Molecular and Cellular Biochemistry

, Volume 335, Issue 1, pp 119–125

Increased liver oxidative stress and altered PUFA metabolism precede development of non-alcoholic steatohepatitis in SREBP-1a transgenic spontaneously hypertensive rats with genetic predisposition to hepatic steatosis

Authors

  • Hana Malínská
    • Institute for Clinical and Experimental Medicine
  • Olena Oliyarnyk
    • Institute for Clinical and Experimental Medicine
  • Miriam Hubová
    • Institute for Clinical and Experimental Medicine
  • Václav Zídek
    • Institute of PhysiologyCzech Academy of Sciences
  • Vladimír Landa
    • Institute of PhysiologyCzech Academy of Sciences
  • Miroslava Šimáková
    • Institute of PhysiologyCzech Academy of Sciences
  • Petr Mlejnek
    • Institute of PhysiologyCzech Academy of Sciences
  • Ludmila Kazdová
    • Institute for Clinical and Experimental Medicine
  • Theodore W. Kurtz
    • University of California
    • Institute of PhysiologyCzech Academy of Sciences
Article

DOI: 10.1007/s11010-009-0248-5

Cite this article as:
Malínská, H., Oliyarnyk, O., Hubová, M. et al. Mol Cell Biochem (2010) 335: 119. doi:10.1007/s11010-009-0248-5

Abstract

The temporal relationship of hepatic steatosis and changes in liver oxidative stress and fatty acid (FA) composition to the development of non-alcoholic steatohepatitis (NASH) remain to be clearly defined. Recently, we developed an experimental model of hepatic steatosis and NASH, the transgenic spontaneously hypertensive rat (SHR) that overexpresses a dominant positive form of the human SREBP-1a isoform in the liver. These rats are genetically predisposed to hepatic steatosis at a young age that ultimately progresses to NASH in older animals. Young transgenic SHR versus SHR controls exhibited simple hepatic steatosis which was associated with significantly increased hepatic levels of oxidative stress markers, conjugated dienes, and TBARS, with decreased levels of antioxidative enzymes and glutathione and lower concentrations of plasma α- and γ-tocopherol. Transgenic rats exhibited increased plasma levels of saturated FA, decreased levels of n−3 and n−6 polyunsaturated FA (PUFA), and increased n−6/n−3 PUFA ratios. These results are consistent with the hypothesis that excess fat accumulation in the liver in association with increased oxidative stress and disturbances in the metabolism of saturated and unsaturated fatty acids may precede and contribute to the primary pathogenesis of NASH.

Keywords

Hepatic steatosisNon-alcoholic steatohepatitisOxidative stressFatty acid compositionSREBP-1a transgenic spontaneously hypertensive rat

Introduction

Non-alcoholic fatty liver disease (NAFLD) affects approximately 20–30% of the population in developed countries. It is often associated with obesity and insulin resistance and is a common finding in patients with metabolic syndrome [1, 2]. NAFLD is characterized by a wide spectrum of liver pathologies, ranging from simple steatosis that might progress to non-alcoholic steatohepatitis (NASH), advanced fibrosis, and cirrhosis [3]. The pathophysiology of NAFLD is not fully understood and little is known about the factors that are responsible for the transition from benign steatosis to steatohepatitis and why this happens in only some individuals. According to the two-hit hypothesis, hepatic steatosis represents only the first hit that is not per se sufficient to cause severe hepatic damage but predisposes fatty liver to various second hits, such as oxidative stress or proinflammatory cytokines [46]. However, it is also known that the severity of steatosis is well correlated with progression to NASH in humans [7] and several recent reports suggest that accumulation of lipids in the liver and especially disturbances in the composition of hepatic fatty acids might play a causal role in the development of NASH [810]. In the current study, we analyzed the role of oxidative stress and fatty acid composition in a new animal model of hepatic steatosis and NASH, transgenic spontaneously hypertensive rats (SHRs) that overexpress a dominant positive form of the human SREBF1 gene (sterol regulatory element factor 1 coding for SREBP-1a isoform) under control of the PEPCK promoter [11]. In our original studies of this model in young 10-week-old SHR, expression of the SREBF1 transgene was associated with simple hepatic steatosis, dyslipidemia, and insulin resistance. However, there was no apparent increase in the amount of hepatic inflammation compared with young, age-matched SHR controls. In contrast, 16-month-old transgenic rats exhibited distinct features of steatohepatitis [11]. Therefore, in the current study, we studied young and old transgenic SHR with hepatic steatosis to determine the time course between changes in hepatic oxidative stress and FA composition in relationship to the development of NASH.

Materials and methods

Animals and diets

Derivation of SREBP-1a transgenic rats was described in detail previously [11]. In the current experiments, we studied young (age 10 weeks) and old (age 16 months) male transgenic SHR and male SHR controls after feeding them a high-fructose diet (60% fructose) (K4102.0 diet; Hope Farms, the Netherlands) for 2 weeks. All experiments were performed in agreement with the Animal Protection Law of the Czech Republic (311/1997) and were approved by the Ethics Committee of the Institute of Physiology, Czech Academy of Sciences, Prague.

Histological analysis

Liver specimens from 2-month-old and 16-month-old SREBP-1a transgenic and control rats were fixed in 10% neutral formalin solution and processed according to the routine protocol. The 5-μm cut sections were stained with hematoxylin and eosin.

Biochemical analysis

Animals were killed by decapitation. Sera and livers were collected and stored at −80°C until analyses. Livers were quickly homogenized with Potter-Elvejhem homogenizer at 0–4°C. The activity of superoxide dismutase (SOD) was analyzed using the reaction of blocking nitrotetrazolium blue reduction and nitroformazan formation [12]. Catalase (CAT) activity measurement is based on the ability of H2O2 to produce with ammonium molybdate a color complex detected spectrophotometrically [13]. The activity of seleno-dependent glutathione peroxidase (GSH-Px) was monitored by oxidation of gluthathione by Ellman reagent (0.01 M solution of 5,5′-dythiobis-2 nitrobenzoic acid) [14]. The level of GSH was determined in the reaction of SH-groups using Ellman reagent [15]. Glutathione reductase (GR) activity was measured by the decrease of absorbance at 340 nm using a millimolar extinction coefficient of 6220 M−1cm−1 for NADPH (using Sigma assay kit). The levels of conjugated dienes were analyzed by extraction in the media (heptane: isopropanol = 2:1) and measured spectrophotometrically in heptane’s layer [16]. The levels of TBARS were determined by the reaction with thiobarbituric acid [17]. Serum concentrations of α- and γ-tocopherols were determined by reverse-phase high performance liquid chromatography (HPLC) with fluorescence detection according to the modified method of Catignani and Biery [18, 19] and were corrected to serum triglyceride levels.

Serum lipids were extracted according to Folch [20]. Lipid classes were separated by thin layer chromatography using hexane-diethylether-acetic acid (80:20:3, v/v) as a solvent system. FA in serum phospholipids was converted to methyl esters using 1% solution of Na in methanol and the FA methyl esters were eluted with hexane. Gas chromatography of the FA methyl esters was performed on a GS 5890A (Hewlett Packard, USA) instrument equipped with a flame-ionization detector. A carbowax-fused silica capillary column (25 m × 0.25 mm i.d.) was used. The column temperature was 150.225°C (2°C/min), hydrogen was used as the carrier gas. Individual peaks of FA methyl esters were identified by comparing retention times with those of authentic standards (Sigma, Czech Republic). The composition of serum FA (spectrum of 17 main FA) was analyzed. The product/precursor ratios of the serum FA were used to calculate indices reflecting the activities of enzymes involved in hepatic FA metabolism: elongase (18:0/16:0), Δ6 desaturase (18:3n8/18:2n6), Δ5 desaturase (20:4n6/20:3n6), and Δ9 desaturase (16:1n7/16:0).

Statistical analysis

The data are expressed as means ± SEM. Individual groups were compared by unpaired Student t-test. Statistical significance was defined as P < 0.05.

Results

As can be seen in Fig. 1b, liver isolated from young transgenic rats showed centrolobular steatosis without any signs of inflammation. In contrast, livers isolated from old transgenic rats showed prominent fatty change, focal liver-cell necrosis, and associated inflammatory infiltrate consistent with NASH (Fig. 1c, d).
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Fig. 1

Liver histology in SREBP-1a transgenic and control rats. a Normal portal tract and liver parenchyma in 16-month-old non-transgenic rats. b Liver isolated from young 2-month-old SREBP-1a rats showed centrolobular steatosis without any signs of inflammation. c Livers isolated from 16-months-old SREBP-1a transgenic rats showed prominent fatty changes and steatohepatitis. d Detailed features of steatohepatitis with focal liver-cell necrosis and associated inflammatory infiltrate (indicated by arrows)

Figure 2 shows that lipid peroxidation products reflected by measurements of conjugated dienes and TBARS, were markedly elevated in livers of young SREBP-1a transgenic SHR when compared to their age-matched SHR controls. Increased lipid peroxidation was accompanied by decreased activities of the antioxidant enzymes, SOD and GSH-Px in young transgenic rats (Fig. 3). Hepatic levels of the GSH-Px cofactor, reduced glutathione, were also significantly decreased in young transgenic SHR rats (Fig. 3). GR and CAT activities were not significantly different in young transgenic versus control rats (data not shown). Serum levels of α-tocopherol were significantly decreased in young transgenic SHR when compared to non-transgenic controls (Fig. 3). Serum levels of γ-tocopherol were also significantly lower in transgenic rats versus controls (0.137 ± 0.014 vs. 0.485 ± 0.029 μmol/mmol triglycerides, P < 0.001). Lipid peroxidation products were significantly greater in 16-month-old transgenic rats versus age-matched controls and also greater than in young 10-week-old transgenic rats (Fig. 2). In addition, activities of antioxidant enzymes in old rats were significantly decreased compared to age-matched controls and to young transgenic rats (Fig. 3). There were no significant differences in levels of α-tocopherol between young and old rats. However, the levels of α-tocopherol were significantly lower in old transgenic rats versus age-matched controls (Fig. 3).
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Fig. 2

Markers of oxidative stress in livers of young and old transgenic SHR overexpressing human SREBP-1a protein (solid bars) and non-transgenic age-matched controls (open bars). Lipoperoxidation products, thiobarbiturate reactive substances (TBARS), and conjugated dienes (CD) were significantly increased in young transgenic rats with a simple hepatic steatosis when compared to SHR controls. Concentrations of lipoperoxidation products were higher in old rats and old transgenic rats exhibited higher levels of TBARS and conjugated dienes when compared to age-matched controls. * and ** denote P < 0.05 and P < 0.005, respectively

https://static-content.springer.com/image/art%3A10.1007%2Fs11010-009-0248-5/MediaObjects/11010_2009_248_Fig3_HTML.gif
Fig. 3

Parameters of antioxidant defense in livers and serum levels of α-tocopherol in young and old transgenic SHR overexpressing human SREBP-1a protein (solid bars) and non-transgenic age-matched controls (open bars). Liver SOD (superoxide dismutase), GSH-Px (seleno-dependent glutathione peroxidase), and GSH (reduced glutathione) were decreased in young transgenic rats suggesting derangement in the antioxidant status of the liver. Activities of antioxidant enzymes were lower in old rats and old transgenic rats exhibited decreased levels of enzyme activities compared to age-matched controls. Serum α-tocopherol was significantly decreased in young and old transgenic rats when compared to controls. * and ** denote P < 0.005 P < 0.0001, respectively

The FA composition in serum phospholipids in young and old transgenic and age-matched controls is shown in Table 1. Young transgenic rats compared to age-matched controls, exhibited significantly increased levels of saturated FA by 15%, mainly due to a higher content of stearic (18:0) acid. Old rats exhibited increased levels of saturated FA, however, there was no significant difference in saturated FA between old transgenic rats and age-matched controls. Concentrations of monounsaturated FA (MUFA) in young and old transgenic rats were similar to their age-matched controls. Young transgenic rats showed a significant depletion in polyunsaturated n−6 and n−3 FA (PUFA), including linoleic (18:2n6) by 18%, eicosadienoic (20:2n6) by 46%, dihomo-γ-linolenic (20:3n6) by 42%, adrenic (22:4n6) by 67%, α-linolenic (18:3n3) by 60%, and eicosapentaenoic (20:5n3) acid by 77%. The n−6/n−3 ratio was significantly increased in transgenic rats when compared to controls. Young transgenic SHR also exhibited significantly increased saturated/unsaturated FA ratios when compared to non-transgenic controls. Old rats showed decreased levels of polyunsaturated n−6 and n−3 FA when compared to young rats, however, there were no significant differences in total n−6 and n−3 PUFA between old transgenic and age-matched controls. Table 2 shows FA enzyme activities as estimated from product/precursor ratios of serum phospholipids in young and old rats that predominantly reflect hepatic desaturase activity. Young transgenic rats exhibited a significant decrease in Δ6 desaturase activity while the activity of Δ5 desaturase was increased when compared to age-matched controls. The activity of Δ6 desaturase was also decreased in old transgenic rats when compared to their age-matched controls.
Table 1

Fatty acid composition (%) in serum phospholipids of young and old rats

Fatty acid

Young SHR

Young SHR-SREBP-1a

P

Old SHR

Old SHR-SREBP-1a

P

14:0

0.03 ± 0.01

0.03 ± 0.01

N.S.

0.07 ± 0.01

0.08 ± 0.03

N.S.

16:0

5.29 ± 1.32

7.87 ± 1.81

N.S.

7.05 ± 0.84

8.75 ± 1.16

N.S.

18:0

24.44 ± 0.44

28.86 ± 0.36

<0.0

28.20 ± 0.31

29.35 ± 0.48

N.S.

20:0

0.40 ± 0.08

0.27 ± 0.04

N.S.

0.47 ± 0.04

0.63 ± 0.17

<0.05

Σ SFA

30.12 ± 1.17

37.03 ± 2.05

<0.02

46.10 ± 1.42

48.81 ± 0.76

N.S.

16:1n7

0.12 ± 0.05

0.24 ± 0.07

N.S.

0.22 ± 0.04

0.15 ± 0.02

N.S.

18:1n9

3.67 ± 0.33

4.45 ± 0.38

N.S.

3.68 ± 0.52

3.67 ± 0.18

N.S.

18:1n7

2.65 ± 0.10

3.09 ± 0.20

N.S.

2.86 ± 0.16

2.62 ± 0.02

N.S.

20:1n9

0.55 ± 0.10

0.26 ± 0.03

<0.05

0.61 ± 0.12

0.68 ± 0.09

N.S.

Σ MUFA

6.99 ± 0.46

8.04 ± 0.62

N.S.

7.36 ± 0.04

7.11 ± 0.28

N.S.

18:2n6

13.13 ± 0.65

10.71 ± 0.74

<0.05

22.73 ± 0.67

23.37 ± 0.46

N.S.

18:3n6

0.53 ± 0.23

0.22 ± 0.04

N.S.

0.36 ± 0.19

0.15 ± 0.02

<0.02

20:2n6

1.02 ± 0.15

0.55 ± 0.04

<0.02

0.55 ± 0.05

0.59 ± 0.04

N.S.

20:3n6

1.16 ± 0.12

0.67 ± 0.10

<0.01

1.65 ± 0.23

1.48 ± 0.08

N.S.

20:4n6

37.92 ± 2.44

39.17 ± 2.55

N.S.

15.01 ± 0.65

14.46 ± 2.46

N.S.

22:4n6

1.88 ± 0.48

0.61 ± 0.16

<0.05

2.16 ± 0.38

0.90 ± 0.16

<0.05

Σn−6 PUFA

55.64 ± 0.07

51.92 ± 2.17

N.S.

42.45 ± 1.21

40.95 ± 3.46

N.S.

18:3n3

0.65 ± 0.27

0.26 ± 0.04

<0.05

0.47 ± 0.17

0.10 ± 0,04

<0.01

20:5n3

0.78 ± 0.21

0.18 ± 0.07

<0.05

0.18 ± 0.10

0.05 ± 0.01

<0.05

22:5n3

2.01 ± 0.28

1.50 ± 0.23

N.S.

1.16 ± 0.14

0.89 ± 0.07

N.S.

22:6n3

3.83 ± 1.14

1.09 ± 0.17

N.S.

2.29 ± 0.55

2.09 ± 0.25

N.S.

Σn−3 PUFA

7.27 ± 1.5

3.03 ± 0.47

<0.05

4.10 ± 0.34

3.12 ± 0.23

N.S.

ΣSFA/ΣPUFA

0.43 ± 0.15

0.55 ± 0.04

<0.05

0.57 ± 0.03

0.64 ± 0.02

N.S.

n−6/n−3

9.10 ± 1.45

18.96 ± 2.04

<0.01

14.04 ± 2.18

15.04 ± 2.13

N.S.

Table 2

Indices of fatty acid enzyme activities estimated from serum phospholipid measurements

Enzyme

Young SHR

Young SHR-SREBP-1a

P

Old SHR

Old SHR-SREBP-1a

P

Elongase

6.38 ± 1.51

6.93 ± 2.78

N.S.

5.02 ± 1.13

4.89 ± 1.56

N.S.

Δ5 desaturase

33.46 ± 1.82

63.15 ± 5.80

<0.001

12.60 ± 4.60

10.03 ± 2.53

N.S.

Δ6 desaturase

0.04 ± 0.01

0.02 ± 0.01

<0.05

0.02 ± 0.01

0.01 ± 0.00

<0.05

Δ9 desaturase

0.02 ± 0.00

0.01 ± 0.00

N.S.

0.03 ± 0.01

0.02 ± 0.1

N.S.

Discussion

In the current study, we used a new model of hepatic steatosis and metabolic syndrome, the SREBP-1a transgenic SHR to investigate potential mechanisms involved in the transition of simple hepatic steatosis to NASH. In this model, transgenic expression of SREBP-1a promotes fat accumulation in the liver by increasing expression of genes regulating hepatic lipid synthesis [11]. Thus, genetic induction of hepatic steatosis is the primary factor initiating development of pathophysiological second hits such as oxidative stress, changes in FA composition, and insulin resistance in this model. Accordingly, the current model may be useful for exploring the role of excess accumulation of hepatic lipids in triggering the sequence of events leading to development of NASH [810]. We have found evidence that in young SREBP-1a transgenic SHR compared to age-matched SHR controls, genetically induced severe hepatic steatosis is associated with increased liver oxidative stress and alterations in serum FA composition that precede the subsequent development of histologic features of NASH in older animals. Transgenic SHR also showed deficiency of vitamin E (α- and γ-tocopherol) which might further contribute to oxidative stress. This observation is consistent with previous reports of decreased serum levels of vitamin E in humans with NASH [21].

In the current study, hepatic steatosis induced by transgenic expression of SREBP-1a was associated with marked changes in the FA composition of serum phospholipids. FA composition in serum phospholipids is routinely measured in humans and reflects both the effects of dietary FA intake and endogenous FA metabolism including synthesis of FA, β-oxidation, desaturation, elongation, and lipoperoxidation. We found that primary hepatic steatosis in the liver of 10-week-old transgenic rats was associated with a depletion of n−3 and n−6 PUFA in serum phospholipids as well as increased ratios of n−6/n−3 and saturated/unsaturated FA. It has been suggested that increased n−6/n−3 ratios may affect lipid metabolism and predispose to inflammation and steatohepatitis since decreased hepatic levels of PUFA, especially n−3 PUFA, favor lipid synthesis [2224] and increased n−6/n−3 ratios are associated with insulin resistance [25, 26]. A possible cause of PUFA depletion, particularly of n−3 PUFA, is the deranged antioxidant status of the liver since long-chain PUFA is sensitive to lipoperoxidation. In insulin-resistant individuals, the composition of fatty acid in plasma phospholipids is usually associated with increased Δ9 and Δ6 desaturase activities and decreased Δ5 desaturase activity [27]. In the current study, we observed an opposite relationship in which the estimated activity of Δ6 desaturase was decreased in transgenic rats while the activity of Δ5 desaturase was increased compared to insulin sensitive, non-transgenic SHR controls. In a model of hypertension and insulin resistance, the SHR fed a high-fructose diet, Comte et al. [28] observed increased Δ9 desaturase activity while there were no differences in Δ6 desaturase activity versus SHR controls fed standard chow. In another study, sucrose fed rats exhibited significantly increased activities of Δ9, Δ5, and Δ6 desaturases when compared to controls [29]. In a model of hypertriglyceridemia and insulin resistance, the hereditary hypertriglyceridemic (hHTg) rat, the levels of Δ6 desaturase were decreased by 80% compared to insulin sensitive controls [30]. Thus, the relationship of individual desaturase activities to hepatic steatosis and insulin resistance is not fully understood and may vary depending on the associated experimental circumstances [31].

In humans, conflicting results have been published on the role of oxidative stress and antioxidant defense in the pathogenesis of NAFLD and NASH [3235] and there are only several reports on the role of FA composition in the pathogenesis of NAFLD and NASH. Araya et al. [9] and Elizondo et al. [36] reported depletion of long-chain PUFA, increased n−6/n−3 long-chain PUFA ratio in liver phospholipids, and increased hepatic lipid peroxidation in patients with NAFLD compared to controls. In addition, Allard et al. [8] reported depletion of hepatic n−3 and n−6 PUFA without significant changes in the n−6/n−3 ratio together with increased levels of lipid peroxidation products and decreased antioxidant capacity of the liver in patients with NAFLD. These findings suggest an important role for oxidative stress and depletion of long-chain PUFA in the pathogenesis of NASH and are very similar to those reported in the current study. It has also been suggested that marked steatosis and increased saturated/unsaturated FA ratios might promote the transition of simple fatty liver to NASH [10]. The results of the current study support this possibility because genetically induced severe hepatic steatosis was accompanied by increased saturated/unsaturated FA ratios in young SREBP-1a transgenic rats prior to the development of NASH in older animals.

In summary, the current results are consistent with the possibility that excess fat accumulation in the liver associated with increased oxidative stress, depletion of PUFA, and disturbances in the ratio of saturated/unsaturated fatty acids may play a causal role in the pathophysiology of NASH in our animal model. Thus, the SREBP-1a transgenic rat could provide new opportunities for: (a) investigating mechanisms of antioxidant defense failure and depletion of long-chain PUFA in the pathogenesis of NASH and (b) testing new approaches to the prevention and treatment of this increasingly common disorder.

Acknowledgments

This work was supported by National Institutes of Health grants HL35018, HL56028, and HL63709 (T.W.K) and by grant IAA500110604 from the Czech Academy of Sciences, grants NR9387 and NR9359 from the Ministry of Health of the Czech Republic and the European Commission within the Sixth Framework Programme through the Integrated Project EURATools (contract no. LSHG-CT-2005-019015) (M.P.). M.P. is an international research scholar of the Howard Hughes Medical Institute.

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