Expression of apoptosis on rat liver by hepatic vagus hyperactivity after ventromedial hypothalamic lesioning

Takayoshi Kiba1, Satoru Saito1, Kazushi Numata1, Yasuhiro Kon2, Tetsuya Mizutani3, and Hisahiko Sekihara1

1 Third Department of Internal Medicine, Yokohama City University, School of Medicine, Yokohama 236-0004; and 2 Laboratory of Experimental Animal Science and 3 Laboratory of Public Health, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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We examined whether the Fas (APO-1/CD95)/Fas ligand system mediates apoptosis in rats with ventromedial hypothalamus (VMH) lesions. Northern and Western blotting indicated that VMH lesions lead to a significant increase in Fas mRNA and protein expression from day 1 to day 7 and in Fas ligand mRNA and protein expression from day 2 to day 7. Immunohistochemistry indicated that the region of strongest Fas expression shifted from acinar zone 1 to zones 2 and 3 by day 7 after VMH lesioning and that at days 2-7 Fas-ligand-positive hepatocyte cell membranes and cytoplasm were randomly distributed in acinar zones 1-3. We also analyzed activation of caspase 3-like proteases in hepatocytes, Kupffer cells, and sinusoidal endothelial cells. Spectrofluorometric assay demonstrated that caspase 3-like activity significantly increased only in hepatocytes after VMH lesioning. Moreover, electron microscopy and TUNEL assay showed that VMH lesions induced apoptosis. All of these effects were completely inhibited by hepatic vagotomy and administration of atropine. Vagal firing after VMH lesioning may stimulate Fas/Fas ligand system-mediated apoptosis through the cholinergic system in the rat liver.

Fas; Fas ligand; vagus nerve; ventromedial hypothalamus


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

APOPTOSIS IS A PROCESS of cell death that is morphologically distinct from necrosis and may be induced by a number of factors in both normal and pathological states (30, 54). Liver cells may undergo apoptosis both during liver development and during hepatocyte renewal in the adult liver (19). Apoptosis may also play an important role in liver pathology such as viral hepatitis (23, 47), acute liver failure (13), or alcohol-induced liver injury (13).

The diverse conditions in which programmed cell death may occur suggest that various mechanisms may lead to the activation of apoptosis-regulating genes (30). When ligated and trimerized in vitro, Fas (APO-1/CD95) induces apoptosis in a variety of sensitive cell types (17, 63, 69). The Fas ligand belongs to the tumor necrosis factor (TNF) family, and Fas is a member of the TNF receptor family (52). The Fas-Fas ligand complex is composed of two membrane proteins: the Fas ligand, a 31-kDa glycoprotein located on the plasma membrane of lymphocytes, and Fas, a 45-kDa glycoprotein located on the plasma membrane of several cells, in particular hepatocytes, in which it is abundant (52). Fas engagement activates a cascade of caspase proteases (5, 9, 18, 42, 50), as found in most apoptotic processes (28).

There is a paucity of information on the expression of Fas in the murine liver. Fas mRNA is expressed in a limited number of tissues including the thymus, liver, heart, lung, and ovary of normal mice (66), but its role in these cells remains unclear. Moreover, hepatocytes are sensitive to Fas antibodies (53) and Fas is upregulated in acute liver failure and chronic hepatitis B but not in alcoholic cirrhosis (23). Liver cell apoptosis can be induced by T lymphocytes, which express Fas ligand on activation (45).

The hypothalamus plays a vital role in the integration of neurohumoral information and possesses autonomic centers that are connected to the viscera via the autonomic nervous system. Use of the pair-feeding method showed that lesions of the ventromedial hypothalamus (VMH) produce hepatocyte proliferation (34, 37) and that vagal firing produced by VMH lesions stimulates DNA synthesis (32, 35). However, it is unclear whether apoptosis occurs in rat liver after VMH lesioning. In this study, we investigated the expression of Fas and Fas ligand mRNA and protein in rat liver by Northern and Western blot analysis and immunohistochemistry after VMH lesioning and assessed whether VMH lesions induce apoptosis by electron microscopy and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay. Using spectrofluorometric assay, we also investigated activation of caspase 3-like proteases, an important mediator of apoptosis, in hepatocytes, Kupffer cells, and sinusoidal endothelial cells. In addition, we examined the specific role of vagal hyperactivity on this effect by treating animals with hepatic vagotomy and atropine, a cholinergic blocker.


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ABSTRACT
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Animals. Female Sprague-Dawley rats weighing 230-250 g were used in this study. They were maintained in a constant-temperature environment (23 ± 2°C) in light-controlled cages with a 12-h light-dark cycle (lights on 7:00 AM) and were given free access to food and water. Tissue samples were taken from the liver of pair-fed VMH-lesioned rats, sham VMH-lesioned rats, hepatic-vagotomized VMH-lesioned rats, and atropine-treated VMH-lesioned rats up to day 7 after VMH lesioning (n = 6 in each group), and serum glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), and lactate dehydrogenase (LDH) levels were measured (n = 6 in each group). The livers were divided into three groups. One group was fixed with 10% formalin, embedded in paraffin, and cut into 3-µm-thick sections for histochemical studies. The second group was fixed with 3% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.3) for electron microscopy, and the third group was frozen at -80°C for subsequent Northern and Western blot analysis. We also prepared six animals from each group for measurement of caspase 3 activity in hepatocytes, Kupffer cells, and sinusoidal endothelial cells.

VMH lesions. VMH lesions or simulated operations were performed as previously described (33, 36). The stereotaxic coordinates were at the bregma anteriorly, 0.75 mm lateral to the midsagittal line, and 1.0 mm dorsal from the base of the skull, according to the atlas of De Groot (16). Sham operations were performed in an identical manner except that no current was applied. After the operations, the rats were returned to their cages and given free access to food and water. Localization of the VMH lesions was verified by microscopic examination of the brain at the end of the experiment.

Hepatic vagotomy. Hepatic vagotomy was achieved by sectioning the hepatic branch of the vagus nerve (32). The hepatic branch, which leaves the main vagal trunk a few millimeters proximal to the cardia, was exposed and completely transected near the vagal trunk under a dissecting microscope. Simulated denervation was achieved by laparotomy without hepatic vagotomy.

Administration of atropine, a cholinergic blocker. Atropine methylbromide (Sigma Chemical, St. Louis, MO) was injected 30 min before VMH lesioning and then at 10 mg/kg intraperitoneally twice a day at 8:00 AM and 8:00 PM.

Pair feeding. Pair-fed controls were used to discriminate the effects of VMH lesions, hepatic vagotomy, and administration of atropine. Food consumption in each group was measured by subtracting remaining pellets and spillage from initial weight values. The measurements were performed for 7 days. The results of food consumption are shown in Table 1. Animals with VMH lesions consumed more food (3 days, 2.2-fold increase; 7 days, 2.4-fold increase) than simulated-surgery controls. Hepatic vagotomy did not alter food consumption in the VMH-lesioned animals. However, the administration of atropine decreased food consumption less (3 days, 26% of controls; 7 days, 22% of controls) than the administration of saline to VMH-lesioned rats. There were no differences in food consumption between saline-treated VMH-lesioned rats and untreated VMH-lesioned rats. To exclude the effects of reduced food intake by atropine administration, other groups were pair fed with VMH-lesioned rats with atropine. One-third of the total volume consumed was given to each group at 8:00 AM, and the remaining two-thirds was given at 8:00 PM because delivery of several rations of food throughout the day conforms to the natural diurnal rhythm.

                              
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Table 1.   Food consumption of the study groups

TUNEL assay. TUNEL was carried out as previously described (24). Briefly, after fixing with 10% formalin buffer, sections were deparaffinized and hydrated. They were then treated with proteinase K, rinsed in purified water containing 3% H2O2 to inactivate endogenous peroxidase activity, and then rinsed with terminal deoxynucleotidyl transferase (TdT) buffer (100 mM potassium cacodylate, 2 mM CoCl2, and 0.2 mM dithiothreitol, pH 7.2) and incubated with TdT solution (0.3 equivalent U/µl TdT, 0.04 nmol/µl biotinylated uridine triphosphate). The sections were then reacted with peroxidase-labeled streptavidin for 30 min at room temperature. Positive reactions were visualized with 3-amino-9-ethylcarbazole mixed solution, and the nuclei were stained with hematoxylin. Treatment with DNase 1 (0.7 µg/ml potassium cacodylate buffer, pH 7.2) before the addition of TdT reaction solution was used as a positive control, and TUNEL was conducted by using solution without TdT as a negative control.

Measurement of caspase 3-like proteases in hepatocytes, Kupffer cells, and sinusoidal endothelial cells. Cells were isolated essentially according to Deaciuc et al. (15). VMH-lesioned rats or sham VMH-lesioned rats were anesthetized with pentobarbital sodium (Nembutal; 5 mg/100 g body wt ip), the abdominal cavity was opened, and the portal vein was exposed for cannulation. After 10 min of flow-through perfusion with Ca2+-free Hanks' bicarbonate buffer (25 mM) at 36.5°C and a flow rate of 40 ml/min, the perfusion system was switched to recirculation mode and the liver was perfused for another 4-5 min with 50 ml of Hanks' bicarbonate buffer containing Ca2+ (4 mM) and 25 mg of collagenase type IV (clostridiopeptidase A, EC 3.424.3 from Clostridium histolyticum; Sigma Chemical). The perfusion medium was gassed with O2-CO2 (19:1 vol/vol) throughout the entire perfusion period. At the end of perfusion, the liver was minced with scissors and the resulting mixture was filtered through a nylon mesh to remove undigested tissue. Centrifugation was performed at 80 g for 2 min at 20°C to separate parenchymal from nonparenchymal cells. Parenchymal cells were washed twice with Gey's buffer [containing 2% (wt/vol) dialyzed BSA] and finally resuspended in RPMI 1640 medium (25 mM HEPES, pH 7.4, supplemented with 2% BSA) at a density of 5 × 106 cells/ml. Kupffer cells and sinusoidal endothelial cells were isolated by centrifugal elutriation using a Beckman J2-21 centrifuge equipped with a JE-6B elutriation system (Spinco Division, Beckman Instruments, Palo Alto, CA). Kupffer cells and sinusoidal endothelial cells were washed twice and finally resuspended in RPMI 1640 at a density of 5 × 106 cells/ml. The viability of all cell types was determined by trypan blue exclusion and was found to be >= 94%. Cross-contamination of cell preparations never exceeded 6% for Kupffer cells and sinusoidal endothelial cells. The caspase 3-like activity was measured using a commercially available kit (Clontech Laboratories, Palo Alto, CA) according to the manufacturer's instructions. A total of 2 × 106 hepatocytes, Kupffer cells, or sinusoidal endothelial cells from VMH-lesioned rats or sham VMH-lesioned rats were lysed (200 µg protein), and a fluorescent substrate, DEVD-7-amino-4-trifluoromethylcoumarin (AFC; 50 µM final concn), was incubated with cell lysate at 37°C for 60 min. The fluorescence of cleaved substrates was measured using a spectrophotometer (Hitachi F2000; Hitachi, Tokyo, Japan) at an excitation wavelength of 400 nm and an emission wavelength of 505 nm. An AFC calibration curve was established by serial dilution of a pure AFC solution provided in the kit.

DNA fragmentation assay. High-molecular-weight hepatic DNA obtained on day 3 from sham VMH-lesioned (n = 6) and pair-fed VMH-lesioned (n = 6) rats was prepared with proteinase K and extracted by the phenol-chloroform method (8). DNA fragmentation was determined by the Staley method (58) using an ApoAlert LM-PCR ladder assay kit (Clontech Laboratories). Genomic DNA (0.5 µg) was mixed with 1 nmol each of 24-bp and 12-bp unphosphorylated oligonucleotides in 35 ml of T4 DNA ligation buffer (Clontech Laboratories). Oligonucleotides were annealed by heating at 55°C for 10 min, cooling to 10°C over 55 min, and then incubating at 10°C for 10 min. T4 DNA ligase (200 units; Clontech Laboratories) was added, and ligations were incubated at 16°C for 16 h. The 24-bp and 12-bp sequences of the blunt-end linkers used to amplify nucleosomal ladders were previously described (58). Ligated DNA (150 ng) was amplified by PCR in a 100-µl volume containing 124 pmol of 24-bp linker primer, 67 mM Tris · HCl, pH 8.8, 2 mM MgCl2, 16 mM (NH4)2SO4, 10 mM beta -mercaptoethanol, 100 µg/ml BSA, and dATP, dCTP, and dTTP (each 320 µM). The tubes were heated to 72°C for 3 min, 5 units of Taq polymerase (Clontech Laboratories) was added per 100 ml of reaction, and the 5' protruding ends of the ligated adapters were filled in by incubation at 72°C for an additional 5 min. Samples were amplified for 25 cycles of 1 min at 94°C and 3 min at 72°C. PCR products (15 µl) were analyzed by 1.2% agarose gel electrophoresis.

Electron microscopy. Liver tissue samples were prepared for electron microscopy by standard procedures, and the ultrathin sections were examined and photographed with a JEOL 1200 EX electron microscope.

Purification of mRNA and Northern blot analysis. Total cellular RNA was isolated from the livers of the VMH-lesioned and sham-lesioned rats by the guanidinium thiocyanate-phenol-chloroform method using TRIzol reagent (Life Technologies, Tokyo, Japan) according to the manufacturer's instructions. Briefly, tissue was homogenized in TRIzol reagent, extracted with a phenol-chloroform mixture, and centrifuged to recover the aqueous sample, which was then precipitated with isopropanol. The RNA precipitate was collected by centrifugation, washed, and reprecipitated. Total cellular RNA was collected by centrifugation and resuspended in water.

A rat Fas cDNA clone (38) was kindly provided by Prof. M. Yamamoto (National Defense Medical College, Tokorozawa, Japan). The subclone, which carries the full-length cDNA for rat Fas ligand in pBluescript, was kindly provided by Prof. S. Nagata (Osaka University Medical School, Suita, Japan) (59). Two oligonucleotide primers were designed based on the nucleotide sequence of the rat Fas antigen (38) and rat Fas ligand (59), and cRNA probe was synthesized with a T7 promoter (5'-TAATACGACTCACTA). The primer sequences were Fas sense primer, 5'-AGGAAAAGAACAATCCACC; antisense primer, 5'-TCTCACAGACTGCCTAGGCG; Fas ligand sense primer, 5'-TATATAAGCCAAAAAAGGTC; and antisense primer, 5'-ATGCAGCAGCCCGTGAATTA. PCR was performed for 34 cycles at 94°C for 45 s, at 55°C for 45 s, and at 72°C for 2 min. Digoxigenin (Dig)-labeled cRNA was synthesized by in vitro transcription of the PCR product using T7 RNA polymerase (Boehringer Mannheim, Mannheim, Germany). A human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cRNA (Clontech Laboratories) was used as an internal standard on all filters. The Fas, Fas ligand, and GAPDH cRNA probes were ~1.3, 0.9, and 1.0 in size, respectively.

RNA was separated on denaturing formaldehyde 1.0% agarose gels (10 µg RNA/lane) (56) and transferred and cross-linked to Hybond-N+ nylon membrane (Amersham Life Science, Little Chalfont, UK). Membranes were stored at 4°C until use (56).

Filters were hybridized with Dig-labeled rat Fas or Fas ligand cRNA probe at 65°C overnight in hybridization buffer [7% SDS, 500 mM Na phosphate (pH 7.0), 2% blocking reagent (Boehringer Mannheim), and 1 mM EDTA]. At the end of the incubation, the filter was washed three times at 65°C in 40 mM Na phosphate (pH 7.2) with 1% SDS for 20 min and once at 65°C in 0.2× sodium saline citrate (SSC; 1× SSC is 150 mM sodium chloride and 15 mM sodium citrate, pH 7.4) with 0.1% SDS. After washing, the filters were rinsed and then blocked for 1 h in Tris-NaCl buffer with 1% blocking solution. After blocking, filters were incubated with antidigoxigenin-alkaline phosphatase (AP) (1:10,000 vol/vol) in blocking solution for 30 s at room temperature and then washed three times. Filters were incubated in disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]- decan}-4-yl)phenyl phosphate (1:100 vol/vol; Boehringer Mannheim) in AP buffer for 15 min at 37°C. Filters were then exposed to XAR-5 film (Eastman Kodak, Tokyo, Japan), and signals were quantitated using the Macbeth image analysis system (Kollmorrgen, Newburgh, NY). The ratios of Fas and Fas ligand mRNA to GAPDH mRNA were used for statistical analysis.

Protein isolation and Western blot analysis. Livers were homogenized in 0.25 M saccharose solution. The protein content of the preparation was determined by the Smith method (57), using Protein dotMETRIC (Geno Technology, St. Louis, MO). For Western blot analysis, equal amounts of protein (100 µg) were separated by 15% SDS-PAGE according to the Laemmli method (39) before electrophoretic transfer to a polyvinylidene difluoride membrane (Immobilon; Millipore, Bedford, MA). The membrane was blocked for 1 h in PBS-5% skim milk and incubated for 2 h with 1:100,000 anti-Fas (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-Fas ligand (Santa Cruz Biotechnology) and then for 1 h with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Sigma Chemical) as the second antibody at room temperature. Bands were visualized with ECL Plus (Amersham Pharmacia Biotech, Little Chalfont, UK). Filters were exposed to XAR-5 film, and the signals were quantitated using the Macbeth image analyzing system.

Immunohistochemical analysis. Immunohistochemical analysis was performed as previously described (36, 37). The monoclonal antibodies used for immunohistochemical staining were anti-Fas (Pharmingen, San Diego, CA), anti-Fas ligand (Transduction Laboratories, Lexington, KY), and anti-proliferating cell nuclear antigen (PCNA) (DAKO, Glostrup, Denmark). The sample was deparaffinized in xylene, hydrated through decreasing concentrations of ethyl alcohol, and washed in distilled water. Endogenous peroxidase activity was reduced by incubating the samples for 20 min in methanol and 0.3% hydrogen peroxide. After being rinsed with water, the slides were placed in a glass dish filled with 10 mM sodium citrate buffer, pH 6.0. Tissue sections were boiled in a microwave oven (650 W) twice for 5 min each. After being washed in water, the sections were immersed in 5% normal swine serum (DAKO) to abolish nonspecific antibody binding. The slides were then incubated with anti-Fas, anti-Fas ligand, or anti-PCNA antibody overnight at 4°C, rinsed, and incubated with biotinylated anti-hamster, anti-mouse, or anti-goat immunoglobulins (Vector Laboratories, Burlingame, CA). Antibody complexes were visualized by the avidin-biotin-peroxidase method. The peroxidase color reaction was developed with Tris buffer (0.05 mM, pH 7.6) containing 3,3'-diaminobenzidine tetrahydrochloride (0.02%), hydrogen peroxide (0.006%), and sodium azide (0.01%) and counterstained with hematoxylin (Muto Chemical, Tokyo, Japan). More than 1,000 hepatocytes were examined at random. Hepatocytes with brown cytoplasm were considered positive for the protein stained.

Statistical analysis. All data are expressed as means ± SE. For statistical analysis, the data were first assessed by a two-way analysis and groups were compared by the Bonferroni method for multiple comparisons. A P value of <0.05 was considered significant.


    RESULTS
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MATERIALS AND METHODS
RESULTS
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Effect of VMH lesions on total body weight and liver weight. The body weights of the pair-fed animals with VMH lesions (241.1 ± 9.3 g at 3 days, 243.9 ± 11.8 g at 7 days) and animals with sham VMH lesions (238.9 ± 10.0 g at 3 days, 240 ± 9.6 g at 7 days) were not significantly different on days 3 and 7 after VMH lesioning. The liver weights of the pair-fed animals with VMH lesions and the sham-surgery animals were also not significantly different (0 days, 8.20 ± 0.23 vs. 8.28 ± 0.16 g; 3 days, 8.28 ± 0.24 vs. 8.27 ± 0.18 g; 7 days, 8.57 ± 0.33 vs. 8.60 ± 0.25 g).

Expression of Fas (APO-1/CD95) and Fas ligand mRNA in liver after VMH lesioning. The time course of changes in Fas and Fas ligand gene expression in the livers of pair-fed VMH lesioned rats was examined by Northern blotting (Fig. 1). Fas expression was markedly increased by day 1, and the increase persisted for up to 7 days, whereas Fas ligand expression increased on day 2 and persisted for up to 7 days. In the pair-fed VMH-lesioned rats, Fas and Fas ligand mRNA levels reached a maximum between day 3 and day 5 but had not decreased to initial levels by day 7. Quantitative evaluation of Fas and Fas ligand mRNA expression 3 days after VMH lesioning showed ~10-fold and 4-fold increases, respectively, over expression on day 0. In the liver of sham-operated rats, the basal level of Fas and Fas ligand expression was unaffected up to day 7.


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Fig. 1.   Time course of expression of Fas (A) and Fas ligand (B) mRNA in pair-fed ventromedial hypothalamus (VMH)-lesioned rats, hepatic-vagotomized VMH-lesioned rats, and atropine-treated VMH-lesioned rats. Total cellular RNA (10 µg) was blotted and hybridized with digoxigenin (Dig)-labeled Fas and Fas ligand probes. To exclude the effects of reduced food intake by atropine administration, other groups were pair fed with VMH-lesioned rats treated with atropine. The graph depicts the changes in levels of Fas (A) and Fas ligand (B) mRNA relative to pre-VMH lesions up to 7 days after lesioning. All values are expressed as means ± SE (n = 6). *P < 0.05 vs. baseline; Dagger P < 0.05 compared with animals on other days after VMH lesioning.

Expression of Fas (APO-1/CD95) and Fas ligand protein in liver after VMH lesioning. Western blot analyses identified a band with a molecular mass of ~45 kDa (Fig. 2), which is characteristic of the Fas protein in rodent cells (52, 53). Fas was expressed at 1 day after VMH lesioning but showed markedly increased expression in the liver 3-5 days after VMH lesioning. The anti-Fas ligand antibody detected a single band of ~31 kDa, which corresponded to Fas ligand (27). Fas ligand was not expressed until day 1 after VMH lesioning but was expressed on day 2. Fas ligand protein levels peaked on days 3 and 4 and then gradually decreased until day 7. Quantitative evaluation of Fas protein expression on day 3 showed an ~3-fold increase from day 1, whereas quantitative evaluation of Fas ligand protein expression on day 3 showed an ~1.2-fold increase from day 2. Fas and Fas ligand expression was not detected in the liver of sham-operated rats up to day 7.


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Fig. 2.   Time course of expression of Fas (A) and Fas ligand (B) protein in pair-fed VMH-lesioned rats, hepatic-vagotomized VMH-lesioned rats, and atropine-treated VMH-lesioned rats. Protein (100 µg) was blotted and examined by Western blotting analysis. The protein molecular masses are indicated on right. To exclude the effects of reduced food intake by atropine administration, other groups were pair fed with VMH-lesioned rats treated with atropine. The graph depicts the changes in protein levels of Fas (A) relative to days 1-7 after VMH lesions and in protein levels of Fas ligand (B) relative to days 2-7 after lesioning. All values are expressed as means ± SE (n = 6). *P < 0.05 vs. baseline; Dagger P < 0.05 compared with animals on other days after VMH lesioning.

The distribution of Fas immunoreactivity in the cytoplasm of cells was analyzed by the double-step avidin-biotin-peroxidase method with anti-avidin antibody in pair-fed VMH lesioned rats (Figs. 3c and 4C). Fas expression was identified in the cytoplasm of randomly distributed hepatocytes in the remaining liver parenchyma of pair-fed VMH lesioned rats. Although some cells stained more strongly than others, all identifiable staining was regarded as positive. One day after VMH lesioning, sections from the liver treated with anti-Fas antibody showed numerous positive cell nuclei in the cells of acinar zones 1-3. In pair-fed VMH-lesioned rats, the number of Fas-positive hepatocytes in acinar zones 1-3 of the lobule increased significantly, peaking in acinar zone 1 at 1-3 days and in acinar zones 2 and 3 at 3 days and decreasing thereafter. Moreover, the strongest Fas expression shifted from acinar zone 1 to zones 2 and 3 by day 7 after VMH lesioning. Sham VMH-lesioned rats showed no significant increases over the 7 days.


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Fig. 3.   a and b: Hepatocytes from sham VMH-lesioned (a) and VMH-lesioned (b) rats stained with hematoxylin-eosin at 3 days after VMH lesioning; magnification, ×200. Three days after VMH lesioning, typical fatty changes were detected in the liver tissue. However, we could not detect any classic apoptotic cells. c: Fas staining in hepatocytes in VMH-lesioned rats at 3 days; magnification, ×200. Arrows indicate Fas-positive cell cytoplasm. d: Fas ligand staining in hepatocytes in VMH-lesioned rats at 3 days; magnification, ×200. Arrows indicate Fas-ligand-positive hepatocyte cell cytoplasm. e: TUNEL staining in hepatocytes in VMH-lesioned rats at 3 days after VMH lesioning; magnification, ×250. Hepatocytes show apoptosis (arrows). f: Agarose gel electrophoresis; left, 100-bp DNA ladder (Life Technologies); right, DNA ladders were detected from rat liver 3 days after VMH lesioning. g and h: Electron micrographs of representative apoptotic hepatocytes from sham VMH-lesioned (g) and VMH-lesioned (h) rats at 3 days after VMH lesioning; magnification, ×7,000. An electron micrograph shows the presence of condensed nucleus and mitochondrial clustering in VMH-lesioned rats.



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Fig. 4.   Time course of proliferating cell nuclear antigen (PCNA; A), TUNEL (B), and Fas (C) positivity in hepatocytes in acinar zones 1-3 up to 7 days after VMH lesion formation. Values are means ± SE (n = 6). The original microscopic magnification was ×200. The hepatic acinus was defined according to Lamers et al. (40). More than 1,000 hepatocytes were examined at random. Hepatocytes with brown cytoplasm were considered positive. *P < 0.05 compared with acinar zone 1 of animals on other days after VMH lesioning; Dagger P < 0.05 compared with acinar zone 2 of animals on other days after VMH lesioning; §P < 0.05 compared with acinar zone 3 of animals on other days after VMH lesioning.

Fas ligand was not expressed in the liver of pair-fed VMH-lesioned rats on days 0 and 1. At days 2-7 after VMH lesioning, Fas ligand-positive cell membranes and cytoplasm of hepatocytes were randomly distributed in acinar zones 1-3 (Fig. 3d). Sham VMH-lesioned rats showed no expression of Fas ligand in liver tissue over the 7 days.

Cell proliferation and apoptosis in liver after VMH lesioning. VMH lesions induce an increase in the number of PCNA-positive hepatocytes (37). Changes in PCNA expression in hepatocytes up to 7 days after VMH lesioning were determined by immunohistochemistry. Proliferation of hepatocytes in acinar zones 1-3 began to increase on day 1 and reached a maximum at day 3 (Fig. 4A). The area of most intense proliferation progressively shifted from acinar zone 1 to zone 3 over several days. Apoptosis was identified as nuclear staining by the TUNEL assay. Apoptosis gradually shifted from acinar zone 1 to zone 3 between days 2 and 7 after VMH lesioning (Figs. 3e and 4B), whereas no TUNEL-positive cells were observed in the sham VMH-lesioned rats. The liver at 3 days after VMH lesioning demonstrated internucleosomal DNA fragmentation consistent with apoptosis (Fig. 3f), which was not evident in sham VMH-lesioned rats. Consistent with this, electron micrographs also showed the presence of condensed nucleus and mitochondrial clustering at 3 days after VMH lesioning (Fig. 3h) but not in sham VMH-lesioned rats (Fig. 3g). However, no apoptotic cells could be identified in hematoxylin and eosin-stained paraffin sections of liver after VMH lesioning according to Bursch et al. (12), although typical fatty changes were seen (Fig. 3b).

Activation of caspase 3-like proteases in liver after VMH lesioning. Caspase 3 (CRP32/Yama) functions in the apoptotic process as a key cysteine aspartase (1, 18, 51). We assessed apoptosis based on activation of caspase 3-like proteases detected by spectrofluorometric assay and determined whether the apoptotic response was due to cell apoptosis in the hepatocytes, Kupffer cells, and sinusoidal endothelial cells of the liver of VMH-lesioned rats and sham VMH-lesioned rats (Fig. 5). Caspase 3-like activity was assayed based on its ability to cleave a fluorescent substrate (DEVD-AFC). The caspase 3-like activity of hepatocytes in the liver was increased by VMH lesioning at day 2, peaked at days 3 and 4, and then decreased. VMH lesioning of pair-fed animals induced an 11.0-fold increase in caspase 3-like activity in hepatocytes. However, increases in caspase 3-like activity were not detected in Kupffer cells and sinusoidal endothelial cells in the liver. Moreover, sham VMH lesioning did not increase caspase 3-like activity in the liver.


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Fig. 5.   Activation of caspase 3-like proteases by VMH lesions in hepatocytes (A), Kupffer cells (B), and sinusoidal cells (C) up to 7 days after VMH lesion formation. Values are means ± SE (n = 6). *P < 0.05 vs. baseline; Dagger P < 0.05 compared with animals on other days after VMH lesioning.

Effect of hepatic vagotomy and administration of cholinergic blocker atropine on weight, Fas and Fas ligand expression, and apoptosis in liver after VMH lesioning. Liver weight in pair-fed VMH-lesioned rats was not affected by the administration of atropine (day 0, 8.22 ± 0.20 g; day 3, 7.89 ± 0.28 g; day 7, 8.31 ± 0.27 g) or hepatic vagotomy (day 0, 8.17 ± 0.23 g; day 3, 8.09 ± 0.17 g; day 7, 8.49 ± 0.31 g) compared with pair-fed sham VMH-lesioned rats (day 0, 8.20 ± 0.23 g; day 3, 8.28 ± 0.24 g; day 7, 8.57 ± 0.33 g). Hepatic vagotomy or administration of atropine in VMH-lesioned rats did not increase the levels of Fas or Fas ligand mRNA and protein expression (Figs. 1 and 2). Both of these treatments prevented the appearance of apoptosis shown by electron microscopy and TUNEL assay and the increase in caspase 3-like activity detected in hepatocytes in the liver in the pair-fed VMH-lesioned rats (Fig. 5).

Serum GOT, GPT, and LDH levels after VMH lesioning. Pair-feeding methods were used, and levels of the serum enzymes GOT, GPT, and LDH were determined on days 0, 1, 3, 5, and 7 after VMH lesioning (Table 2). It was previously reported that the administration of atropine (10 mg/kg ip) did not affect the levels of GOT, GPT, or LDH in normal control rats (4). We therefore determined the levels of GOT, GPT, and LDH in atropine-treated VMH-lesioned rats. In the pair-fed VMH-lesioned rats, GOT and GPT increased significantly at day 3 and then continued to increase until day 7 after VMH lesioning. In hepatic-vagotomized VMH-lesioned and atropine-treated VMH-lesioned rats, GOT and GPT did not increase until day 7. In the sham VMH-lesioned rats, serum levels of these transaminases showed no change, which suggests that hepatocyte function in the sham VMH-lesioned rat is normal. No significant increases in LDH were observed up to day 7 in any of the groups.

                              
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Table 2.   Time course of biochemical parameters in liver after VMH lesion formation


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The functions of Fas and Fas ligand in the liver have been a recent focus of research interest. In the present study, VMH lesions led to significant increases in Fas mRNA and protein expression from day 1 and in Fas ligand expression from day 2, with both reaching a maximum on day 3. TUNEL assay showed that apoptotic cells were scattered throughout the liver tissue of the VMH-lesioned rats. Therefore, we speculate that the Fas-Fas ligand complex may play an important role in the VMH-lesioned rat liver.

We detected an increase of caspase 3-like activity in the hepatocytes and TUNEL-positive cells and DNA ladders in the liver after VMH lesioning, but no classic apoptotic cells were seen on hematoxylin-eosin staining. Willingham (67) suggested that apoptotic cells may not "live" long in vivo. Dying cells in vivo are removed by phagocytic cells such as Kupffer cells, a process that emphasizes the usefulness of the apoptotic mechanism for normal tissue remodeling. However, the removal of cells can occur with different efficiencies in different tissues. Unlike cell culture, in which the cell remnants remain undisturbed, evidence of apoptosis in vivo can be cleared rapidly in tissues with active phagocytes. Consistent with this, it was reported that the degeneration of germ cells in rat testis involved apoptosis (7, 48). However, Blanco-Rodriguez and Martinez-Garcia (7) reported that the probability of cutting an apoptotic cell is increased in paraffin sections, whereas it was more difficult to appreciate the early morphological signs and the affected cell types with this method. This suggests that apoptotic cells may not be detected by hematoxylin-eosin staining of tissues. Moreover, we also speculate that it is difficult to detect the classic apoptotic cells by the remarkable fatty changes in the liver after VMH lesioning.

The hypothalamus plays a vital role in the integration of neurohumoral information and possesses autonomic centers that are connected to the viscera via the autonomic nervous system. In studies using the pair-feeding method, lesions of the VMH produced hepatocyte proliferation (34, 37) and vagal firing produced by VMH lesions stimulated DNA synthesis (32, 35). It was determined whether there is an injurious stimulus that leads to apoptosis and then is later followed by reparative proliferation or there is no injurious stimulus, as in fetal development (62, 68), and the apoptosis occurs late in the course as part of remodeling. Therefore, we compared the timing of these two phenomena by simultaneously measuring PCNA- and TUNEL-positive hepatocytes. Mitotic changes occurred from acinar zone 1 to zone 3 in hepatocytes and were then followed by apoptosis. This suggests that VMH lesion-mediated hepatocyte proliferation progresses via coordinated cell proliferation and apoptosis.

Although Fas is a cell surface protein, it was strongly expressed in the cytoplasm of hepatocytes in the VMH-lesioned rats. Several other studies support this finding. Nerve growth factor receptor, a member of the TNF receptor family to which Fas also belongs, has also been detected in the cytoplasm of Schwann cells (61). When Fas cDNA was transfected into COS cells, Fas expression was detected in the cytoplasm as well as the membrane by indirect immunofluorescence analysis (14). Moreover, Fas expression was detected immunohistochemically in the cytoplasm of hepatocytes in liver samples from patients with hepatitis C virus infection (29). In addition, in samples from hepatitis B virus patients, Fas was mainly expressed in the cytoplasm of hepatocytes (47). These data support our finding of Fas expression mainly localized to the cytoplasm of hepatocytes.

In the present study, Fas ligand-positive cell membranes and cytoplasm of hepatocytes were randomly distributed in acinar zones 1-3. Although Fas ligand is functional on activated T cells, it is also expressed in several nonlymphoid cells and tissues including epithelial cells, macrophages, and dendritic cells (6, 20, 21, 25, 43, 60). The presence of Fas ligand in nonlymphoid tissue has been shown to delete reactive lymphoid cells during viral infections and is responsible for protecting immune-privileged sites from cellular immune-mediated damage (25, 26). Moreover, Bonfoco et al. (10) reported that nonlymphoid T cells minimized potential tissue damage as a consequence of inflammation. These data are consistent with our finding of Fas ligand in the cytoplasm of hepatocytes. We speculate that the expression of Fas ligand in the cytoplasm of hepatocytes may be related to protection from some specific cellular damage in VMH-lesioned rats.

It would be interesting to determine whether vagal firing after VMH lesioning directly induces Fas ligand expression in hepatocytes. Fas ligand expression was originally thought to be restricted to activated T cells and natural killer cells (23). Because vagal stimulation increases the release of lymphocytes from the thymus into the circulation (3), vagal hyperactivity associated with VMH lesions may induce this release of lymphocytes from the thymus. Fas ligand is not expressed on resting T cells but is rapidly induced by activation (65). We therefore speculate that vagal firing after VMH lesioning may initially induce Fas expression and then Fas ligand expression in lymphocytes and/or hepatocytes by the activation of Fas expression, based on the time lag between the expression of mRNA and protein of Fas and Fas ligand in the liver after VMH lesioning.

VMH lesions induce hepatic vagal firing (70). In this study, hepatic vagotomy and administration of atropine prevented the increase in Fas and Fas ligand expression and in apoptosis that occurs after formation of VMH lesions. Therefore, we speculate that VMH lesions may trigger the induction of Fas/Fas ligand-mediated apoptosis through the cholinergic system of the hepatic vagus nerve. The cholinergic system modulates growth and proliferation in a number of cells, including gastric cells (49) and hepatocytes after partial hepatectomy (31). Consistent with this, LeSage et al. (41) recently reported that the cholinergic system moderates growth and apoptosis of cholangiocytes in bile duct-ligated rats. Moreover, very recently, Frucht et al. (22) reported that the cholinergic system mediates human colon cancer cell proliferation. However, we cannot discount the possibility that VMH lesions may induce apoptosis via other mechanisms such as TNF-alpha and free radical injury that might be induced by ischemia from vagally induced changes in vascular tone. Lynch et al. (44) reported that the TNF-alpha concentrations in VMH-lesioned rats and sham-lesioned rats were typically below the level of detection (<15 pg/ml) for their assay. This indicated that there is little relationship between TNF-alpha and the appearance of apoptosis in the liver of VMH-lesioned rats. In addition, if free radical injury may mediate the appearance of apoptosis in VMH-lesioned rats, TUNEL-positive cells may be evident in the liver within 1 day after VMH lesioning, because vagal hyperactivity was induced immediately after VMH lesioning (70). However, we did not detect any TUNEL-positive cells in the liver at day 1 after VMH lesioning. Future experiments in a Fas knockout mouse would provide conclusive for the specific importance of the Fas-Fas ligand role in this phenomenon.

The mechanism of reduction in Fas/Fas ligand-mediated apoptosis at 7 days after VMH lesioning is also unknown. Other mechanisms may suppress Fas/Fas ligand-mediated apoptosis while maintaining the increased vagal firing rate (70). Apoptosis may be mediated by several inhibitory proteins, including bcl-2 and neuronal apoptosis inhibitory protein (55, 64). Further studies are needed to examine whether these inhibitory proteins also moderate VMH lesion-induced apoptosis.

At days 3-7, the serum levels of GOT and GPT were increased in VMH-lesioned rats. Consistent with this, VMH lesions are known to cause fatty changes in the liver with hyperphagia (11). Therefore, these fatty changes might have affected the enzyme levels of GOT and GPT. However, it was reported that a 28-day high-fat, hypercaloric diet induced an increase in liver weight and fatty liver change but did not change GOT and GPT levels (2). This indicated that fatty changes do always not induce changes in these transaminases. Many investigators have suggested that the Fas-Fas ligand system is a major mechanism in hepatic cytotoxicity (23, 45, 46). In the present study, hepatic vagotomy and administration of atropine abrogated the increase in GOT and GPT at days 3, 5, and 7. Therefore, we speculate that the expression of Fas and Fas ligand by vagal firing through the cholinergic system after VMH lesioning may induce hepatic cytotoxicity.

In conclusion, the present study suggests that Fas/Fas ligand-mediated apoptosis in the liver after VMH lesioning may be induced through the cholinergic system of the hepatic vagus system.


    ACKNOWLEDGEMENTS

We thank Y. Nagoshi for technical advice and assistance.


    FOOTNOTES

This study was supported in part by Grant 09670562 from the Ministry of Education of Japan.

Address for reprint requests and other correspondence: T. Kiba, Third Dept. of Internal Medicine, Yokohama City Univ. School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan (E-mail: takkiba{at}earthlink.net).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 7 July 2000; accepted in final form 13 December 2000.


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Am J Physiol Gastrointest Liver Physiol 280(5):G958-G967
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