Open Access

Erythropoietin suppresses hepatic steatosis and obesity by inhibiting endoplasmic reticulum stress and upregulating fibroblast growth factor 21

  • Authors:
    • Ting Hong
    • Zhijuan Ge
    • Bingjie Zhang
    • Ran Meng
    • Dalong Zhu
    • Yan Bi
  • View Affiliations

  • Published online on: May 28, 2019     https://doi.org/10.3892/ijmm.2019.4210
  • Pages: 469-478
  • Copyright: © Hong et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

Erythropoietin (EPO), known primarily for its role in erythropoiesis, was recently reported to play a beneficial role in regulating lipid metabolism; however, the underlying mechanism through which EPO decreases hepatic lipid accumulation requires further investigation. Endoplasmic reticulum (ER) stress may contribute to the progression of hepatic steatosis. The present study investigated the effects of EPO on regulating ER stress in fatty liver. It was demonstrated that EPO inhibited hepatic ER stress and steatosis in vivo and in vitro. Interestingly, these beneficial effects were abrogated in liver‑specific sirtuin 1 (SIRT1)‑knockout mice compared with wild‑type littermates. In addition, in palmitate‑treated hepatocytes, small interfering RNA‑mediated SIRT1 silencing suppressed the effects of EPO on lipid‑induced ER stress. Additionally, EPO stimulated hepatic fibroblast growth factor 21 (FGF21) expression and secretion in a SIRT1‑dependent manner in mice. Furthermore, the sensitivity of hepatocytes from obese mice to FGF21 was restored following treatment with EPO. Collectively, the results of the present study revealed a new mechanism underlying the regulation of hepatic ER stress and FGF21 expression induced by EPO; thus, EPO may be considered as a potential therapeutic agent for the treatment of fatty liver and obesity.

Introduction

Non-alcoholic fatty liver disease (NAFLD) has become a major public health concern, affecting over half a billion people worldwide, and has been associated with a variety of metabolic comorbidities (1). Hepatic steatosis, as a key metabolic hallmark of NAFLD, is mainly caused by excessive fat accumulation. An overload of free fatty acids, particularly saturated free fatty acids, may induce the endoplasmic reticulum (ER) stress response (2,3). Additionally, the presence of non-functional unfolded or misfolded proteins and subsequent ER stress may result in reduced lipid droplet stability and increased lipogenesis, aggravating NAFLD (4). Importantly, in the livers of patients with NAFLD and animal models of diet-induced obesity, chronic ER stress and prolonged activation of the unfolded protein response (UPR) was observed; these factors play a key role in the development of hepatic steatosis (5-7).

Erythropoietin (EPO), a glycoprotein hormone, has been traditionally considered as a key regulator of erythropoiesis (8,9); however, its protective role has recently been extended to ameliorating metabolic disorders (10). Specifically, EPO was demonstrated to promote brown fat-like characteristics to increase fatty acid oxidation in white adipose tissue from obese mice (11); EPO was reported to decrease adipose tissue mass by increasing fat utilization in skeletal muscles from obese mice or humans during aerobic exercise (12,13). In liver tissues, EPO was found to reduce insulin resistance via peroxisome proliferator-activated receptor γ (PPARγ)-dependent protein kinase B (AKT) activation, and alleviate steatosis, partially via lipophagy (14,15); however, the mechanism underlying the role of EPO in hepatic lipid metabolism, ER stress in particular, remains unknown.

Sirtuin 1 (SIRT1), a member of the sirtuin family of nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylases, regulates key aspects of lipid metabolism (16,17). SIRT1 can deacetylate protein kinase-like ER kinase (PERK) and inhibit the PERK-eukaryotic initiation factor 2α (eIF2α) axis of the UPR pathway, thereby suppressing hepatic steatosis in mice (18-20). Our previous study revealed that the NAD+/NADH ratio and SIRT1 activity increased in response to EPO in HepG2 cells in vitro (15). However, the role of SIRT1 in association with EPO in ER stress remains unclear. In addition, SIRT1-induced fibroblast growth factor 21 (FGF21) expression was found to play a critical role in controlling obesity and energy balance (21,22).

The aim of the present study was to investigate the effects of EPO on hepatic ER stress and FGF21 expression, and determine whether this mechanism involves SIRT1.

Materials and methods

Animal model

All animal studies were performed in accordance with the National Institutes of Health guidelines and were approved by the Nanjing University Medical School Institutional Animal Care and Use Committee. Male hepatocyte-specific SIRT1-deleted (SIRT1-LKO) mice and wild-type (WT, C57BL/6J) littermates (8 weeks old) were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China). The mice were housed under controlled temperature (20-22°C) and humidity (50%) conditions, with a 12-h light/dark cycle. All mice were given free access to high-fat diet (HFD; 60% calories from fat, 25% calories from carbohydrates and 15% calories from protein; Yangzhou University, Yangzhou, China) or standard chow (NC; 10% calories from fat, 75% calories from carbohydrates and 15% calories from protein; Yangzhou University), as described below. The mice were intraperitoneally injected with 3,000 U/kg of recombinant human EPO (Sunshine Pharmaceutical) or an equivalent volume of PBS every other day (n=6 per group). For the first experiment, SIRT1-LKO mice and WT littermates were fed HFD for 12 weeks, and then divided into two groups prior to treatment with EPO or PBS for 5 weeks. For the second experiment, C57BL/6J mice were treated with EPO or PBS for 7 or 14 days; the mice were given free access to NC and water. Finally, for the third experiment, C57BL/6J mice were fed HFD or NC for 12 weeks; the HFD group was intraperitoneally injected with EPO or PBS for 5 weeks. Lean littermate control mice were intraperitoneally injected with an equivalent volume of PBS. Body weight and fat were measured weekly (AccuFat-1050, MAG-MED). After 5 weeks, the mice were fasted for 8 h and underwent an intraperitoneal glucose tolerance test (IPGTT), as previously described (14). Glucose levels were determined from blood samples obtained via the tail veil at 0, 30, 60 and 120 min following glucose administration (1.5 g/kg). Subsequently, all the mice were deprived of food for 8 h prior to sacrifice for the collection of blood samples and liver tissues for further examination. After being weighed, fresh liver tissues were subjected to hematoxylin and eosin (H&E; Goodbio) and Oil Red O staining (Goodbio) (15). For immunofluorescence, the sections were incubated with rabbit anti-protein disulfide isomerase (PDI) antibody (1:500; Cell Signaling Technology, Inc.; cat. no. 3501). For immunohistochemistry (IHC), the sections were incubated with rabbit anti-FGF21 antibody (1:200; Abcam; cat. no. ab66564) and quantitated by ImageJ plugin IHC profiler (National Institutes of Health), as previously described (23-25). Liver triglyceride (TG) content was measured using an ELISA kit according to the manufacturer's instructions (BioVision, Inc.). All samples were stored at −80°C for further analysis.

Cell culture

Primary hepatocytes were isolated from 8-12-week-old C57BL6/J mice (20-25 g) using a two-step perfusion method, and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), as described previously (26). For pharmacological studies, cells were treated with 40 U/ml EPO, 0.3 mmol/l palmitate (PA; Sigma-Aldrich; Merck KGaA) or 1 µmol/l thapsigargin (Thp; Sigma-Aldrich; Merck KGaA) for 24 h. Where indicated, the cells were pre-treated with 40 µmol/l resveratrol (Sigma-Aldrich; Merck KGaA) or 2 µmol/l EX-527 (MedChem Express, LLC) for 1 h prior to EPO treatment. Primary hepatocytes isolated from EPO-treated and control mice were incubated with 1, 2 and 10 nmol/l human recombinant FGF21 (Abcam) for 4 h; vehicle treatment served as control.

Small interfering RNA (siRNA) transfection

Primary hepatocytes were transfected with siRNA against SIRT1 (GenePharma Co., Ltd.) using Lipofectamine® 3000 (Thermo Fisher Scientific, Inc.). PA and EPO were added to the cells at 36 h post-transfection. The siRNA sequences were as follows: SIRT1 siRNA, 5′-GAUGAAGUUGACCUCCUCA-3′; and negative control, 5′-UUCUCCGAACGUGUCACGUTT-3′.

RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qRCR) analysis

Total RNA was extracted from mouse liver tissue using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). RT of RNA into cDNA was performed with the RT reagent kit (Takara Bio, Inc.), followed by qPCR analysis on an ABI StepOne Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) using SYBR Green (Roche Diagnostics) at a final volume of 20 µl, according to the manufacturer's protocol. The primers used were as follows: FGF21, forward 5′-CCTCCAGTTTGGGGGTCAAG-3′ and reverse 5′-CTGGTTTGGGGAGTCCTTCT-3′; peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), forward 5′-GTCCTTCCTCCATGCCTGAC-3′ and reverse 5′-TAGCTGAGCTGAGTGTTGGC-3′; FGF receptor 1 (FGFR1), forward 5′-GTGGAGAATGAGTATGGGAGC-3′ and reverse 5′-GGATCTGGACATACGGCAAG-3′; single-pass transmembrane protein βKlotho, forward 5′-ACGAGGGCTGTTTTATGTGG-3′ and reverse 5′-CAGGTGAGGATCGGTAAACTG-3′; acyl-CoA oxidase 1 (Acox1), forward 5′-TGCCATTCGATACAGTGCTG-3′ and reverse 5′-CAGGAGCGGGAAGAGTTTATAC-3′; pyruvate dehydrogenase kinase 4, isoenzyme 4 (Pdk4), forward 5′-GGATTACTGACCGCCTCTTTAG-3′ and reverse 5′-GGGAGCTTTTCTACAGACTCAG-3′; enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase (Ehhadh), forward 5′-AGCTGTTTATGTACCTTCGGG-3′ and reverse 5′-CTGCTTTGGGTCTGACTCTAC-3′; glyceraldehyde 3-phosphate dehydrogenase (GAPDH), forward 5′-TGGCCTTCCGTGTTTCCTAC-3′ and reverse 5′-GAGTTGCTGTTGAAGTCGCA-3′. Expression was normalized to GAPDH and determined via the 2−ΔΔCq method (27).

Western blot analysis

Liver tissues or cultured hepatocytes were lysed with radioimmunoprecipitation assay lysis buffer (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with protease inhibitor cocktail (Roche Diagnostics). Total protein concentration was measured using the bicinchoninic acid method (BCA protein assay kit; Thermo Fisher Scientific, Inc.). Subsequently, 20 µg of each sample was separated via 8-15% Bis-Tris NuPAGE gels (Goodbio) and transferred to polyvinylidene difluoride membranes (Invitrogen; Thermo Fisher Scientific, Inc.). The membranes were blocked with 5% non-fat milk or bovine serum albumin, and incubated overnight at 4°C with specific antibodies. Antibodies against SIRT1 (rabbit monoclonal, cat. no. 9475, 1:1,000), PERK (rabbit monoclonal, cat. no. 3192, 1:1,000), phosphorylated (p)-eIF2α (Ser51, rabbit monoclonal, cat. no. 3398, 1:1,000), eIF2α (rabbit monoclonal, cat. no. 5324, 1:1,000) and GAPDH (rabbit monoclonal, cat. no. 5174, 1:2,000) were obtained from Cell Signaling Technology, Inc. The anti-FGF21 antibody (rabbit polyclonal, cat. no. ab66564, 1:1,000) and glucose-regulated protein 78 (GRP78; rabbit polyclonal, cat. no. ab21685, 1:1,000) were purchased from Abcam. The anti-p-PERK antibody (Thr981, rabbit polyclonal, cat. no. YP1055, 1:1,000) was obtained from ImmunoWay Biotechnology. The membranes were then washed and incubated with a secondary antibody (goat anti-rabbit IgG, ZSGB-BIO; OriGene Technologies, Inc.) at room temperature for 1 h. The antibody-antigen complexes were visualized by enhanced chemiluminescence (ECL; EMD Millipore). Band intensities were quantified using ImageJ software (National Institutes of Health). All antibodies were validated by the manufacturer.

FGF21 levels

According to the manufacturer's instructions, the levels of FGF21 in mouse serum and cell culture supernatant were determined using a FGF21 ELISA kit (cat. no. ab212160, Abcam). Briefly, after equilibrating all reagents at room temperature, 50 µl of samples or standards were added to the wells, followed by the antibody cocktail. After 1 h of incubation, the wells were washed three times to remove unbound material. Subsequently, 100 µl 3,3′,5,5′-tetramethylbenzidene substrate was added to each well and incubated for 15 min at room temperature in the dark. The reaction was terminated by adding 50 µl stop solution and the optical density was recorded at 450 nm.

Statistical analysis

All analyses were performed using SPSS software (version 22.0, IBM Corp.). All data are expressed as the mean ± standard error of the mean. Differences between multiple groups were determined by performing one-way ANOVA followed by the least significant difference or Dunnett's T3 post hoc test. Student t-tests were used to assess differences between two groups. P<0.05 was considered to indicate a statistically significant difference.

Results

EPO-induced suppression of hepatic steatosis and reductions in body weight are compromised by hepatic-specific SIRT1 deletion in HFD-fed mice

After 5 weeks of treatment, EPO-treated HFD-fed mice exhibited weight loss and reduced body fat compared with control mice (Fig. 1A and B). Additionally, IPGTTs revealed that the blood glucose levels were lower in EPO-treated mice compared with those in the PBS-treated group (Fig. 1C). Furthermore, histological analysis, including H&E and Oil Red O staining, demonstrated that EPO markedly alleviated hepatic steatosis in HFD-fed WT mice (Fig. 1D). Consistently, liver TG levels and liver weight were decreased in response to EPO treatment in the WT group (Fig. 1E and F). Compared with WT mice, these beneficial effects on body weight and hepatic steatosis were not observed in SIRT1-LKO mice. These results suggested a critical role for hepatic SIRT1 in mediating the protective effects of EPO in attenuating obesity and hepatic lipid accumulation.

EPO suppresses hepatic ER stress in a SIRT1-dependent manner

ER stress plays an important role in the development of hepatic steatosis (6). To investigate whether EPO alleviates metabolic ER stress, we measured the levels of the specific ER stress markers PDI and GRP78, and investigated PERK/eIF2α signaling in the UPR pathway. In HFD-fed mice, EPO intervention notably inhibited the expression of PDI, GRP78, p-PERK and p-eIF2α (Fig. 2A and B). In vitro, EPO-treated primary hepatocytes were incubated with PA or the ER stress inducer Thp. Treatment with PA or the ER stress agonist promoted the expression of GRP78, p-PERK and p-eIF-2α, in parallel with an increase in TG content, suggesting that activation of ER stress may induce hepatic steatosis. Conversely, the upregulation of these ER stress markers and increased TG content induced by Thp and PA were suppressed by EPO (Figs. 3A and S1).

SIRT1, a potent regulator of energy metabolism and stress response, may play an important role in fatty liver and obesity (18,28). The present study investigated whether SIRT1 mediates the effects of EPO on hepatic ER stress and steatosis. As shown in Fig. 2B, no notable alterations in the expression of GRP78, p-PERK or p-eIF2α were observed in SIRT1-LKO mice following EPO treatment. In addition, the effects of EPO on decreasing the protein expression levels of GRP78, p-PERK and p-eIF2α were attenuated following SIRT1 inhibition by siRNA-mediated silencing in PA-treated primary hepatocytes (Figs. 3B and S2). Collectively, these findings indicate that SIRT1 may mediate the effects of EPO on alleviating hepatic ER stress and lipid accumulation.

EPO increases hepatic FGF21 expression

In view of the notable effects of EPO treatment on the liver and whole-body metabolism, it was hypothesized that a hepatokine may mediate the effects of EPO on alleviating hepatic lipid accumulation and obesity. Thus, the expression of FGF21, a hormone secreted by the liver that acts as a potent regulator of lipid metabolism, was analyzed (29). The results revealed that the mRNA and protein expression levels of FGF21 were increased in primary hepatocytes following treatment with 20-40 U/ml EPO (Fig. 4A and B), which was consistent with the alterations in FGF21 expression in cell culture (Fig. S3A). The results of PCR, western blotting and IHC revealed that short-term EPO treatment for 7 or 14 days in mice fed NC promoted FGF21 mRNA and protein expression in liver tissues (Fig. 4C-F), which was accompanied by increased levels of circulating FGF21 (Fig. S3B). Moreover, previous evidence indicated that SIRT1-mediated activation of FGF21 prevented liver steatosis and obesity (21). Therefore, whether SIRT1 is required for EPO-induced FGF21 expression was investigated. As shown in Fig. 5A, EPO induced the expression of SIRT1 and FGF21 in hepatocytes, mimicking the effects of the SIRT1 agonist resveratrol. Conversely, the SIRT1 inhibitor EX-527 suppressed the upregulated expression of FGF21 mediated by EPO. In addition, EPO treatment increased the expression levels of FGF21 in the circulation and liver tissues of HFD-fed WT mice, but this effect was mitigated in mice harboring a hepatic-specific deletion of SIRT1 (Figs. 5B and S3C). Collectively, these results suggest that SIRT1 is required for EPO-stimulated FGF21 production in hepatocytes.

EPO partially restores sensitivity of hepatocytes to FGF21 in HFD-fed mice

An HFD diet was reported to suppress the expression of hepatic FGF21 receptors, including FGFR1 and βKlotho, and suggested that obesity occurs under FGF21-resistant conditions (30). In the livers of HFD-fed mice, EPO treatment partially restored the expression of the aforementioned receptors (Fig. 6A and B). Additionally, Acox1, Pdk4 and Ehhadh have been proposed as downstream target genes of the FGF21/PGC-1α axis associated with hepatic lipid β-oxidation (30,31). The mRNA expression levels of these genes were decreased in the livers of mice administered an HFD-fed challenge. Conversely, EPO intervention restored the expression of PGC-1α, Acox1 and Ehhadh, but not Pdk4 (Fig. 6C and D). In addition, FGF21-induced phosphorylation of eIF2α was inhibited in mouse hepatocytes isolated from HFD mice; however, FGF21 intervention enhanced eIF2α phosphorylation (Fig. 6E). Collectively, these results demonstrated that EPO treatment enhanced FGF21 sensitivity in response to alterations in metabolism.

Discussion

EPO has been attracting increasing attention for its potential beneficial effects against the progression of obesity, diabetes and fatty liver disease. However, the underlying mechanism mediating these effects remains unclear. In the present study, a novel mechanism by which EPO alleviates obesity-induced ER stress and hepatic steatosis in a SIRT1-dependent manner was proposed. Additionally, EPO was reported to serve as a positive regulator of hepatic FGF21 expression, further supporting the hypothesis that EPO may be a promising agent for the treatment of hepatic steatosis and obesity.

Hepatic ER stress, induced by pharmacological agents or metabolic dysregulation, promotes hepatic lipid accumulation by increasing lipogenesis (32). Genetic ablation studies revealed that the phosphorylation of eIF2α, a key downstream target of PERK, exacerbates the progression of hepatic steatosis in mice subjected to pharmacologically induced ER stress (33). Conversely, transgenic mice with inactivated eIF2α via dephosphorylation were protected from HFD-induced hepatic steatosis (34). In the present study, in addition to the protective effects of EPO against weight gain and fat accumulation, EPO was observed to decrease lipid content and alleviate ER stress in the livers of HFD-fed mice and PA-induced hepatocytes. These observations were associated with decreases in the expression levels of ER stress markers, including PDI, GRP78, p-PERK and p-eIF2α. Consistently, previous studies have reported the ability of EPO to protect rats against cardiac dysfunction and nephrotoxicity by attenuating intracellular ER stress (35,36). Furthermore, SIRT1 overexpression was proposed to attenuate lipid-induced ER stress and hepatic accumulation by decreasing mammalian target of rapamycin complex 1 activity (18,37). Our previous study reported that EPO was able to increase hepatic SIRT1 activity in vitro (15). The results of the present study suggested the presence of in vivo cross-talk between EPO and SIRT1 in liver tissue. As the alleviating effect of EPO on ER stress and fatty liver was abrogated in SIRT1-LKO mice, SIRT1 may regulate the effects of EPO in the liver. Additionally, SIRT1 loss-of-function approaches in hepatocytes via siRNA further indicated that SIRT1 is required for EPO to attenuate hepatic ER stress and lipid deposition. Collectively, these findings revealed a novel mechanism through which EPO alleviates ER stress in hepatic steatosis in a SIRT1-dependent manner.

Furthermore, the effects of EPO on alleviating metabolic dysregulation were eliminated in SIRT1-LKO mice. Previous studies reported that hepatic SIRT1 markedly induced FGF21 expression and secretion to control whole-body energy metabolism (21,38). The hepatokine FGF21 has been considered as a promising therapeutic agent that acts by promoting hepatic fatty acid oxidation, the browning of white adipose tissue and the thermogenesis of brown adipose tissue (39-41). The present study suggested that EPO could increase the expression of hepatic FGF21 in vivo and in vitro. However, SIRT1 deficiency (genetic or pharmacologically induced) inhibited the effects of EPO on the induction of FGF21, which was accompanied by diminished effect on increased expression of FGF21-targeted PGC-1α. These results suggested that SIRT1-induced FGF21 expression may contribute to the effects of EPO on alleviating hepatic steatosis and obesity. Of note, obese animals or human subjects, or those with fatty liver, had elevated FGF21 levels, indicating that the function of FGF21 is impaired under conditions of fat accumulation (30,42). The expression of FGFR1 and βKlotho, which serve as co-receptors of FGF21, was down-regulated by HFD feeding (43). Additionally, EPO treatment restored the expression of these receptors, which was accompanied by the upregulation of FGF21/PGC-1α-axis target genes, including Acox1 and Pdk4. In addition, eIF2α is a molecular target of FGF21 in the regulation of ER stress in the liver (44); the present study demonstrated that eIF2α phosphorylation in response to FGF21 in mouse hepatocytes was restored by EPO treatment, compared with PBS-treated obese mice. As regards the feedback mechanism underlying the effects of FGF21 on lipid metabolism related to ER stress (45), further investigation is required to determine the role of FGF21 in association with the effects of EPO on alleviating ER stress. Collectively, the results of the present study indicated that EPO treatment induced FGF21 expression and restored its sensitivity under conditions of obesity, contributing to improved systemic metabolism.

In summary, the results of the present study demonstrated that EPO ameliorated hepatic steatosis and obesity by inducing SIRT1-mediated inhibition of ER stress and promoting FGF21 expression (Fig. 7). These findings may improve our understanding of this mechanism and provide more experimental evidence on the therapeutic value of EPO in fatty liver and obesity.

Supplementary Data

Abbreviations:

EPO

erythropoietin

ER

endoplasmic reticulum

eIF2α

eukaryotic initiation factor 2α

FGF21

fibroblast growth factor 21

HFD

high-fat diet

IPGTT

intraperitoneal glucose tolerance test

NAFLD

non-alcoholic fatty liver disease

PA

palmitate

PDI

protein disulfide isomerase

GRP78

glucose-regulated protein 78

PERK

protein kinase-like ER kinase

PGC-1α

peroxisome proliferator-activated receptor-γ coactivator α

PPARγ

proliferator-activated receptor γ

AKT

protein kinase B

siRNA

small interfering RNA SIRT1, sirtuin 1

TG

triglyceride

UPR

unfolded protein response

NAD

nicotinamide adenine dinucleotide

DMEM

Dulbecco's modified Eagle's medium

FBS

fetal bovine serum

BCA

bicinchoninic acid

Pdk4

pyruvate dehydrogenase kinase 4, isoenzyme 4

Acox1

acyl-CoA oxidase 1

Ehhadh

enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

Acknowledgments

Not applicable.

Funding

The present study was supported by the National Natural Science Foundation of China Grant Awards (grant nos. 81600637, 81770819, 81570736, 81570737, 81370947, 81500612, 81400832, 81600632 and 81703294), the National Key Research and Development Program of China (grant no. 2016YFC1304804), the Natural Science Foundation of Jiangsu Province of China (grant no. BK20160116), the Jiangsu Provincial Key Medical Discipline (grant no. ZDXKB2016012), the Key Project of Nanjing Clinical Medical Science, the Key Research and Development Program of Jiangsu Province of China (grant nos. BE2015604 and BE2016606), the Jiangsu Provincial Medical Talent (grant no. ZDRCA2016062), and the Nanjing Science and Technology Development Project (grant no. 201605019).

Availability of data and materials

The datasets generated and/or analyzed during the present study are available from the corresponding author on reasonable request.

Authors' contributions

TH contributed to the study design, data acquisition and interpretation, drafting of the article, and revision of the manuscript. ZG contributed to the study design, data acquisition, data analysis, and the drafting and revision of the manuscript. BZ and RM contributed to the acquisition and analysis of the data and the drafting of the manuscript. YB contributed to the study design, data interpretation, and the drafting and revision of the manuscript. DZ contributed to the study design, data interpretation, and the revision of the manuscript. All authors have read and approved the final version of this manuscript for publication.

Ethics approval and consent to participate

All animal experiments were approved by the Nanjing University Medical School Institutional Animal Care and Use Committee, and were performed in accordance with the National Institutes of Health guidelines.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests associated with this publication.

References

1 

Younossi Z, Anstee QM, Marietti M, Hardy T, Henry L, Eslam M, George J and Bugianesi E: Global burden of NAFLD and NASH: Trends, predictions, risk factors and prevention. Nat Rev Gastroenterol Hepatol. 15:11–20. 2018. View Article : Google Scholar

2 

Deng X, Pan X, Cheng C, Liu B, Zhang H, Zhang Y and Xu K: Regulation of SREBP-2 intracellular trafficking improves impaired autophagic flux and alleviates endoplasmic reticulum stress in NAFLD. Biochim Biophys Acta Mol Cell Biol Lipids. 1862:337–350. 2017. View Article : Google Scholar

3 

Leamy AK, Egnatchik RA and Young JD: Molecular mechanisms and the role of saturated fatty acids in the progression of non-alcoholic fatty liver disease. Prog Lipid Res. 52:165–174. 2013. View Article : Google Scholar

4 

Fu S, Watkins SM and Hotamisligil GS: The role of endoplasmic reticulum in hepatic lipid homeostasis and stress signaling. Cell Metab. 15:623–634. 2012. View Article : Google Scholar : PubMed/NCBI

5 

Basseri S and Austin RC: ER stress and lipogenesis: A slippery slope toward hepatic steatosis. Dev Cell. 15:795–796. 2008. View Article : Google Scholar : PubMed/NCBI

6 

Ashraf NU and Sheikh TA: Endoplasmic reticulum stress and Oxidative stress in the pathogenesis of non-alcoholic fatty liver disease. Free Radic Res. 49:1405–1418. 2015. View Article : Google Scholar : PubMed/NCBI

7 

Jang MK, Nam JS, Kim JH, Yun YR, Han CW, Kim BJ, Jeong HS, Ha KT and Jung MH: Schisandra chinensis extract ameliorates nonalcoholic fatty liver via inhibition of endoplasmic reticulum stress. J Ethnopharmacol. 185:96–104. 2016. View Article : Google Scholar : PubMed/NCBI

8 

Yien YY, Shi J, Chen C, Cheung JTM, Grillo AS, Shrestha R, Li L, Zhang X, Kafina MD, Kingsley PD, et al: FAM210B is an erythropoietin target and regulates erythroid heme synthesis by controlling mitochondrial iron import and ferrochelatase activity. J Biol Chem. 293:19797–19811. 2018. View Article : Google Scholar : PubMed/NCBI

9 

Jelkmann W: Erythropoietin: Back to basics. Blood. 115:4151–4152. 2010. View Article : Google Scholar : PubMed/NCBI

10 

She J, Yuan Z, Wu Y, Chen J and Kroll J: Targeting erythropoietin protects against proteinuria in type 2 diabetic patients and in zebrafish. Mol Metab. 8:189–202. 2018. View Article : Google Scholar :

11 

Kodo K, Sugimoto S, Nakajima H, Mori J, Itoh I, Fukuhara S, Shigehara K, Nishikawa T, Kosaka K and Hosoi H: Erythropoietin (EPO) ameliorates obesity and glucose homeostasis by promoting thermogenesis and endocrine function of classical brown adipose tissue (BAT) in diet-induced obese mice. PLoS One. 12:e01736612017. View Article : Google Scholar : PubMed/NCBI

12 

Caillaud C, Connes P, Ben Saad H and Mercier J: Erythropoietin enhances whole body lipid oxidation during prolonged exercise in humans. J Physiol Biochem. 71:9–16. 2015. View Article : Google Scholar : PubMed/NCBI

13 

Hojman P, Brolin C, Gissel H, Brandt C, Zerahn B, Pedersen BK and Gehl J: Erythropoietin over-expression protects against diet-induced obesity in mice through increased fat oxidation in muscles. PLoS One. 4:e58942009. View Article : Google Scholar : PubMed/NCBI

14 

Ge Z, Zhang P, Hong T, Tang S, Meng R, Bi Y and Zhu D: Erythropoietin alleviates hepatic insulin resistance via PPARγ-dependent AKT activation. Sci Rep. 5:178782015. View Article : Google Scholar

15 

Hong T, Ge Z, Meng R, Wang H, Zhang P, Tang S, Lu J, Gu T, Zhu D and Bi Y: Erythropoietin alleviates hepatic steatosis by activating SIRT1-mediated autophagy. Biochim Biophys Acta Mol Cell Biol Lipids. 1863:595–603. 2018. View Article : Google Scholar : PubMed/NCBI

16 

Sathyanarayan A, Mashek MT and Mashek DG: ATGL promotes autophagy/lipophagy via SIRT1 to control hepatic lipid droplet catabolism. Cell Rep. 19:1–9. 2017. View Article : Google Scholar : PubMed/NCBI

17 

Kong Q, Zhang H, Zhao T, Zhang W, Yan M, Dong X and Li P: Tangshen formula attenuates hepatic steatosis by inhibiting hepatic lipogenesis and augmenting fatty acid oxidation in db/db mice. Int J Mol Med. 38:1715–1726. 2016. View Article : Google Scholar : PubMed/NCBI

18 

Li Y, Xu S, Giles A, Nakamura K, Lee JW, Hou X, Donmez G, Li J, Luo Z, Walsh K, et al: Hepatic overexpression of SIRT1 in mice attenuates endoplasmic reticulum stress and insulin resistance in the liver. FASEB J. 25:1664–1679. 2011. View Article : Google Scholar : PubMed/NCBI

19 

Pan Q, Ren Y, Liu W, Hu Y, Zheng J, Xu Y and Wang G: Resveratrol prevents hepatic steatosis and endoplasmic reticulum stress and regulates the expression of genes involved in lipid metabolism, insulin resistance, and inflammation in rats. Nutr Res. 35:576–584. 2015. View Article : Google Scholar : PubMed/NCBI

20 

Kang X, Yang W, Wang R, Xie T, Li H, Feng D, Jin X, Sun H and Wu S: Sirtuin-1 (SIRT1) stimulates growth-plate chondrogenesis by attenuating the PERK-eIF-2α-CHOP pathway in the unfolded protein response. J Biol Chem. 293:8614–8625. 2018. View Article : Google Scholar : PubMed/NCBI

21 

Li Y, Wong K, Giles A, Jiang J, Lee JW, Adams AC, Kharitonenkov A, Yang Q, Gao B, Guarente L and Zang M: Hepatic SIRT1 attenuates hepatic steatosis and controls energy balance in mice by inducing fibroblast growth factor 21. Gastroenterology. 146:539–549.e7. 2014. View Article : Google Scholar :

22 

Han HS, Choi BH, Kim JS, Kang G and Koo SH: Hepatic Crtc2 controls whole body energy metabolism via a miR-34a-Fgf21 axis. Nat Commun. 8:18782017. View Article : Google Scholar : PubMed/NCBI

23 

Zheng Q, Tong M, Ou B, Liu C, Hu C and Yang Y: Isorhamnetin protects against bleomycin-induced pulmonary fibrosis by inhibiting endoplasmic reticulum stress and epithelial-mesenchymal transition. Int J Mol Med. 43:117–126. 2019.

24 

Varghese F, Bukhari AB, Malhotra R and De A: IHC Profiler: An open source plugin for the quantitative evaluation and automated scoring of immunohistochemistry images of human tissue samples. PLoS One. 9:e968012014. View Article : Google Scholar : PubMed/NCBI

25 

Shah KN, Bhatt R, Rotow J, Rohrberg J, Olivas V, Wang VE, Hemmati G, Martins MM, Maynard A, Kuhn J, et al: Aurora kinase A drives the evolution of resistance to third-generation EGFR inhibitors in lung cancer. Nat Med. 25:111–118. 2019. View Article : Google Scholar

26 

Seglen PO: Hepatocyte suspensions and cultures as tools in experimental carcinogenesis. J Toxicol Environ Health. 5:551–560. 1979. View Article : Google Scholar : PubMed/NCBI

27 

Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar

28 

Zhang W, Sun Y, Liu W, Dong J and Chen J: SIRT1 mediates the role of RNA-binding protein QKI 5 in the synthesis of triglycerides in non-alcoholic fatty liver disease mice via the PPARα/FoxO1 signaling pathway. Int J Mol Med. 43:1271–1280. 2019.PubMed/NCBI

29 

Babaknejad N, Nayeri H, Hemmati R, Bahrami S and Esmaillzadeh A: An overview of FGF19 and FGF21: The therapeutic role in the treatment of the metabolic disorders and obesity. Horm Metab Res. 50:441–452. 2018. View Article : Google Scholar : PubMed/NCBI

30 

Fisher FM, Chui PC, Antonellis PJ, Bina HA, Kharitonenkov A, Flier JS and Maratos-Flier E: Obesity is a fibroblast growth factor 21 (FGF21)-resistant state. Diabetes. 59:2781–2789. 2010. View Article : Google Scholar : PubMed/NCBI

31 

Gasparin FRS, Carreño FO, Mewes JM, Gilglioni EH, Pagadigorria CLS, Natali MRM, Utsunomiya KS, Constantin RP, Ouchida AT, Curti C, et al: Sex differences in the development of hepatic steatosis in cafeteria diet-induced obesity in young mice. Biochim Biophys Acta Mol Basis Dis. 1864:2495–2509. 2018. View Article : Google Scholar : PubMed/NCBI

32 

Henkel AS: Unfolded protein response sensors in hepatic lipid metabolism and nonalcoholic fatty liver disease. Semin Liver Dis. 38:320–332. 2018. View Article : Google Scholar : PubMed/NCBI

33 

Rutkowski DT, Wu J, Back SH, Callaghan MU, Ferris SP, Iqbal J, Clark R, Miao H, Hassler JR, Fornek J, et al: UPR pathways combine to prevent hepatic steatosis caused by ER stress-mediated suppression of transcriptional master regulators. Dev Cell. 15:829–840. 2008. View Article : Google Scholar : PubMed/NCBI

34 

Oyadomari S, Harding HP, Zhang Y, Oyadomari M and Ron D: Dephosphorylation of translation initiation factor 2alpha enhances glucose tolerance and attenuates hepatosteatosis in mice. Cell Metab. 7:520–532. 2008. View Article : Google Scholar : PubMed/NCBI

35 

Kong D, Zhuo L, Gao C, Shi S, Wang N, Huang Z, Li W and Hao L: Erythropoietin protects against cisplatin-induced nephrotoxicity by attenuating endoplasmic reticulum stress-induced apoptosis. J Nephrol. 26:219–227. 2013. View Article : Google Scholar

36 

Lu J, Dai Q, Ma G, Zhu Y, Chen B, Li B and Yao Y: Erythropoietin attenuates cardiac dysfunction in rats by inhibiting endoplasmic reticulum stress-induced diabetic cardiomyopathy. Cardiovasc Drugs Ther. 31:367–379. 2017. View Article : Google Scholar : PubMed/NCBI

37 

Jung TW, Lee KT, Lee MW and Ka KH: SIRT1 attenuates palmitate-induced endoplasmic reticulum stress and insulin resistance in HepG2 cells via induction of oxygen-regulated protein 150. Biochem Biophys Res Commun. 422:229–232. 2012. View Article : Google Scholar : PubMed/NCBI

38 

Matsui S, Sasaki T, Kohno D, Yaku K, Inutsuka A, Yokota- Hashimoto H, Kikuchi O, Suga T, Kobayashi M, Yamanaka A, et al: Neuronal SIRT1 regulates macronutrient-based diet selection through FGF21 and oxytocin signalling in mice. Nat Commun. 9:46042018. View Article : Google Scholar : PubMed/NCBI

39 

Jimenez V, Jambrina C, Casana E, Sacristan V, Muñoz S, Darriba S, Rodó J, Mallol C, Garcia M, Leó X, et al: FGF21 gene therapy as treatment for obesity and insulin resistance. EMBO Mol Med. 10:pii: e8791. 2018. View Article : Google Scholar : PubMed/NCBI

40 

Liu M, Cao H, Hou Y, Sun G, Li D and Wang W: Liver plays a major role in FGF-21 mediated glucose homeostasis. Cell Physiol Biochem. 45:1423–1433. 2018. View Article : Google Scholar : PubMed/NCBI

41 

Fisher FM, Kleiner S, Douris N, Fox EC, Mepani RJ, Verdeguer F, Wu J, Kharitonenkov A, Flier JS, Maratos-Flier E and Spiegelman BM: FGF21 regulates PGC-1α and browning of white adipose tissues in adaptive thermogenesis. Genes Dev. 26:271–281. 2012. View Article : Google Scholar : PubMed/NCBI

42 

Dushay J, Chui PC, Gopalakrishnan GS, Varela-Rey M, Crawley M, Fisher FM, Badman MK, Martinez-Chantar ML and Maratos-Flier E: Increased fibroblast growth factor 21 in obesity and nonalcoholic fatty liver disease. Gastroenterology. 139:456–463. 2010. View Article : Google Scholar : PubMed/NCBI

43 

Rusli F, Deelen J, Andriyani E, Boekschoten MV, Lute C, van den Akker EB, Müller M, Beekman M and Steegenga WT: Fibroblast growth factor 21 reflects liver fat accumulation and dysregulation of signalling pathways in the liver of C57BL/6J mice. Sci Rep. 6:304842016. View Article : Google Scholar : PubMed/NCBI

44 

Jiang S, Yan C, Fang QC, Shao ML, Zhang YL, Liu Y, Deng YP, Shan B, Liu JQ, Li HT, et al: Fibroblast growth factor 21 is regulated by the IRE1α-XBP1 branch of the unfolded protein response and counteracts endoplasmic reticulum stress-induced hepatic steatosis. J Biol Chem. 289:29751–29765. 2014. View Article : Google Scholar : PubMed/NCBI

45 

Ye M, Lu W, Wang X, Wang C, Abbruzzese JL, Liang G, Li X and Luo Y: FGF21-FGFR1 coordinates phospholipid homeostasis, lipid droplet function, and ER stress in obesity. Endocrinology. 157:4754–4769. 2016. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

August 2019
Volume 44 Issue 2

Print ISSN: 1107-3756
Online ISSN:1791-244X

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
APA
Hong, T., Ge, Z., Zhang, B., Meng, R., Zhu, D., & Bi, Y. (2019). Erythropoietin suppresses hepatic steatosis and obesity by inhibiting endoplasmic reticulum stress and upregulating fibroblast growth factor 21. International Journal of Molecular Medicine, 44, 469-478. https://doi.org/10.3892/ijmm.2019.4210
MLA
Hong, T., Ge, Z., Zhang, B., Meng, R., Zhu, D., Bi, Y."Erythropoietin suppresses hepatic steatosis and obesity by inhibiting endoplasmic reticulum stress and upregulating fibroblast growth factor 21". International Journal of Molecular Medicine 44.2 (2019): 469-478.
Chicago
Hong, T., Ge, Z., Zhang, B., Meng, R., Zhu, D., Bi, Y."Erythropoietin suppresses hepatic steatosis and obesity by inhibiting endoplasmic reticulum stress and upregulating fibroblast growth factor 21". International Journal of Molecular Medicine 44, no. 2 (2019): 469-478. https://doi.org/10.3892/ijmm.2019.4210