26 mice aged 3 month.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4400387&req=5

Figure 5: (A) The images of immunofluorescent staining for detection of LC3BII expression. (B-D) The expressions of LC3BII, P62, T-p70 and P-P70 were detected by Western blotting assay. Data are presented as mean ± SD. m: mouse age in months. OVE: OVE26. P-p70: phospho-p70 S6 Kinase; T-p70: total p70 S6 kinase. * P<0.05 compared to FVB controls. & P <0.05 compared to OVE26 mice aged 3 month.
Mentions: The clearance routes for the deleterious unfolded/misfolded proteins are not only endoplasmic reticulum-associated degradation (ERAD) but also ER stress-activated autophagy. If not timely removed, the unfolded/misfolded proteins can be toxic to cells to trigger cell death. Therefore, autophagy could play a very important role in keeping cellular homeostasis in the compromised liver caused by metabolic abnormalities of diabetes. The autophagy signaling components including LC3BII, P62, phospho-p70 S6 Kinase (P-p70) and total p70 S6 kinase (T-p70) were further evaluated. LC3BII, an important autophagy effector associate with lipid droplets movement, was reported being concentrated in autophagosome membranes during the autophagic process 22. Therefore, we firstly investigated the cytoplasmic distribution of LC3BII in the hepatocytes by immunofluorescent staining. As shown in the Fig 4A, positive staining of LC3BII represented by red fluorescence extensively diffused in the hepatocytes of OVE26 mice, whereas no fluorescence signaling of LC3BII was detected in the same age FVB controls (Fig 5A). To study the dynamic changes, the protein levels of LC3BII were further quantified by Western blot. The hepatic LC3BII protein levels in the all OVE26 mice were higher than that in the same age FVB controls, which consisted to the result of fluorescence staining. Interestingly, the hepatic LC3BII level in the OVE26 mice was significantly increased at month 5 compared to that at month 3, but the increase was blunted at month 8 (Fig 5B), implying a compromised autophagy function. Since changes in LC3BII levels could be caused by either autophagosome formation or degradation in lysosomes, and this issue needed to be clarified. P62 is an important component in the autophagy signaling. It has been shown that the aggregating p62 and ubiquitinylated proteins serve as a nucleating scaffold for autophagosome biogenesis 23. In addition, p62 can bind directly to LC3 proteins via a specific sequence motif, and acts as autophagy receptors for ubiquitinated proteins 24. P62 is also required for the aggregation of ubiquitinylated proteins and delivers ubiquitinylated cargos to the proteasome 25. Therefore, p62 was further evaluated by Western blot in the OVE26 mice and FVB controls. Our results showed decreased P62 expressions in the OVE26 mice at month 5 and month 8 compared to the same aged FVB controls (Fig 5C), which provided further evidence of autophagy dysfunction because of loss of autophagosome biogenesis. mTOR/p70S6K signaling pathway plays an important role in regulation of autophagy 26. It has been shown that an mTOR independent p70S6K inhibitor prevents LC3-I conversion to LC3-II, a critical process in autophagosome formation, in the situation of massive autophagy 27. Therefore, we next examined the mTOR substrates p70S6K levels in the hepatic tissues of OVE26 mice. The expressions of phospho-p70S6K and total p70S6K proteins were analyzed by Western blot and the ratio of phospho-p70S6K and total p70S6K was determined. The results showed that phospho-p70S6K protein level was up-regulated significantly at month 5 but the increase was blunted at month 8 in the liver tissues of OVE26 mice (Fig 5D), implying less autophagosomes formation in the liver at later stage of diabetes. All these results suggested suppression of hepatic autophagy in the later stage of OVE26 mice, and the dysfunction of autophagy was associated with the lipid metabolic abnormalities by diabetes.

Bottom Line: Likewise, autophagy functioned well in the early stage but suppressed in the later stage.The inactivation of unfolded protein response and suppression of autophagy were positively related to the development of steatohepatitis, which linked to metabolic abnormalities in the compromised hepatic tissues in diabetic condition.We conclude that the adaption of ER stress and impairment of autophagy play an important role to exacerbate lipid metabolic disorder contributing to steatohepatitis in diabetes.

View Article: PubMed Central - PubMed

Affiliation: 1. Department of Infectious Diseases, Affiliated Hospital of Guiyang Medical College, Guiyang, Guizhou, China, 550004 ; 4. Kosair Children's Hospital Research Institute, the Department of Pediatrics of the University of Louisville, Louisville, KY 40202, USA.

ABSTRACT
Diabetes mellitus and nonalcoholic fatty liver disease (NAFLD) are often identified in patients simultaneously. Recent evidence suggests that endoplasmic reticulum (ER) stress and autophagy dysfunction play an important role in hepatocytes injury and hepatic lipid metabolism, however the mechanistic interaction between diabetes and NAFLD is largely unknown. In this study, we used a diabetic mouse model to study the interplay between ER stress and autophagy during the pathogenic transformation of NAFLD. The coexist of inflammatory hepatic injury and hepatic accumulation of triglycerides (TGs) stored in lipid droplets indicated development of steatohepatitis in the diabetic mice. The alterations of components for ER stress signaling including ATF6, GRP78, CHOP and caspase12 indicated increased ER stress in liver tissues in early stage but blunted in the later stage during the development of diabetes. Likewise, autophagy functioned well in the early stage but suppressed in the later stage. The inactivation of unfolded protein response and suppression of autophagy were positively related to the development of steatohepatitis, which linked to metabolic abnormalities in the compromised hepatic tissues in diabetic condition. We conclude that the adaption of ER stress and impairment of autophagy play an important role to exacerbate lipid metabolic disorder contributing to steatohepatitis in diabetes.

Show MeSH
Related in: MedlinePlus