Nonalcoholic fatty liver disease (NAFLD) is a serious health problem in developed countries. We documented the effects of feeding with a NAFLD-inducing, methionine- and choline-deficient (MCD) diet, for 1–4 weeks on rat liver oxidative stress, with respect to a control diet. Glycogen, neutral lipids, ROS, peroxidated proteins, and SOD2 were investigated using histochemical procedures; ATP, GSH, and TBARS concentrations were investigated by biochemical dosages, and SOD2 expression was investigated by Western Blotting. In the 4-week-diet period, glycogen stores decreased whereas lipid droplets, ROS, and peroxidated proteins expression (especially around lipid droplets of hepatocytes) increased. SOD2 immunostaining decreased in poorly steatotic hepatocytes but increased in the thin cytoplasm of macrosteatotic cells; a trend towards a quantitative decrease of SOD expression in homogenates occurred after 3 weeks. ATP and GSH values were significantly lower for rats fed with the MCD diet with respect to the controls. An increase of TBARS in the last period of the diet is in keeping with the high ROS production and low antioxidant defense; these TBARS may promote protein peroxidation around lipid droplets. Since these proteins play key roles in lipid mobilization, storage, and metabolism, this last information appears significant, as it points towards a previously misconsidered target of NAFLD-associated oxidative stress that might be responsible for lipid dysfunction.
Nonalcoholic fatty liver disease (NAFLD), the most recurrent liver disorder in Western countries, is commonly associated with obesity and progression of metabolic syndrome [
For investigating the etiology and regulation of NAFLD and NASH and for testing potential therapeutic approaches, several experimental models were developed. These are based on the application of a diet, on the administration of drugs to laboratory animals, or on the exposure of hepatic cell lines to these drugs; genetically modified rodents or zebrafish have also been introduced [
The MCD diet contains high levels of sucrose and fat but lacks methionine and choline. Choline is an essential nutrient fundamental for cell membrane integrity, transmembrane signaling, phosphatidylcholine synthesis, neurotransmission, and methyl metabolism [
A rich literature is available documenting oxidative stress associated with liver steatosis (e.g., [
Histochemistry provides a unique opportunity to document
Our approach successfully demonstrated a lobular-zone dependent evolution of ROS production and effects throughout the 4-week period of the diet. In particular, it revealed oxidative-induced alteration of proteins in the coat of lipid droplets in hepatocytes.
Male Wistar rats were fed with methionine- and choline-deficient (MCD) diet (Laboratorio Dottori Piccioni, Gessate, Italy) [
The use of the animal model was approved by the Italian Ministry of Health and Pavia University Animal Care Commission.
Tissue samples were fixed in 2% p-formaldehyde in 0.1 M phosphate buffer (pH 7.4) for 24 h, dehydrated, and included in Paraplast. Glycogen was visualized with the periodic acid-Schiff reaction on liver sections of 7
Frozen sections (8
The filter set 09 (BP450-490, FT510, and LP515), which includes an excitation filter BP 450–490 nm (blue light), a dichroic mirror 510 nm, and a barrier filter 520 nm, was used to excite and reveal Nile Red-induced yellow-gold fluorescence.
Frozen sections (8
Frozen tissue sections (8
Methacarn solution consisting of 60% (vol/vol) absolute methanol, 30% chloroform, and 10% glacial acetic acid was freshly prepared before fixation. Tissue samples were fixed in Methacarn overnight at 4°C. For embedding, tissue samples were dehydrated three times for one hour in fresh 100% ethanol at 4°C, immersed in xylene once for one hour and then thrice for 1 h at room temperature, and embedded in Paraplast. Paraplast-embedded sections (6
The slides were observed with a Zeiss Axioskop 2 Plus light microscope (Carl Zeiss Microimaging, Jena, Germany) equipped with Differential Interference Contrast (DIC) and with the 09 Zeiss filter set to demonstrate Nile Red-induced yellow-gold fluorescence of neutral lipids: BP 450–490 excitation filter, FT 510 beam splitter, and LP515 emission filter. Photomicrography was made using a Canon EOS 1100D digital camera (Tokyo, Japan) set to a resolution of 6 megapixels.
Tissue ATP was measured by the luciferin-luciferase method with the ATP Bioluminescence Assay Kit CLS II (Roche Molecular Biochemicals, Milan, Italy). The hepatic concentration of reduced glutathione (GSH) was measured by an enzymatic method (Cayman Chemical Co., Ann Arbor, MI, USA). Hepatic lipid peroxidation was assessed by measuring the amount of thiobarbituric acid reactive substances (TBARS) formation, which was measured according to the method of Esterbauer et al. [
Statistical analysis of data concerning ATP, GSH, and TBARS concentration was performed using R software (R Development Core Team). Data normally distributed were analyzed by one-way ANOVA, followed by Tukey’s multiple comparisons test. Data not normally distributed were analyzed by Kruskal-Wallis test, followed by Dunn’s multiple comparisons test. The value of
40 mg of liver was lysed in 1 mL of RIPA buffer (50 mM Tris-HCl, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, and 1 mM Na3VO4, pH 7.4) supplemented with Protease Inhibitor Cocktail. Lysates were then centrifuged for 10 min at 4°C, avoiding rescuing the lipids ring in the samples. Protein concentration was measured with a spectrophotometer (UV-1601, Shimadzu, Tokyo, Japan) set to 562 nm, using Pierce BCA Protein Assay Kit (bicinchoninic acid, Thermo Fisher Scientific Inc., Rockford, IL, USA). Sample buffer (3x) consisting of 6% SDS, 1.5% DTT, 30% glycerol, 0.03% bromophenol blue, and 0.5 M Tris-Gly buffer (1x, containing SDS 0.1%, pH 8.3) was added to the appropriate aliquots of supernatants. After boiling for 10 min at 95°C, equal aliquots of protein were separated by 12% SDS-PAGE under standard conditions, followed by wet transfer onto PVDF membrane (Bio-Rad Laboratories, Hercules, CA, USA) at 4°C for 2 h. After blocking with 0.1% Tween-20-TBS containing 5% BSA for 1 h at room temperature, membranes were incubated overnight with rabbit polyclonal anti-SOD2 primary antibody (NB100-1992; Novus Biologicals, Littleton, CO 80120, USA) diluted 1 : 2000 and, at a later stage, with a monoclonal anti-
Digital images were analyzed using Quantity One 1-D Analysis Software, Bio-Rad Laboratories. The optical density (OD) of each band was calculated following the manufacturer’s instructions. The OD values of SOD2 were normalized versus own relative band of
The liver of rats fed, for two weeks, with a diet isocaloric with the MCD diet but containing methionine and choline was considered representative and herewith described as “control.”
Glycogen deposits, evidenced by a fuchsia precipitate with the PAS reaction, were present in hepatocytes throughout the lobule; they were particularly abundant in periportal and centrolobular cells (Figure
Liver of rats submitted to control diet for 2 weeks. (a) Representative photomicrographs of PAS reaction for glycogen, abundant in all hepatocytes. (b) Nile Red-induced fluorescence of small neutral lipid droplets in the perisinusoidal cytoplasm of hepatocytes. (c) Diaminobenzidine-Mn2+-Co2+ reaction for Reactive Oxygen Species (ROS); intense reaction in periportal hepatocytes and occasional Kupffer cells in the midzone or pericentral regions. (d) Immunoreaction against dinitrophenyl (DNP) groups for demonstrating carbonyl groups derivatized with dinitrophenyl hydrazine (DNPH); these are present in perisinusoidal and canalicular membrane domains of hepatocytes. (e) Immunoreaction for visualizing the expression of Mn-dependent Superoxide Dismutase 2 (SOD2); moderately intense staining in the cytoplasm of periportal and pericentral hepatocytes. P: branch of portal vein; CL: branch of centrolobular vein. Scale bar: 50
Small neutral lipid droplets, evidenced by the yellow-gold fluorescence induced by Nile Red, were present in all hepatocytes; these were polarized towards the sinusoidal domains (Figure
Lobular zonation of ROS, demonstrated by a blue precipitate with the Mn-DAB-Co reaction, was observed. The reaction was particularly intense in the cytoplasm of hepatocytes in the periportal/midzone region, where it was polarized towards the sinusoidal domains, and very low in the pericentral region. Occasional sinusoidal cells displayed strong staining (Figure
Resulting dinitrophenyl (DNP) groups were demonstrated with an immunoperoxidase method giving a brown final reaction product. Intense immunoreactivity was seen in sinusoidal and canalicular membrane domains of hepatocytes; diffuse lighter staining was also seen in the cytoplasm of periportal cells (Figure
Immunoreactivity for SOD2 was present in hepatocytes throughout the lobule, being intense in periportal and pericentral cells and moderate in hepatocytes of the midzone; it was present as a granular brown product in the cytoplasm partially polarized towards sinusoidal and canalicular domains (Figure
Figure
Comparison between glycogen accumulation (PAS reaction; (a), (c), (e), and (g)) and neutral lipid accumulation (Nile Red reaction; (b), (d), (f), and (h)) in the liver of rats fed with the MCD diet for 1–4 weeks. Representative photomicrographs. It is shown that whereas the glycogen content of the liver sharply decreases in the 2nd week of the diet and vanishes altogether in the 3rd and 4th weeks, the neutral lipid content dramatically increases from the 2nd to the 4th week. P: branch of portal vein; CL: branch of centrolobular vein. Scale bar: 50
Figure
Comparison between ROS formation (DAB-Mn-Co reaction; (a), (c), (e), and (g)) and carbonyl groups derivatized with DNPH (immunoreaction against DNP; (b), (d), (f), and (h)) in the liver of rats fed with the MCD diet for 1–4 weeks. Representative photomicrographs. Whereas in the 1st week of the diet the lobular ROS pattern is similar to the control liver, from the 2nd week onwards, ROS formation is intense in all hepatocytes, being concentrated in the cytoplasm surrounding large lipid droplets. The patterns of peroxidized proteins follow the ROS trend. P: branch of portal vein; CL: branch of centrolobular vein. Scale bar: 50
Figure
Expression of Mn-dependent Superoxide Dismutase 2 (SOD2) in the liver of rats fed with the MCD diet for 1–4 weeks. Representative photomicrographs. Immunoreactivity is concentrated in the thin rim of cytoplasm surrounding the large lipid droplets. P: branch of portal vein; CL: branch of centrolobular vein. Scale bar: 50
Figure
Evaluation of SOD2 expression in livers of the rats fed with control or MCD diet for 1–4 weeks. (a) Comparison of SOD2 expression in the liver of rats fed with control and MCD diet for 1–4 weeks. Histograms representing the ratio between optical density (OD) values of homogenates of the liver of rats fed with the MCD diet for 1 to 4 weeks and the OD of control livers (b).
A marked decrease in the hepatic levels of ATP and GSH was found already at the 1st week in rats fed with a MCD diet as compared with the control group (Figures
Changes in tissue ATP, TBARS, and GSH concentration in rats fed with a MCD diet with respect to values concerning a control diet. The results are reported as the mean ± SE. MCD diet groups versus control group.
The high content of glycogen in hepatocytes is in keeping with the high caloric diet (45% sucrose) to which the animals were submitted.
The droplets emitting bright-yellow fluorescence are ascribed to intracellular triacylglycerols (TAGs) depots and/or to lipoproteins [
The histochemical reaction used is based on the ROS-induced oxidation of Mn2+ to Mn3+, which in turn oxidizes DAB, causing its polymerization; intensification of the colored polymer is achieved by Co3+ ions. This reaction proved to demonstrate mainly superoxide anion (
Protein carbonyl (CO) groups are considered as early biomarkers of oxidative stress [
Superoxide Dismutases are key enzymes, which protect cells from oxidative stress by scavenging superoxide radicals through the dismutation into O2 and H2O2 [
The progressive increase of neutral lipid droplets along the 4-week period of the MCD diet, visualized by the yellow-gold fluorescence of Nile Red, confirms the efficacy of the diet in inducing steatosis. The fluorochrome Nile Red has been used to document steatosis in rats fed with a diet rich in fructose and alcohol [
In parallel with the increase in neutral lipids, the amount of stored glycogen sharply decreased. A similar observation was made in the liver of Wistar rats fed with a choline-deficient diet [
A further effect of the MCD diet was an increase in ROS production by hepatocytes throughout the whole lobule. As already mentioned, the main sources of ROS are complexes I and III of the mitochondria electron transport chain [
In normal conditions, antioxidant systems control and inactivate ROS produced by mitochondria, with residual species serving signaling purposes [
Protein carbonyl groups are considered as early markers of proteins altered by oxidative stress since they may be introduced in proteins by reaction of nucleophilic side chains of Cysteine, Histidine, and Lysine residues with aldehydes produced during lipid peroxidation (e.g., 4-hydroxy-2-nonenal and malondialdehyde, TBARS-positive) [
The presence of peroxidated proteins surrounding lipid droplets observed in this study may be an important feature contributing to the dysfunction of hepatocytes in fatty liver. Triglyceride accumulation in lipid droplets is believed to be a mechanism of defense against cytotoxicity induced by free fatty acids [
A correlation between liver steatosis and oxidative stress has been often reported in the literature (e.g., [
The immunohistochemical analysis of SOD2 revealed a change in protein expression pattern in hepatocytes along the 4-week period of the MCD diet that was similar, in particular in the last weeks, to that of ROS and peroxidated proteins patterns. In particular, large steatotic cells showed intense immunoreactivity in the thin rim of cytoplasm surrounding the lipid droplets. The correspondence between strong ROS production and intense SOD2 expression in the same areas of the lobule suggests an antioxidant attempt to compensate for the high ROS production. Such attempt, associated with the fact that GSH concentration was much lower than normal, might not be entirely successful, thus explaining the formation of peroxidated proteins in the same areas where the ROS production was high. The general trend towards decrease of SOD2 expression of liver homogenates in the last 2 weeks detected by Western Blotting is here ascribed to the much lower cytoplasm area occupied by the enzyme as hepatocytes acquire an adipocyte-like morphology.
As concerns the GSH concentration, though we found it to be not significantly different along the 4-week period of the diet, it is much lower with respect to the control. These data are in keeping with data reported in the literature concerning depletion of antioxidant defenses and in particular of GSH in NAFLD [
In conclusion, the histochemical approach herewith illustrated and whose main results are summarized in Table
Summary of the relevant observations regarding metabolic and oxidative stress alterations induced by a 1–4-week period of MCD diet, with respect to control diet.
Parameter | Diet-induced modifications |
---|---|
Glycogen content | Sharp decrease |
|
|
Neutral lipid content | Progressive increase |
|
|
Reactive Oxygen Species | Progressive increase especially surrounding lipid droplets |
|
|
Peroxidated proteins | Progressive increase especially surrounding lipid droplets |
|
|
Superoxide Dismutase 2 expression | Concentrated in thin rim of cytoplasm surrounding lipid droplets; trend towards decreased expression in the last 2 weeks |
Dinitrophenyl
Glutathione
Lipid droplet
Methionine- and choline-deficient
Nonalcoholic fatty liver disease
Nonalcoholic steatohepatitis
Reactive Oxygen Species
Perilipin, adipose differentiation-related protein, and tail-interacting protein of 47 kDa
Superoxide Dismutase
Very-low-density lipoproteins
S-Adenosylmethionine
Thiobarbituric acid reactive substances.
The authors declare that they have no conflict of interests.
This work was supported by Fondazione Cariplo, Grant no. 2011-0439, and by the University of Pavia (FAR).