Glucose regulated protein 78 is potentially an important player in the development of nonalcoholic steatohepatitis

doi:10.1016/j.gene.2017.09.051

Gene

Volume 637, 30 December 2017, Pages 138-144

https://doi.org/10.1016/j.gene.2017.09.051 Get rights and content

Highlights

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A rat model of nonalcoholic steatohepatitis (NASH) was successfully established by induction of both a high sucrose and a high fat diet.
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The plasma levels of LPS, ALT, FFA, and TG in and the contents of FFA, TG, TNFα, and MDA in the liver of NASH rat were gradually increased.
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Macrophage infiltration and hepatocytic apoptosis was significantly increased in the livers of the rats from the NASH group compared to the control group.
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The expression levels of GRP78 and CD68 in the liver of the NASH of rats group were increased compared to the control group.
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ER stress leads to GRP78 upregulation in HepG2 cells.

Abstract

Endoplasmic reticulum stress (ERS) plays an important role in metabolic diseases. Glucose regulated protein 78 (GRP78) is a molecular chaperone in the ER where it is a marker for ERS activation. This study investigates the role of GRP78 in the pathogenesis of nonalcoholic steatohepatitis (NASH) in rats. Our rat model of NASH was induced by both a high sucrose and a high fat diet. The expression levels of LPS, ALT, FFA, and TG in the serum and FFA, TG, MDA, and TNFα in the liver were assessed. H&E, TUNEL and IHC staining were performed to examine histological changes, apoptosis and macrophage infiltration in the NASH liver tissue, respectively. The expression level of GRP78 in the liver was evaluated by Western blot and RT-PCR. The plasma levels of LPS, ALT, FFA, and TG in and the contents of FFA, TG, TNFα, and MDA in the liver were gradually increased. Macrophage infiltration and hepatocytic apoptosis was significantly increased in the livers of the rats from the NASH group compared to the control group. The protein and mRNA levels of GRP78 in the liver of rats from the NASH group were also upregulated. In addition, GRP78 expression levels were positively correlated with the levels of ALT, TNFα, CD68 and hepatocytic apoptosis. Thus, our results suggest that GRP78 may be an important player in the pathogenesis of NASH.

Introduction

Nonalcoholic fatty liver disease (NAFLD) is characterized by an excessive deposition of triglycerides in the liver, which can lead to chronic liver disease without history of excessive alcohol consumption. The NAFLD spectrum starts from fatty liver, progressing through nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis, liver failure and finally leads to end-stage liver disease and liver cancer (Chalasani et al., 2012, Onyekwere et al., 2015, Liu et al., 2016). NASH is a critical intermediate stage in the NAFLD spectrum and the pathogenesis of NASH may follow the classic “two-hit theory” (Day and James, 1998) in which the first hit refers to an accumulation of fatty acids and triglycerides within the liver that sensitizes it to additional proinflammatory insults and the second hit is chronic stress, such as enhanced lipid peroxidation (Sanyal et al., 2001) and endoplasmic reticulum stress (ERS) (Zhang et al., 2014b). Induction of ERS can enable liver cells to mount a resistant and adaptive response to prevent damage using the unfolded protein response pathways. However, if the ERS is sustained and serious, it will induce apoptosis, inflammation and hepatocyte injury (Hotamisligil, 2010, Gentile et al., 2011). Glucose regulated protein 78 (GRP78) is known to be a marker for ERS (Ji et al., 2011). GRP78 upregulation has been observed in various diseases, such as metabolic syndrome, alcoholic liver disease and CCL4-induced liver cirrhosis (Cheng, 2008, Sage et al., 2012, Galligan et al., 2014, Zhang et al., 2014a). Clinical observations and experimental studies show that patients with NASH often concomitantly present with intestinal endotoxemia (IETM) (Brun et al., 2007, Harte et al., 2010), which is interesting as endotoxin is a potential inducer of ERS (Hiramatsu et al., 2006).

In this study, we have successfully established an animal model of NASH, which was induced by high fat and high sucrose (Zhou et al., 2014). The GRP78 expression at both RNA transcription and protein levels were examined in the development of NASH in this murine model. Based on our results, we propose that GRP78 may play an important role in the pathogenesis of NASH. Additionally, our study attempts to establish a relationship between GRP78 expression and IETM.

Section snippets

Animal model

30 SD male rats, weighing 220–250 g, were obtained from the Experimental Animal Center of Shanxi Medical University. Experiments were carried out in accordance with the guidelines issued by the Ethical Committee of Shanxi Medical University. After a week of adaptive feeding, the rats were randomly divided into two groups: the normal control group and the experimental group which was treated with sucrose and fat-enriched diet (70% normal fodder + 20% lard + 10% sucrose + 1% cholesterol + 0.25% Cholic

Biochemical and inflammation indexes in the plasma and liver tissues of the NASH rats

To assess the biochemical indexes in NASH models, we examined the levels of ALT, TG, FFA and LPS in the plasma and TG, FFA, TNFα and MDA in the liver. The levels of ALT, TG and FFA in the plasma (Fig. 1A, B & D), and TG, FFA and MDA (Fig. 1C, E & H) in the liver samples were elevated at 4 weeks with disease progression in the NASH mice while the levels of LPS in the plasma and TNFα in the liver began to increase at 8 weeks (Fig. 1F&G), when compared to plasma and liver samples from the control

Discussion

In this study, the NASH rat model successfully recapitulated elevated levels of ALT in the plasma and pathological changes in the liver. The elevation of GRP78 is a critical component of the ER stress response and the elevation of GRP78 can adjust the homeostasis of ERS stress (Than and Newsome, 2015). GRP78 has been shown to play a critical role in the pathogenesis of many diseases (Pizzo, 2009, Ji et al., 2011). Our study showed that GRP78 expression at both the protein and mRNA levels began

Acknowledgments

This work was supported by the Dr. Start Fund of Shanxi Medical University (Grant No. B03201202 and B03201203), the Youth Science and Technology Research Fund, Basic Research Projects in Shanxi (Grant No. 2014021040-3), Shanxi Province Higher Education Science and Technology Innovation Project (Grant No. 2013121), and Shanxi Province Natural and Science Foundation (Grant No. 2014011046-3).

Conflict of interest

The authors declare that they have no any conflict of interest.

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This study analyzed the formation of goose fatty liver due to endoplasmic reticulum stress (ERS) caused by 3 types of sugar. Transcriptome analysis was performed for liver tissues from geese fed a traditional diet (maize flour), geese overfed with traditional diet, and geese overfed with diet supplemented with glucose, fructose, or sucrose. Correlation analysis of the liver tissue transcriptomes showed that differentially expressed genes (DEGs) involved in ERS were significantly negatively correlated with DEGs involved in inflammation response in the sucrose overfeeding group, and significantly positively correlated with the DEGs involved in lipid metabolism in fructose overfeeding group. Goose primary hepatocytes were isolated in vitro and then treated with glucose or fructose. Some were also treated with ERS inhibitor 4-phenylbutyric acid (4-PBA). In the hepatocytes, mRNA expression of X-Box Binding Protein 1 (XBP1), activating transcription factor 6 (AFT6) and glucose-regulated protein 78 (GRP78) genes increased in the two sugar groups (glucose and fructose), but were suppressed by adding 4-PBA. The mRNA expression data, protein kinase contents, and triglyceride (TG) and very low-density lipoprotein (VLDL) concentrations all suggest that ERS regulates lipid deposition induced by glucose and fructose via elevating lipid synthesis, inhibiting fatty acid oxidation, and decreasing lipid transportation. In conclusion, glucose, or fructose cause ERS and then ERS causes lipid deposition in goose primary hepatocytes. Three types of sugar cause lipid accumulation and then lipid accumulation prevents ERS during goose fatty liver formation, which suggests a potential mechanism protects goose livers from ERS. The different sugars may induce lipid deposition in different ways.
Decoding the roles of heat shock proteins in liver cancer
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Hepatocellular carcinoma (HCC) is one of the most common gastrointestinal malignancies, characterized by insidious onset and high propensity for metastasis and recurrence. Apart from surgical resection, there are no effective curative methods for HCC in recent years, due to resistance to radiotherapy and chemotherapy. Heat shock proteins (HSP) play a crucial role in maintaining cellular homeostasis and normal organism development as molecular chaperones for intracellular proteins. Both basic research and clinical data have shown that HSPs are crucial participants in the HCC microenvironment, as well as the occurrence, development, metastasis, and resistance to radiotherapy and chemotherapy in various malignancies, particularly liver cancer. This review aims to discuss the molecular mechanisms and potential clinical value of HSPs in HCC, which may provide new insights for HSP-based therapeutic interventions for HCC.
Chicoric acid (CA) induces autophagy in gastric cancer through promoting endoplasmic reticulum (ER) stress regulated by AMPK
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ER stress ensues through prolonged activation of the pro-death ER stress pathways regulated by CHOP [56]. The increase of BIP expression is often used as indicator of ERS [57]. Accumulating studies have suggested that ER stress is a potential therapeutic target for developing drugs that interfere with specific signaling pathways to prevent tumor growth [58,59].
Gastric cancer is one of the most common cancers leading to tumor-related deaths worldwide. Chicoric acid (CA) exhibits a variety of protective effects in different diseases. However, its role in regulating tumor progression has not been reported. Autophagy, as a conserved catabolic process, sustains cellular homoeostasis responding to stress to modulate cell fate. In the study, the effects of CA on gastric cancer were investigated. The results indicated that CA treatment markedly reduced the cell viability and induced apoptosis in gastric cancer cells, and prevented tumor growth in an established xenograft gastric cancer model. Furthermore, CA exposure significantly induced autophagy both in gastric cancer cells and tumor samples, as evidenced by the up-regulated expression of LC3II. Moreover, phosphorylated AMP-activated protein kinase (AMPK) and p70S6 kinase (p70s6k) expression were obviously promoted by CA in vitro and in vivo. Importantly, blocking AMPK activation abrogated CA-induced expression of LC3II in gastric cancer cells. In addition, endoplasmic reticulum (ER) stress in tumor samples or cells was markedly induced by CA treatment through promoting the expression of associated signals such as Parkin, protein kinase RNA-like ER kinase (PERK), activating transcription factors 4 (ATF4) and ATF6. Importantly, these effects were abolished by the inhibition of AMPK signaling. Collectively, our findings indicated that CA prevents human gastric cancer progression by inducing autophagy partly through the activation of AMPK, and represents an effective therapeutic strategy against gastric cancer development.
Endoplasmic reticulum stress-induced iRhom2 up-regulation promotes macrophage-regulated cardiac inflammation and lipid deposition in high fat diet (HFD)-challenged mice: Intervention of fisetin and metformin
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Further, IRE1 activation leads to unconventional splicing of XBP1 [83]. The increase of GRP78 expression is often used as indicator of ERS [84]. Here in our study, HFD-induced ERS in heart tissue samples was again verified, as evidenced by the up-regulated expression of ERS-associated markers, including p-PERKT980, p-eIF2αS51, p-IRE1S724, XBP1, ATF6, ATF4, CHOP and GRP78.
Endoplasmic reticulum stress (ERS) has been implicated in obesity-associated cardiac remodeling and dysfunction. Inactive rhomboid protein 2 (iRhom2), also known as Rhbdf2, is an inactive member of the rhomboid intramembrane proteinase family, playing an essential role in regulating inflammation. Nevertheless, the role of ERS-meditated iRhom2 pathway in metabolic stress-induced cardiomyopathy remains unknown. In the study, we showed that 4-PBA, as an essential ERS inhibitor, significantly alleviated high fat diet (HFD)-induced metabolic disorder and cardiac dysfunction in mice. Additionally, lipid deposition in heart tissues was prevented by 4-PBA in HFD-challenged mice. Moreover, 4-PBA blunted the expression of iRhom2, TACE, TNFR2 and phosphorylated NF-κB to prevent HFD-induced expression of inflammatory factors. Further, 4-PBA restrained HFD-triggered oxidative stress by promoting Nrf-2 signaling. Importantly, 4-PBA markedly suppressed cardiac ERS in HFD mice. The anti-inflammation, anti-ERS and anti-oxidant effects of 4-PBA were verified in palmitate (PAL)-incubated macrophages and cardiomyocytes. In addition, promoting ERS could obviously enhance iRhom2 signaling in vitro. Intriguingly, our data demonstrated that PAL-induced iRhom2 up-regulation apparently promoted macrophage to generate inflammatory factors that could promote cardiomyocyte inflammation and lipid accumulation. Finally, interventions by adding fisetin or metformin significantly abrogated metabolic stress-induced cardiomyopathy through the mechanisms mentioned above. In conclusion, this study provided a novel mechanism for metabolic stress-induced cardiomyopathy pathogenesis. Therapeutic strategy to restrain ROS/ERS/iRhom2 signaling pathway could be developed to prevent myocardial inflammation and lipid deposition, consequently alleviating obesity-induced cardiomyopathy.
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Research paperGlucose regulated protein 78 is potentially an important player in the development of nonalcoholic steatohepatitis

Highlights

Abstract

Introduction

Section snippets

Animal model

Biochemical and inflammation indexes in the plasma and liver tissues of the NASH rats

Discussion

Acknowledgments

Conflict of interest

Gastroenterology

Gastroenterology

Free Radic. Biol. Med.

Cell

Gastroenterology

Atherosclerosis

Gene

Increased intestinal permeability in obese mice: new evidence in the pathogenesis of nonalcoholic steatohepatitis

Am. J. Physiol. Gastrointest. Liver Physiol.

Dissection of endoplasmic reticulum stress signaling in alcoholic and non-alcoholic liver injury

J. Gastroenterol. Hepatol.

Immunoglobulin heavy-chain-binding protein (BiP): a stress protein that has the potential to be a novel therapy for rheumatoid arthritis

Biochem. Soc. Trans.

Endoplasmic reticulum stress and the unfolded protein response in nonalcoholic fatty liver disease

Antioxid. Redox Signal.

Elevated endotoxin levels in non-alcoholic fatty liver disease

J Inflamm (Lond)

Research paper
Glucose regulated protein 78 is potentially an important player in the development of nonalcoholic steatohepatitis