Chronic intermittent hypobaric hypoxia ameliorates endoplasmic reticulum stress mediated liver damage induced by fructose in rats

Life Sciences 121 (2015) 40–45

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Chronic intermittent hypobaric hypoxia ameliorates endoplasmic reticulum stress mediated liver damage induced by fructose in rats Fang Yuan a,c,1, Xu Teng a,b,1, Zan Guo a, Jing-Jing Zhou a,c, Yi Zhang a,c,⁎, Sheng Wang a,c,⁎⁎ a b c

Department of Physiology, Hebei Medical University, Shijiazhuang, China Hebei Key Laboratory of Laboratory Animal, Hebei Medical University, Shijiazhuang, China Hebei Collaborative Innovation Center for Cardio–cerebrovascular Disease, Shijiazhuang, China

a r t i c l e

i n f o

Article history: Received 26 May 2014 Accepted 12 November 2014 Available online 1 December 2014 Chemical compounds studied in this article: Fructose (PubChem CID: 5984) Keywords: Chronic intermittent hypobaric hypoxia Metabolic syndrome Hepar Apoptosis Endoplasmic reticulum stress

a b s t r a c t Aim: High-fructose intake induces nonalcoholic fatty liver disease (NAFLD) and chronic intermittent hypobaric hypoxia (CIHH) has beneficial effects on the body. We hypothesized that CIHH has protective effects on the impaired hepar in fructose-fed rats. Main methods: Sprague–Dawley rats (male, 160–180 g) were randomly divided into 4 groups: control group (CON), fructose group (FRUC, 10% fructose in drinking water for 6 weeks), CIHH group (simulated 5000 m altitude, 6 h per day for 6 weeks), and CIHH plus fructose groups (CIHH-F). Histopathology of liver, arterial blood pressure, blood biochemicals, hepatocyte apoptosis, and marker proteins of endoplasmic reticulum stress (ERS) were measured. Key findings: The arterial blood pressure, body mass index, abdominal fat weight and liver weight were increased in FRUC rats but not in CIHH-F rats. Likewise, the serum glucose, insulin, insulin C peptide, triglyceride (TG) and total cholesterol (TC) were elevated in FRUC rats but not in CIHH-F rats after fasting 12 h. Meanwhile, the hepatic steatosis and hepatocyte apoptosis occurred in FRUC rats but not in CIHH-F rats. Finally the expression of ERS markers including GRP78 (glucose-regulated protein78), CHOP (C/EBP Homologous Protein), and caspase-12 in hepatic tissue were up-regulated in FRUC rats, but such up-regulation was not observed in CIHH-F rats. Significance: Our results suggest that CIHH protect hepar against hepatic damage through inhibition of ERS in fructose-fed rats. CIHH might be the new therapy for NAFLD. © 2014 Elsevier Inc. All rights reserved.

Introduction Nonalcoholic fatty liver disease (NAFLD), the main expression of the metabolic syndrome (MS) in liver, is a common liver disease and the prevalence is likely to increase in the coming decades. NAFLD includes simple steatosis and nonalcoholic steatohepatitis (NASH). Simple steatosis is a benign pathological change without chronic alcohol consumption. NASH displays necro-inflammation and fibrosis, eventually leading to cirrhosis, hepatocellular carcinoma and end-stage liver disease. NASH is the most general pathogeny of cryptogenic cirrhosis [3]. The collecting evidence suggests that diabetes, obesity, and insulin resistance are tightly associated with ectopic fat accumulation especially in the liver. Hepatic steatosis might be the cause of insulin resistance [28]. However, the mechanism underlying NAFLD remains unknown, and an effective therapy for NAFLD is needed. ⁎ Correspondence to: Y. Zhang, Department of Physiology, Hebei Medical University, Shijiazhuang 050017, China. Tel.: +86 311 8626 5663; fax: +86 311 8626 6811. ⁎⁎ Correspondence to: S. Wang, Department of Physiology, Hebei Medical University, Shijiazhuang 050017, China. Tel.: +86 311 8626 1183; fax: +86 311 8626 6811. E-mail addresses: [email protected] (Y. Zhang), [email protected] (S. Wang). 1 The first two authors contributed equally.

http://dx.doi.org/10.1016/j.lfs.2014.11.019 0024-3205/© 2014 Elsevier Inc. All rights reserved.

Recently, it has been shown that the endoplasmic reticulum (ER) is implicated in both the development of simple steatosis and the progression to NASH [27]. Disruption of ER homeostasis, often termed ER stress, has been demonstrated in liver and adipose tissues of patients with NAFLD [18]. The high fat diet-fed C57BL/6 mice featured enhanced lipogenesis and ER stress [17]. In human normal hepatocytes, L02 and HepG2 cell lines, ER stress induced by saturated fatty acid promoted apoptosis through the PERK (PKR-like Endoplasmic Reticulum Kinase)/ ATF4 (Activating Transcription Factor 4)/CHOP (C/EBP Homologous Protein) signaling pathway [5,14]. Injection of ER stress inducer tunicamycin (TM) could result in NASH in mice demonstrating hepatic steatosis and inflammation. In mice treated with TM, hepatic triglycerides (TG) were increased but plasma lipids, such as TG, total cholesterol (TC), high-density lipoprotein (HDL), and low-density lipoprotein (LDL), were decreased [16]. ER stress was also induced in liver of fructose-fed mice. 4-Phenylbutyric acid (PBA), an ERS inhibitor, was proved to attenuate both ER stress and hepatic lipid accumulation [34]. Chronic intermittent hypobaric hypoxia (CIHH) is also termed as exposure to hypoxia interrupted by normoxia. A lot of researches showed that CIHH had beneficial effects including cardioprotection [10,32], regulation of arterial blood pressure [25,33], prevention of arthritis [23], and facilitation of carotid sinus baroreflex [7]. Recently,

F. Yuan et al. / Life Sciences 121 (2015) 40–45

our study demonstrated that CIHH can prevent cardiac dysfunction and normalize abnormality of plasma glucose, TC, TG, insulin, and insulin C peptide after fasting 12 h [35]. We also found that CIHH suppressed ERS induced by ischemia/reperfusion in myocardium [data unpublished]. So we hypothesized that CIHH might ameliorate hepatic damage of NAFLD through inhibiting ERS in fructose-fed rats.

Materials and methods Chemicals Fructose was purchased from Sigma (St. Louis, MO). The kit for terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and hematoxylin–eosin (HE) was purchased from Roche Applied Science (Indianapolis, IN). Antibodies against GRP78 (glucose-regulated protein78), caspase-12 and CHOP were purchased from Abcam (Cambridge, UK), and antibody against GAPDH and all secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Nitrocellulose (NC) membrane was obtained from Hybond-C (Amersham Life Science, UK) and an enhanced chemiluminescence (ECL) kit was obtained from Beijing Applygen Technologies (Beijing, CN). Other chemicals and reagents were all of analytical grade.

Fructose feeding and CIHH treatment All animal procedures were complied with according to the Animal Management Rule of the Ministry of Health, People's Republic of China (Documentation No. 55, 2001) and EU Directive 2010/63/EU for animal experiments. Sprague–Dawley rats (male, 160–180 g, Hebei Medical University Animal Center) were randomly divided into 4 groups: control (CON), fructose-fed (FRUC), CIHH, and CIHH plus fructose-fed (CIHH-F).The model of MS induced by fructose in rats was the same as our previous studies [35]. Briefly, the rats in the FRUC and CIHH-F groups were fed with 10% fructose in water for 6 weeks. The methods of CIHH were described in our previous studies [10]. Simplified, the rats in the CIHH and CIHH-F groups were in a hypobaric chamber, and were exposed to hypobaric hypoxia (simulated 5000 m altitude for 6 weeks, 6 h per day, PB = 404 mm Hg, PO2 = 84 mm Hg). All rats drank water freely, fed a standard laboratory diet, and were housed in a temperature-controlled room (22 ± 1 °C) with a 12 h/12 h light/ dark cycle (lights on at 06:00 am). The volume of water intake was counted each day and body weight was measured once a week.

Measurement of blood pressure The measurement of systolic arterial blood pressure (SAP) of tail artery was as described previously [6]. Briefly, SAP was determined in conscious rats by a tail-cuff pressure meter (LE5001, Pressure Meter, Powerlab, ADInstruments Company, AUS). SAP was determined 3 times in each rat and the mean was counted.

Measurement of biochemicals in blood and tissue 6 weeks later, 2.5 ml blood for each rat was collected from the angular vein after a 12 h fasting. The blood sample was centrifuged at 3000 rpm for 15 min and plasma was collected. Plasma glucose was determined by glucose oxidation method (Beijing Kemeidongya Company, CN), plasma TC was determined by cholesterase method (Shangdong 3V Biology Company, CN), plasma TG was determined by hydrolase method (Shangdong 3V biology company, CN), and the concentration of plasma insulin and insulin C peptide was determined by enzyme linked immunosorbent assay (ELISA) (Beijing North Biology Technique Institute, CN).

41

HE and TUNEL staining A small segment of liver was fixed in 4% paraformaldehyde for 12 h, embedded in paraffin, cut into 6-μm-thick sections, stained with HE, and then observed under an optical microscope (BX 50, Olympus Optical, Japan). Apoptosis of hepatocytes was determined by TUNEL staining according to the manufacturer's instructions. Simplified, after deparaffinization and fixation, the sections were immersed in 20 mg/ml proteinase K for 20 min. After refixation and equilibration, sections were incubated at 37 °C for 60 min with biotinylated nucleotide and the terminal deoxynucleotidyl transferase recombinant enzyme, blocked in 0.3% H2O2, then incubated with streptavidin horseradish peroxidase (HRP) solution, and then detected with 0.05% diaminobenzidine in PBS containing 0.03% H2O2. Quantitative analysis was operated by determining the ratio of TUNEL-positive/total hepatocytes in 10 random high-power fields for each section. Caspase-3 activity Liver samples were collected and rinsed with PBS, homogenized and lysed in lysis buffer for 15 min on ice, then centrifuged at 12,000 ×g for 10 min at 4 °C. The caspase-3 activity was detected by a commercial kit (Applygen Technologies Inc., Beijing, CN). The operation was made in accordance with the manufacturer's instruction. The quantification of caspase-3 activity was normalized by protein content. Western blot analysis 100 mg of hepatic samples were frozen by liquid nitrogen, homogenized in 1 ml lysis buffer, and then centrifuged at 10,000 rpm for 10 min at 4 °C. Protein extracts were resuspended in a 6× sample buffer, and boiled in water for 15 min at 100 °C. 100 μg of samples were electrophoresed on 10% SDS-PAGE and transferred to NC membranes. The blots were then incubated with primary antibodies anti-GRP78 (1:1000), anti-caspase-12 (1:300), and anti-CHOP (1:1000) respectively at 4 °C overnight, and then with secondary antibody for 1 h at room temperature. The reaction was visualized by the ECL method, and the bands were analyzed by NIH image software three times. The protein contents were normalized to that of GADPH. Statistical analysis Data are expressed as mean ± SD. One-way analysis of variance was used to compare more than two groups, and Tukey's honestly significant difference test was used to test for differences between-individual groups. A P b 0.05 was considered statistically significant. Results Body mass index (BMI), intra-abdominal fat, and liver weights Compared with CON rats, the BMI, intra-abdominal fat weight, and liver weight of FRUC rats were all significantly increased (all P b 0.05, Table 1), and CIHH could inhibit completely the increase induced by fructose (all P b 0.05, Table 1). However, there were no differences in BMI, intra-abdominal fat, and liver weights among CON, CIHH, and CIHH-F rats (all P N 0.05, Table 1). SAP and blood biochemicals Compared with CON rats, SAP in FRUC rats was significantly increased, and CIHH completely inhibited the increase of SAP induced by fructose (P b 0.05, Table 2). However, there were no differences in SAP among CON, CIHH, and CIHH-F rats (P N 0.05, Table 2). Similarly, the levels of plasma glucose, TC, TG, insulin and insulin C peptide after the 12 h fasting were increased in FRUC rats compared with CON rats,

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F. Yuan et al. / Life Sciences 121 (2015) 40–45

Table 1 Effect of daily administration of CIHH on body mass index and relative intra-abdominal fat and liver weights in fructose-fed rats. Groups

Body mass index (g/cm2)

Intra-abdominal fat weight (g/100 g BW)

Liver weight (g/100 g BW)

CON FRUC CIHH CIHH-F

0.62 ± 0.03 0.68 ± 0.03* 0.63 ± 0.02# 0.63 ± 0.03#

0.7 ± 0.1 1.2 ± 0.1* 0.7 ± 0.1# 0.8 ± 0.1#

3.2 ± 0.1 4.4 ± 0.2* 3.0 ± 0.1# 3.4 ± 0.2#

CON: control group; CIHH: chronic intermittent hypobaric hypoxia treating group; FRUC: fructose-fed group; CIHH-F: both CIHH treating and fructose-fed group. n = 6 for each group. Data were expressed as mean ± SD. *, P b 0.05 vs. CON; #, P b 0.05 vs. FRUC.

and CIHH could totally ameliorate the increase of these biomarkers induced by fructose (P b 0.05, Table 2). However, the level of these biomarkers was not different between CIHH and CON rats.

Histopathology The HE stained liver sections were examined by a light microscope. CON and CIHH rats revealed normal hepatic structure and hepatic lobules. Each lobule was made up of radiating plates, strands of cells forming a net-work around a central vein. In addition, some hepatocytes around the hepatic lobules have a little edema in CIHH group. By contrast, FRUC rats showed distended cells, loose cytoplasm, and loss of normal architecture such as disarrangement of hepatocytes in the liver. And there were fat vacuoles in some hepatocytes of FRUC rats. The fat vacuoles and distended cells were diminished in CIHH-F rats (Fig. 1).

Hepatocyte apoptosis TUNEL result and caspase-3 activity showed that the apoptosis in FRUC and CIHH-F rats were significantly increased compared with CON and CIHH rats. The apoptosis in CIHH-F rats was decreased compared with FRUC rats. (P b 0.05, Fig. 2). As similar as the TUNEL staining and caspase-3 activity, the protein expression of active caspase-12 and CHOP, two key factors that mediated ERS-induced apoptosis, were both up-regulated in FRUC rats. CIHH could decrease the up-regulation of active caspase-12 and CHOP induced by fructose. (P b 0.05, Fig. 3C and D).

Hepatocyte endoplasmic reticulum stress The protein levels of GRP78, active caspase-12 and CHOP were significantly increased in FRUC rats compared with CON rats, and the increase of protein level induced by fructose could completely be inhibited by CIHH (P b 0.05, Fig. 3). However, there were no differences in GRP78, active caspase-12 and CHOP among CON, CIHH, and CIHH-F rats (P N 0.05, Fig. 3).

Discussion The beneficial effect of CIHH on the impaired hepar was investigated in fructose-fed rats. Compared with the control rats, the arterial blood pressure, body mass index, abdominal fat weight and liver weight were increased in FRUC rats, but the increase was slight in CIHH-F rats. Plasma glucose, insulin and insulin C peptide, TG and TC after 12-h fasting were elevated in FRUC rats, but the elevation was blunt in CIHH-F rats. Furthermore, hepatic steatosis and hepatocyte apoptosis appeared in FRUC rats, but were prevented in CIHH-F rats. The expression of ERS markers including GRP78, CHOP and caspase-12 in hepatic tissue were up-regulated in FRUC rats, but the up-regulation was inhibited in CIHH-F rats. Collectively, these data suggest that CIHH ameliorated hepatic damage through inhibition of ERS in fructose-fed rats. Fructose plays an important role in the development of NAFLD [1,24]. A high fructose diet is associated with deleterious change of plasma lipid profiles and metabolic changes in mice with obesity syndrome induced by the American lifestyle, which included drinking high-fructose corn syrup in high amounts [1]. High fat diet alone generates obesity, insulin resistance (IR), and some degree of fatty liver with minimal inflammation and no fibrosis. However, the fast food diet included both fructose and fats which could significantly up-regulate gene expression, which was associated with hepatic fibrosis, inflammation, ERS and lipoapoptosis [1]. Fructose consumption, even in the absence of obesity, causes serious and adverse alterations in the hepar with the presence of dyslipidemia, IR, and NAFLD with areas of necroinflammation, which are associated with an adverse prognosis [22]. Dietary fructose, rather than dietary fat, increases the morbidity of precancerous hepatocytes induced by treatment of DEN via IR and oxidative stress in rats [13]. Fructose administration, mainly in drinking water, to laboratory rats and mice reproduces almost all of the features of NAFLD and MS, such as overweight, hypertension, hyperglycemia, hypercholesterolemia, and IR [2,8,20]. Here we also discover that fructose-fed rats displayed raised body mass index, abdominal fat increase, liver weight increase, hypertension, hyperinsulinemia, hyperglyceridemia, insulin resistance, hepatic steatosis and hepatocyte apoptosis increase. Several possible hypotheses related to the development of NAFLD by fructose consumption have been pursued, including increased oxidative and inflammatory stress through nitric oxide synthase induction [26] and tumor necrosis factor α production [12]. Recently, more and more evidence have implicated the ERS in the pathogenesis of NAFLD [36]. In the hepatic samples in the fructose-fed rats, the spliced form of XBP-1 mRNA (a key indicator of activation of ERS) was significantly increased [19]. In aging mice, ERS could exacerbate hepatic steatosis [30]. Compared with non-alcoholic steatosis patients, the protein and mRNA levels of ERS markers in the hepatic biopsy were significantly increased in non-alcoholic steatohepatitis patients [9]. In another study, the findings also reveal the presence of a coordinated, adaptive transcriptional response to hepatic ER stress in human NAFLD [15]. ERS-mediated signaling pathways are associated with amounts of downstream genes that promote the pathology of NAFLD, including fibrosis, inflammation, and apoptosis [18]. With tunicamycin (TM, an ER stress inducer)

Table 2 Effect of CIHH on SAP and blood biochemicals in fructose-fed rats. Groups

SAP (mm Hg)

Ins (nIU)

C-p (ng)

GLU (mM)

TC (mM)

TG (mM)

CON FRUC CIHH CIHH-F

118.2 ± 7.6 151.7 ± 8.3⁎ 113.3 ± 5.9# 115.4 ± 9.1#

10.1 ± 2.9 27.9 ± 8.3⁎ 9.9 ± 1.6# 9.6 ± 1.8#

1.1 ± 0.1 1.6 ± 0.3⁎ 0.9 ± 0.2# 1.0 ± 0.3#

4.1 ± 0.3 6.2 ± 0.5⁎ 4.2 ± 0.6# 4.3 ± 0.4#

1.1 ± 0.2 1.6 ± 0.3⁎ 1.0 ± 0.4# 1.2 ± 0.4#

0.9 ± 0.2 1.5 ± 0.3⁎ 1.0 ± 0.2# 1.0 ± 0.3#

SAP: systolic artery pressure; Ins: insulin; C-P: insulin C peptide; GLU: glucose; TC: total cholesterol; TG: triglycerides; CON: control group; CIHH: chronic intermittent hypobaric hypoxia treating group; FRUC: fructose-fed group; CIHH-F: both chronic intermittent hypobaric hypoxia treating and fructose-fed groups. n = 6 for each group. Data were expressed as mean ± SD. *, P b 0.05 vs. CON. #, P b 0.05 vs. FRUC.

F. Yuan et al. / Life Sciences 121 (2015) 40–45

43

50μm

50μm

50μm

50μm

Fig. 1. Histological analysis of liver of control, CIHH, fructose-fed and CIHH-F groups (magnification, 200×). The arrows show the fat vacuoles in hepatocytes.

treatment, a quick NASH state characterized by hepatic steatosis and inflammation was induced in mice. An increase in hepatic TG and a decrease in plasma lipids, including TG, TC, high-density lipoprotein (HDL), and low-density lipoprotein (LDL), were discovered in the TM-

treated mice [16]. In contrast, phenylbutyric acid (PBA), an ER chemical chaperone, not only significantly ameliorated ERS, but also protected against hepatic lipid accumulation [34]. We also determined presently that the hepatic protein level of GRP78, active caspase-12 and CHOP

b

Ratio of TUNEL positvie/total hepatocytes

a

*#

20 μm

Capase-3 activity (folds difference with control)

20 μm

**

c

20 μm

20 μm

* #

Fig. 2. Effect of CIHH on apoptosis in fructose-fed rats. a. Histological analysis of liver of control, CIHH, fructose-fed and CIHH-F groups (magnification, 400×). The arrows show the TUNEL-positive hepatocytes. b. Quantitative analysis of apoptosis in the four groups (n = 6). c. Activity of caspase-3 in four groups (n = 6 for each group). *P b 0.05, **P b 0.01 vs. Control; # P b 0.05, vs. fructose.

F. Yuan et al. / Life Sciences 121 (2015) 40–45

a

b

Control Fructose CIHH CIHH-F

3.00 Control

GRP78

78 kDa

CHOP

29 kDa

Cas pas e 12

35 kDa

GAPDH

37 kDa

Fructose

GRP78/GAPDH

44

CIHH 2.00

CIHH+F

* #

1.00

0.00

c

d 3.00

3.00

Control

* 2.00

Caspase12/GAPDH

CHOP/GAPDH

Fructose CIHH CIHH+F

# 1.00

Control Fructose CIHH

2.00

1.00

CIHH+F

* #

0.00

0.00

Fig. 3. Effect of CIHH on endoplasmic reticulum stress in fructose-fed rats. a, representative protein expression of GRP78, active caspase-12, CHOP and GAPDH as a control; b–d, quantitative analysis of protein expression of GRP78, active caspase-12, and CHOP (n = 6 for each group). *P b 0.05, vs. Control. #P b 0.05, vs. fructose.

significantly increased in fructose-fed rats. It suggested that fructose might prompt ERS in liver. However, the signaling pathway of ERS induced by fructose was unclear, and needs further investigation. CIHH has been found to have many beneficial effects on the body, such as promotion of health, increase of oxygen utilization, and prevention or treatment of some diseases [31,37]. Our previous study has investigated that CIHH could inhibit the elevation of blood pressure and plasma levels of glucose, insulin and insulin C peptide, TC, and TG induced by fructose feeding in rats after 12 h fasting [35]. In this article, we also confirmed that CIHH treatment normalized the body mass index, abdominal fat increase, liver weight increase, high blood pressure, and metabolic dysfunction induced by fructose feeding. Furthermore, CIHH treatment could decrease hepatic steatosis and impaired liver cells, induced by fructose feeding. These results confirm that CIHH ameliorates hepatic impairment induced by fructose. Interestingly, CIHH could ameliorate ERS in liver induced by fructose, accompanied with an improved pathology of NAFLD. In our other study, we also found that CIHH suppressed ERS induced by ischemia/ reperfusion in myocardium [data not yet published]. These results confirmed that CIHH could actually ameliorate ERS. Considering the key role of ERS in the pathophysiology of NAFLD, these results suggested that CIHH blocked NAFLD by inhibiting ERS. The improvement of ERS-induced apoptosis might be an important signaling pathway through which CIHH plays a beneficial effect on hepatic impairment. CHOP and caspase-12 were the two major signaling pathway mediated ERS-induced apoptosis, which involves in the pathogenesis of NAFLD [4,21]. Especially, while CHOP was depleted, mice were protected from alcohol-induced liver injury [11]. Whole body CHOP-deficiency protects hepatocytes from cholestasis-induced liver damages [29]. Our results also demonstrated that the protein levels of CHOP and active caspase-12 increased in hepatic tissue of rats treated with fructose, and hepatocyte apoptosis accordingly impaired. CIHH

could ameliorate the exacerbation of both protein expression and apoptosis. Taken together, these results suggested that CIHH could block the CHOP and caspase-12 mediated-hepatocyte apoptosis, and result in ameliorating NAFLD. Conclusion In conclusion, our study demonstrates for the first time that CIHH confers effective hepatic protection against hepatic steatosis and apoptosis in fructose-fed rats, which might be related to inhibition of ERS. Conflict of interest The authors declare that there are no conflicts of interest.

Acknowledgments This study was supported by the National Science Foundation of China (No. 81100229), and the Natural Science Foundation of Hebei Province (No. C2012206063). References [1] M. Basaranoglu, G. Basaranoglu, T. Sabuncu, H. Sentürk, Fructose as a key player in the development of fatty liver disease, World J. Gastroenterol. 28 (19) (2013) 1166–1172. [2] J. Bhattacharjee, J.M. Kumar, S. Arindkar, B. Das, U. Pramod, R.C. Juyal, S.S. Majumdar, P. Nagarajan, Role of immunodeficient animal models in the development of fructose induced NAFLD, J. Nutr. Biochem. 25 (2014) 219–226. [3] H. Boppidi, S.R. Daram, Nonalcoholic fatty liver disease: hepatic manifestation of obesity and the metabolic syndrome, Postgrad. Med. 120 (2008) E01–E07. [4] J.W. Brewer, Regulatory crosstalk within the mammalian unfolded protein response, Cell. Mol. Life Sci. 71 (2014) 1067–1079.

F. Yuan et al. / Life Sciences 121 (2015) 40–45 [5] J. Cao, D.L. Dai, L. Yao, H.H. Yu, B. Ning, Q. Zhang, J. Chen, W.H. Cheng, W. Shen, Z.X. Yang, Saturated fatty acid induction of endoplasmic reticulum stress and apoptosis in human liver cells via the PERK/ATF4/CHOP signaling pathway, Mol. Cell. Biochem. 364 (2012) 115–129. [6] F. Cui, L. Gao, F. Yuan, Z.F. Dong, Z.N. Zhou, D.D. Kline, Y. Zhang, D.P. Li, Hypobaric intermittent hypoxia attenuates hypoxia-induced depressor response, PLoS One 7 (2012) e41656. [7] L. Gao, Y. Guan, F. Cui, Y.X. Liu, Z.N. Zhou, Y. Zhang, Facilitation of chronic intermittent hypobaric hypoxia on carotid sinus baroreflex in anesthetized rats, Chin. J. Physiol. 55 (2012) 62–70. [8] J.F. Giani, M.A. Mayer, M.C. Munoz, E.A. Silberman, C. Hocht, C.A. Taira, M.M. Gironacci, D. Turyn, F.P. Dominici, Chronic infusion of angiotensin-(1–7) improves insulin resistance and hypertension induced by a high-fructose diet in rats, Am. J. Physiol. Endocrinol. Metab. 296 (2009) E262–E271. [9] A. González-Rodríguez, R. Mayoral, N. Agra, M.P. Valdecantos, V. Pardo, M.E. Miquilena-Colina, J. Vargas-Castrillón, O. Lo Iacono, M. Corazzari, G.M. Fimia, M. Piacentini, J. Muntané, L. Boscá, C. García-Monzón, P. Martín-Sanz, A.M. Valverde, Impaired autophagic flux is associated with increased endoplasmic reticulum stress during the development of NAFLD, Cell Death Dis. 5 (2014) e1179. [10] Y. Guan, L. Gao, H.J. Ma, Q. Li, H. Zhang, F. Yuan, Z.N. Zhou, Y. Zhang, Chronic intermittent hypobaric hypoxia decreases beta-adrenoceptor activity in right ventricular papillary muscle, Am. J. Physiol. Heart Circ. Physiol. 298 (2010) H1267–H1272. [11] C. Ji, R. Mehrian-Shai, C. Chan, Y.H. Hsu, N. Kaplowitz, Role of CHOP in hepatic apoptosis in the murine model of intragastric ethanol feeding, Alcohol. Clin. Exp. Res. 29 (2005) 1496–1503. [12] G. Kanuri, A. Spruss, S. Wagnerberger, S.C. Bischoff, I. Bergheim, Role of tumor necrosis factor α (TNFα) in the onset of fructose-induced nonalcoholic fatty liver disease in mice, J. Nutr. Biochem. 22 (2011) 527–534. [13] R. Kumamoto, H1. Uto, K. Oda, R. Ibusuki, S. Tanoue, S. Arima, S. Mawatari, K. Kumagai, M. Numata, T. Tamai, A. Moriuchi, H. Fujita, M. Oketani, A. Ido, H. Tsubouchi, Dietary fructose enhances the incidence of precancerous hepatocytes induced by administration of diethylnitrosamine in rat, Eur. J. Med. Res. 18 (2013) 54. [14] T.F. Kuo, H. Tatsukawa, T. Matsuura, K. Nagatsuma, S. Hirose, S. Kojima, Free fatty acids induce transglutaminase 2-dependent apoptosis in hepatocytes via ER stress-stimulated PERK pathways, J. Cell. Physiol. 227 (2012) 1130–1137. [15] A.D. Lake, P. Novak, R.N. Hardwick, B. Flores-Keown, F. Zhao, W.T. Klimecki, N.J. Cherrington, The adaptive endoplasmic reticulum stress response to lipotoxicity in progressive human nonalcoholic fatty liver disease, Toxicol. Sci. 137 (2014) 26–35. [16] J.S. Lee, Z. Zheng, R. Mendez, S.W. Ha, Y. Xie, K. Zhang, Pharmacologic ER stress induces non-alcoholic steatohepatitis in an animal model, Toxicol. Lett. 211 (2012) 29–38. [17] J. Li, J. Huang, J.S. Li, H. Chen, K. Huang, L. Zheng, Accumulation of endoplasmic reticulum stress and lipogenesis in the liver through generational effects of high fat diets, J. Hepatol. 56 (2012) 900–907. [18] M.J. Pagliassotti, Endoplasmic reticulum stress in nonalcoholic fatty liver disease, Annu. Rev. Nutr. 32 (2012) 17–33. [19] A. Rebollo, N. Roglans, M. Alegret, J.C. Laguna, Way back for fructose and liver metabolism: bench side to molecular insights, World J. Gastroenterol. 18 (2012) 6552–6559. [20] C.A. Roncal-Jimenez, M.A. Lanaspa, C.J. Rivard, T. Nakagawa, L.G. Sanchez-Lozada, D. Jalal, A. Andres-Hernando, K. Tanabe, M. Madero, N. Li, C. Cicerchi, K. Mc Fann, Y.Y. Sautin, R.J. Johnson, Sucrose induces fatty liver and pancreatic inflammation in

[21]

[22]

[23]

[24]

[25]

[26]

[27] [28] [29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

45

male breeder rats independent of excess energy intake, Metabolism 60 (2011) 1259–1270. N. Rosso, N.C. Chavez-Tapia, C. Tiribelli, S. Bellentani, Translational approaches: from fatty liver to non-alcoholic steatohepatitis, World J. Gastroenterol. 20 (2014) 9038–9049. A. Schultz, D. Neil, M.B. Aguila, C.A. Mandarim-de-Lacerda, Hepatic adverse effects of fructose consumption independent of overweight/obesity, Int. J. Mol. Sci. 14 (2013) 21873–21886. M. Shi, F. Cui, A.J. Liu, J. Li, H.J. Ma, M. Cheng, J. Yang, Y. Zhang, Protection of chronic intermittent hypobaric hypoxia against collagen-induced arthritis in rat through increasing apoptosis, Sheng Li Xue Bao 63 (2011) 115–123. A.P. Simopoulos, Dietary omega-3 fatty acid deficiency and high fructose intake in the development of metabolic syndrome, brain metabolic abnormalities, and nonalcoholic fatty liver disease, Nutrients 5 (2013) 2901–2923. S.J. Song, Y. Xu, F.F. Li, F. Yuan, Z.N. Zhou, Y. Zhang, Effect of chronic intermittent hypobaric hypoxia on contractile activity of arteries in rats, Sheng Li Xue Bao 63 (2011) 205–210. A. Spruss, G. Kanuri, K. Uebel, S.C. Bischoff, I. Bergheim, Role of the inducible nitric oxide synthase in the onset of fructose-induced steatosis in mice, Antioxid. Redox Signal. 14 (2011) 2121–2135. A. Takaki, D. Kawai, K. Yamamoto, Molecular mechanisms and new treatment strategies for non-alcoholic steatohepatitis (NASH), Int. J. Mol. Sci. 15 (2014) 7352–7379. T. Takamura, H. Misu, T. Ota, S. Kaneko, Fatty liver as a consequence and cause of insulin resistance: lessons from type 2 diabetic liver, Endocr. J. 59 (2012) 745–763. N. Tamaki, E. Hatano, K. Taura, M. Tada, Y. Kodama, T. Nitta, K. Iwaisako, S. Seo, A. Nakajima, I. Ikai, S. Uemoto, CHOP deficiency attenuates cholestasis-induced liver fibrosis by reduction of hepatocyte injury, Am. J. Physiol. Gastrointest. Liver Physiol. 294 (2008) G498–G505. X. Xiong, X. Wang, Y. Lu, E. Wang, Z. Zhang, J. Yang, H. Zhang, X. Li, Hepatic steatosis exacerbated by endoplasmic reticulum stress-mediated downregulation of FXR in aging mice, J. Hepatol. 60 (2014) 847–854. F. Yuan, H.W. Li, S.J. Song, X. Teng, H.J. Ma, Z. Guo, Y. Zhang, Z.N. Zhou, Opioid receptors mediate enhancement of ACh-induced aorta relaxation by chronic intermittent hypobaric hypoxia, Sheng Li Xue Bao 65 (2013) 269–275. Y. Zhang, N. Zhong, Z.N. Zhou, Effects of chronic intermittent hypobaric hypoxia on the L-type calcium current in rat ventricular myocytes, High Alt. Med. Biol. 11 (2010) 61–67. P. Zhang, S.J. Song, W.L. Liu, L.L. Li, W.L. Zhao, Y. Zhang, Eeffects of Coptis chinensis on vasoconstrictive activity of isolated thoracic aorta of normoxic and chronic intermittent hypobaric hypoxic rats, Zhongguo Ying Yong Sheng Li Xue Za Zhi 27 (2011) 420–425. C. Zhang, X. Chen, R.M. Zhu, Y. Zhang, T. Yu, H. Wang, H. Zhao, M. Zhao, Y.L. Ji, Y.H. Chen, X.H. Meng, W. Wei, D.X. Xu, Endoplasmic reticulum stress is involved in hepatic SREBP-1c activation and lipid accumulation in fructose-fed mice, Toxicol. Lett. 212 (2012) 229–240. J.J. Zhou, Y. Wei, L. Zhang, J. Zhang, L.Y. Guo, C. Gao, D.P. Li, Y. Zhang, Chronic intermittent hypobaric hypoxia prevents cardiac dysfunction through enhancing antioxidation in fructose-fed rats, Can. J. Physiol. Pharmacol. 91 (2013) 332–337. X.Q. Zhang, C.F. Xu, C.H. Yu, W.X. Chen, Y.M. Li, Role of endoplasmic reticulum stress in the pathogenesis of nonalcoholic fatty liver disease, World J. Gastroenterol. 20 (2014) 1768–1776. Y. Zhang, Z.N. Zhou, Beneficial effects of intermittent hypobaric hypoxia on the body, Zhongguo Ying Yong Sheng Li Xue Za Zhi 28 (2012) 504–509.