Animal models of obstructive cholestasis and ischemia/reperfusion damage have revealed the functional heterogeneity of liver lobes. This study evaluates this heterogeneity in nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) rat models. Twelve-week-old Obese and Lean male Zucker rats were used for NAFLD. Eight-week-old male Wistar rats fed with 8-week methionine-choline-deficient (MCD) diet and relative control diet were used for NASH. Gelatinase (MMP-2; MMP-9) activity and protein levels, tissue inhibitors of metalloproteinase (TIMPs), reactive oxygen species (ROS), and thiobarbituric acid-reactive substances (TBARS) were evaluated in the left (LL), median (ML), and right liver (RL) lobes. Serum hepatic enzymes and TNF-alpha were assessed. An increase in gelatinase activity in the NASH model occurred in RL compared with ML. TIMP-1 and TIMP-2 displayed the same trend in RL as ML and LL. Control diet RL showed higher MMP-9 activity compared with ML and LL. No significant lobar differences in MMP-2 activity were detected in the NAFLD model. MMP-9 activity was not detectable in Zucker rats. TIMP-1 was lower in LL when compared with ML while no lobar differences were detectable for TIMP-2 in either Obese or Lean Zucker rats. Control diet rats exhibited higher ROS formation in LL versus RL. Significant increases in TBARS levels were observed in LL versus ML and RL in control and MCD rats. The same trend for ROS and TBARS was found in Obese and Lean Zucker rats. An increased serum TNF-alpha occurred in MCD rats. A lobar difference was detected for MMPs, TIMPs, ROS, and TBARS in both MCD and Zucker rats. Higher MMP activation in RL and higher oxidative stress in the LL, compared with the other lobes studied, supports growing evidence for functional heterogeneity among the liver lobes occurring certainly in both NAFLD and NASH rats.
Among emergent metabolic chronic liver diseases, nonalcoholic fatty liver disease (NAFLD) and its more advanced form, nonalcoholic steatohepatitis (NASH), are becoming a major public health problem in industrialized countries [
Animal models are an essential tool for the identification of the mechanisms driving the pathogenesis and progression of NAFLD to NASH. Ideally, experimental models should reflect the etiology, disease progression, and pathology of human NAFLD. Unfortunately, currently available models, MCD diet, Western diet, and high-fat diet, are complementary and each of them partially reflects the real picture of human NAFLD [
The liver parenchyma displays a functional organization known as metabolic zonation: the hepatocytes lined up between the sinusoids along the porto-central axis show structural and functional heterogeneity [
The goal of the present study was to investigate presumed liver lobe heterogeneity in nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) models, in terms of alteration of the ECM, matrix metalloproteinase (MMP) activity, and specific inhibitors (TIMPs) and of oxidative stress content, ROS, and TBARS formation.
Zucker rats represent a well-characterized model of NAFLD. Fourteen 11-week-old male obese (fa/fa) Zucker rats and age-matched lean (fa/-) were used. Animals (n=7 each group) were supplied by Charles River, Italy. The most widely used diet to induce NASH is the methionine-choline-deficient (MCD) diet. Fourteen 8-week-old male Wistar rats were fed with MCD diet (Laboratorio Dottori Piccioni, Milano, Italy), or with an isocaloric diet supplemented by choline and methionine (Control) for 8 weeks. Animals (n=7 each group) were supplied by Charles River, Italy. Animal models used were approved by the Italian Ministry of Health and by the local University Animal Care Commission (Document number 2/2012). At the time of sacrifice, on the basis of rat lobar structure, recently described by Sanger et al. [
Graphic (schematic) representation of hepatic lobes. Liver samples were collected (
Liver injury was assessed by serum level evaluation of alanine transaminase (ALT) and aspartate transaminase (AST) using a commercial kit (Sigma). Serum levels of TNF-alpha were evaluated by a commercial ELISA kit according to the manufacturing procedures (R&D Systems, Minneapolis, MN). Determination of hepatic reactive oxygen species (ROS) was followed by the conversion of 2′,7′-dichlorofluorescein diacetate (H2DCFDA) to fluorescent 2′,7′-dichlorofluorescein (DCF) as previously described [
After sacrifice, hepatic lobes were quickly excised and placed in cold (4°C) buffer (30 mM histidine, 250mM sucrose, 2 mM EDTA, pH 7.2) to remove blood. Liver was weighed and subsequently cut, frozen in liquid nitrogen and stored at -80°C, until use. Hepatic protein was extracted by homogenisation (IKA-Ultraturrax T10) of frozen liver tissue, in an ice-cold extraction buffer (1:10 wt/vol) containing 1% Triton X-100, 500 mmol/L Tris-HCl, 200 mmol/L NaCl, and 10mmol/L CaCl2, pH 7.6 [
In order to detect MMPs lytic activity, the hepatic extracts were normalized to a final concentration of 400
Results are expressed as mean ± standard error. Comparisons between groups were performed by unpaired t test. When data distribution was not normal according to the Kolgonorov-Smrna test, a Mann-Witney test was used. All statistical procedures were performed using the MedCalc statistical software package (11.6.0.0 version). A value of p<0.05 was considered significant.
A general increase in gelatinolytic activity was observed in the NASH model, in the RL. In particular, gelatin zymography revealed a statistical difference between the liver lobes: MMP-2 and MMP-9 activity was significantly increased in the RL compared with the ML in the MCD rats (Figures
Hepatic content in MMP-2 and MMP-9 activity ((a) and (b)) and MMP-2 and MMP-9 protein levels ((c) and (d)) obtained from LL, ML, and RL in NASH and control rats. MMP gelatinase activity is expressed as optical density (OD) for mm2, related to 1 mg/mL protein content. MMP protein content is expressed in ng/mL. Data are shown as mean values ± SE.
The analysis of MMP protein levels revealed comparable MMP-2 levels between lobes in the control and MCD animals (Figure
TIMP-1 and TIMP-2 levels were higher in the RL in the NASH model, when compared with the ML and LL (Figure
Hepatic content in TIMP-1 (a) and TIMP-2 (b) obtained from LL, ML, and RL in MCD and control rats. TIMP levels are expressed in ng/mL. Data are shown as mean values ± SE.
The evaluation of MMP-2 in the NAFLD animals revealed low levels in the ML, though not significantly whereas this activity was significant in Lean Zucker rats (Figure
Hepatic content in MMP-2 activity (a) and MMP-2 and MMP-9 protein levels ((b) and (c)) obtained from LL, ML, and RL in NAFLD and Lean rats. MMP gelatinase activity is expressed as optical density (OD) for mm2, related to 1 mg/mL protein content. MMP content is expressed in ng/mL. Data are shown as mean values ± SE.
The analysis of MMP protein levels in NAFLD animals and their control animals showed a slight decrease in MMP-2 and MMP-9 protein content in the RL when compared with the ML (Figures
TIMP-1 was significantly higher in the ML when compared with the LL in NAFLD rats (Figure
Hepatic content in TIMP-1 (a) and TIMP-2 (b) obtained from LL, ML, and RL in NAFLD and Lean rats. TIMP levels are expressed in ng/mL. Data are shown as mean values ± SE.
In the NASH model, no difference was detectable between lobes in ROS levels (Figure
Hepatic levels of ROS (a) and TBARS (b) obtained from LL, ML, and RL in NASH and control rats. Data are shown as mean values ± SE.
In NAFLD rats, a higher ROS concentration was found in the LL when compared with the ML (Figure
Hepatic levels of ROS (a) and TBARS (b) obtained from LL, ML and RL in NAFLD and Lean rats. Data are shown as mean values ± SE.
Serum AST and ALT increased in NASH animals as compared with the control group (Table
Serum enzymes and TNF-alpha in NASH and NAFLD rats.
ALT | AST | TNF-alpha | ||
---|---|---|---|---|
(U/L) | (U/L) | (pg/mL) | ||
| | 30.8±2 | 97.8±2 | 26.8±2.2 |
| 166.2±23 | 245.1±39 | 36.7±2.6 | |
| ||||
| | 66.2±4.3 | 112.3±2.8 | 10.2±0.5 |
| 114.5±20 | 116.1±10 | 9.5±0.4 |
The rat model used in this study cover the spectrum of liver pathology observed in NASH ranging from hepatic steatosis to inflammation progression to fibrosis. In our study, rats fed the MCD diet for 8 weeks developed steatohepatitis with markers of inflammation. Interestingly, in NASH fibrogenesis, MMPs and TIMPs may play a role not only into the balance between the formation and the degradation of ECM composition [
TNF-alpha, an inflammatory cytokine modulating MMPs involved in repair and remodeling, plays a major role in the progression from steatosis to NASH [
TIMP-1, a natural inhibitor of MMP-9, is the most relevant TIMP in toxic liver injury and dramatically upregulated by inflammatory cytokines such as TNF-alpha [
Using the genetic model of NAFLD, we also detected lobe-heterogenicity for MMP-2 activity; on the contrary, MMP-9 activity was undetectable, in keeping with the findings of other authors in both liver [
In the present study we found that the LL exhibits increased oxidative stress that was superimposable in both models considered. High vulnerability to oxidative stress is responsible for the “second hit” in the spontaneous progression from simple steatosis to NASH [
The lobe-specific heterogeneity could be ascribed to the differential blood supply: recently Sanger et al. described the intrahepatic vascular anatomy in liver rats and mice; of note, the lobar borders of the liver do not always match vascular territorial borders [
Whereas intralobular hepatic heterogeneity is extensively described, only a few studies have reported the difference between hepatic lobes. In particular, the present work exhibits, in control rats, lobe-specific heterogeneity in MMPs, TIMPs, and oxidative stress that persists and appears to be amplified during liver injury such as NASH (Figure
Schematic representation of liver lobes heterogeneity in NASH and NAFLD models (MMPs, matrix metalloproteinases; TIMPs, tissue inhibitors of metalloproteinase ROS, reactive oxygen species; TBARS, thiobarbituric acid-reactive substances).
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare that they have no conflicts of interest.
Giuseppina Palladini and Laura G. Di Pasqua contributed equally.
We thank Mr. Massimo Costa for his skillful technical assistance, Mrs. Nicoletta Breda for her editing assistance, and Professor Anthony Baldry for revising the English.