Ornithine

ABSTRACT. Background: The morbidity and mortality that accompany fatty liver may occur as a result of increased apoptosis of hepatocytes and decreased liver regeneration. We determined the effects of a high-fiber diet on hepatocyte apoptosis and liver regeneration after partial hepatectomy in rats with fatty liver. Methods: Fatty liver was induced in male Wistar rats weighing around 200 g through feeding of a high-fat diet for 4 weeks. The rats were then randomly assigned to 3 groups that received a high-fat diet, a normal diet, or a high-fiber diet for another 4 weeks. Partial hepatectomy (around 70%) was performed, and rats were killed 6, 24, 48, or 72 hours after hepatectomy. We then measured (1) the ratio of remnant liver weight to body weight and assessed the histology of the remnant liver as indicators of fatty liver, (2) caspase-3 activity and in situ cell death detection of DNA fragmentation as indicators of apoptosis, and (3) 5-bromo-2-deoxyuridine (BrdU) activity and (4) ornithine decarboxylase (ODC) contents in remnant livers as markers of regeneration. Results: We found that (1) a high-fat diet for 4 weeks can markedly induce fatty liver, (2) apoptosis of hepatocytes is greater in fatty liver than in normal liver (98 ± 19 us 36 ± 7) at 6 hours after partial hepatectomy (p

Fatty liver is one of the most common hepatic disorders in humans. Nonalcoholic fatty liver disease can be caused by obesity, diabetes mellitus, or an unbalanced diet containing too much energy intake1 or a diet that is high in fat and low in fiber. Although fatty liver is often considered to be a benign condition, steatosis is associated with more frequent mortality and morbidity after liver resection.3 Although the liver has good regenerative ability, hepatic failure can still occur after massive hepatic injury or hepatectomy, especially in fatty liver. The regeneration capacity of the mammalian liver in response to numerous stimuli has been well documented.4 Most studies have focused on the initiation, regulation, metabolic changes, and termination of liver regeneration.5 Several factors, such as hormones, growth factors, nutrition components, immune response, and pharmacologie agents, have been proven to directly or indirectly affect liver regeneration.6,7 However, the effects of fatty liver on regenerative capacity after major liver-tissue loss remain controversial.

Picard et al8 reported that liver regeneration is reduced in rats with fatty liver and that the reduced regeneration may not be due to steatosis itself but to other factors, such as leptin receptor dysfunction. Hussein et al9 found that liver regeneration is not altered in patients with nonalcoholic steatohepatitis and suggested that the delayed postoperative liver failure in these patients may be related to other mechanisms. However, fatty liver is assumed to be an important cause of morbidity in humans and is linked to impaired liver regeneration after liver injury, although the mechanism for this impaired regeneration remains unclear.10,11

Apoptosis is a common form of cell death that occurs in healthy adult tissue under both physiologic and pathologic conditions. Apoptosis and cell proliferation are complementary and account for the maintenance, growth, or involution of a tissue. The regulation of apoptosis is believed to play an important role in hepatic cell regeneration; however, only a few studies of apoptosis and liver regeneration in fatty liver have been reported.12 Yang et al13 studied the vulnerability of fatty liver to endotoxin-induced damage and found that more hepatocyte apoptosis occurred at both early and late stages after liver injury. Clarke1 reported that polyunsaturated fatty acids reduce the risk of enhanced hepatocyte apoptosis due to the excessive cellular lipid accumulation in a fatty liver. However, the role of fatty liver in apoptosis and liver regeneration remains unclear.

Musso et al14 studied patients with nonalcoholic steatohepatitis and found that their dietary intake was rich in saturated fat and cholesterol and poor in polyunsaturated fat, fiber, and the antioxidant vitamins C and E. The insulin sensitivity index in the patients was significantly lower than normal, and postprandial total and very-low-density lipoprotein triglycerides were higher. Therefore, Musso and colleagues14 concluded that dietary habits may promote steatohepatitis directly by modulating hepatic triglyceride accumulation and antioxidant activity and indirectly by affecting insulin sensitivity and postprandial triglyceride metabolism. Barber et al15 experimented in rats and proved that fiber supplementation can maintain a better small bowel mass and, thus, prevent hepatic steatosis. It is agreed that intake of a high-fiber diet is one way to decrease the prevalence of fatty liver. The main purpose of the present study, therefore, was to investigate the effects of a high-fiber diet on hepatocyte apoptosis and liver regeneration in fatty liver after partial hepatectomy.

MATERIALS AND METHODS

Experimental Protocol

Male Wistar rats (purchased from Charles River, Osaka, Japan) weighing about 200 g were used. The rats were randomly assigned to the following groups: (1) control group (C), which was fed a general diet (AIN-93G) for 4 weeks and for 8 weeks; (2) high-fat group (HF), which was fed a high-fat diet for 4 weeks and for 8 weeks; (3) high-fat to normal diet group (FN), which was fed a high-fat diet for 4 weeks and then the AIN-93G diet for 4 weeks; and (4) high-fat to high-fiber diet group (Ff), which was fed a high-fat diet for 4 weeks and then a high-fiber diet (containing watersoluble guar gum) for 4 weeks. The composition of the various diets is shown in Table I. The rats were killed at 4 or 8 weeks for the evaluation of fatty liver.

To measure the effects of fatty liver on hepatocyte apoptosis and liver regeneration, we performed a partial hepatectomy on rats in groups C, HF, FN, and Ff at the end of the 8-week experimental period. Each group of rats was then divided into 4 subgroups (8 rats in each) that were killed 6, 24, 48, or 72 hours after partial hepatectomy. The remnant livers were removed immediately, and indicators of apoptosis and liver regeneration were measured.

Surgical Procedures

All rats were anesthetized by means of intraperitoneal pentobarbital injection (40 mg/kg). A midline laparotomy was performed. Partial hepatectomy was then carried out by means of aseptic extirpation of the median and left lateral lobes according to the procedure of Higgins and Anderson.16 The removed liver samples were weighed immediately. All surgeries were performed between 8 AM and 11 AM only to reduce the influence of diurnal variations.

Measurements

(a) To evaluate fatty liver, the liver was weighed immediately after laparotomy, and the ratio of remnant liver weight to body weight was calculated. Liver color and its edge were observed. Then, the liver was cut into small pieces for histopathologic examination. The liver pieces were fixed in 10% neutral formalin, embedded in paraffin, sectioned, and stained with hematoxylin-eosin for microscopic observation.

(b) Hepatocyte apoptosis was assessed by measuring caspase-3 activity and by using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNED staining method. To measure caspase-3 activity, the remnant liver was homogenized in 25 mmol/L Tris-HCl buffer (pH 7.5) containing 1 mmol/L EGTA, 5 mmol/L MgCl^sub 2^, and 0.5% Triton X-100. After centrifugation at 14,000 × g, the diluted supernatant fluid was assayed for caspase-3 activity with 50 µmol/L of synthetic fluorogenic substrate, Ac-DEVD-α-(4-methyl-coumaryl-7-amide) (Peptide Institute, Osaka, Japan).17 Fluorescence intensity was calibrated with standard concentrations of 7-amino-4-methyl-coumarin, and caspase-3 activity was expressed in pmol per minute per mg of protein. Protein concentrations in the supernatant fluid were assayed with a bicinchoninic acid protein assay kit (Pierce, Rockford, IL).

For the TUNEL method, the remnant liver specimens were fixed in 4% phosphate-buffered paraformaldehyde, kept in this fixative for a few hours, and then embedded in paraffin. Four-micrometer-thick sections were collected on poly-L-lysine-coated glass slides. The nuclear DNA fragmentation of apoptotic hepatocytes was labeled in situ as follows. sections were deparaffinized with xylene and rehydrated with progressively decreasing concentrations of alcohol, followed by phosphate-buffered saline (PBS). Then, each section was treated with 20 µg/mL proteinase K (P2308, Sigma, St. Louis, MO) in 0.1 mol/L Tris-HCl buffer (pH 7.4) for 15 minutes. After the sections were rinsed with PBS, endogenous peroxidase activity was blocked with 3% H^sub 2^O^sub 2^ for 5 minutes. sections were rinsed again with PBS and incubated with 0.5 U/µL terminal deoxynucleotidyl transferase (Boehringer Mannheim, Mannheim, Germany) and 0.05 nmol/µL biotinylated deoxyuridine triphosphate in terminal deoxynucleotidyl transferase buffer (Boehringer Mannheim) for 60 minutes in a humidified chamber at 37°C. Each slide was then observed with a fluorescence microscope under a 200× power field to check staining quality and for acquisition of images (Nikon, Tokyo, Japan). For each rat, liver sections were analyzed by counting cells in 10 different fields chosen at random. After image acquisition, the average numbers of fluorescent apoptotic nuclei were counted by using pattern-recognition software (Bioscan Optimas, Optimas Corporation, Edmonds, WA).

(c) The 5-bromo-2-deoxyuridine (BrdU) -incorporated index and ornithine decarboxylase (ODC) contents were used as indicators of liver regeneration. For measurement of the BrdU-incorporated index, the remnant livers were removed and weighed immediately after resection, and the ratio of remnant liver weight to body was calculated. The remnant livers were then cut into small pieces that were fixed in 10% neutral formalin, embedded in paraffin, and sectioned. BrdU-incorporated hepatocytes were detected by use of an immunocytochemical system18 for monitoring cell proliferation with a monoclonal anti-BrdU cell proliferation kit (Code RPN20, Amersham International PIc., Amersham, UK). The BrdU-incorporated index was calculated by counting the numbers of BrdU-incorporated hepatocytes in 10 randomly selected fields under a magnification of 200×.

ODC contents were measured with an enzymelinked immunosorbent assay kit (ODC Ab-1, Clone MP16-2, # MS-464-PO, Purified, Neomarkers, CA). Briefly, the remnant livers were cut into pieces that were homogenized and then centrifuged at 105,000 × g for 1 hour at 4°C in an ultracentrifuge (Beckman model L5-75, Lorton, VA). The supernatant fractions were obtained, and ODC activity was measured by the method described by Schipper et al.19

Statistical Analysis

All experimental data are expressed as means ± SD. The significant difference among all groups was analyzed by using one-way ANOVA. Ap value of less than .05 was considered statistically significant.

RESULTS

All rats survived well during the experimental periods. By gross inspection at the end of 4 weeks, the livers from rats in the HF group looked larger, greasy, and more yellowish-brown and had a more rounded liver edge than did the livers from rats in the control group. The liver color and edge were somewhat improved in the FN group and were restored significantly in the Ff group compared with the HF group.

Changes in the ratio of liver weight to body weight are shown in Figure 1. The preoperative ratio was around 4.10%. The ratio in the HF group increased to 4.48% at the end of 4 weeks, which was significantly higher than that in the control group. The tendency for the ratio to increase persisted up to 4.58% at the end of 8 weeks in the HF group. In contrast, the ratio was 4.40% at 8 weeks in the FN group and markedly decreased to 4.32% in the Ff group, which was significantly lower than in the HF group.

Histologic pictures of livers from all groups are shown in Figure 2. According to Brunt's grading system,20 the remnant liver of group HF rats was a grade 3 fatty liver with diffuse, mixed microvesicular steatosis and predominantly marked zone 3 ballooning. In the FN group, the fatty liver improved to a grade 2, with scattered acute and chronic inflammations, polymorphs at zone 3 ballooning, and perisinusoidal fibrosis. In the Ff group, the fatty liver recovered to a grade 1, mild fatty liver by the end of the experimental period.

The changes in the remnant liver weight/body weight ratio in each group are shown in Figure 3. The ratio for the control rats was 1.4% ± 0.13% before and 1.52% ± 0.14%, 2.20% ± 0.23%, 2.77% ± 0.29%, and 3.73% ± 0.35% at 6 hours, 24 hours, 48 hours, and 72 hours after partial hepatectomy, respectively. The experimental rats showed a parallel, increasing curve. However, ratios were significantly lower in the HF group than in the control group at 24 hours, 48 hours, and 72 hours after partial hepatectomy (p

The caspase-3 activity of the remnant liver in each group is shown in Table II. In the control group, caspase-3 activity increased to nearly 3 times the value before partial hepatectomy (20 ± 4 pmol/min/mg protein) at 6 hours after partial hepatectomy (58 ± 8 pmol/min/mg protein). It then gradually recovered at 24 hours (30 ± 5 pmol/min/mg protein) and was near normal at 48 hours (24 ± 5 pmol/min/mg protein) and 72 hours (22 ± 5 pmol/min/mg protein). Caspase-3 activity was significantly higher in the HF group than in the control group before (84 ± 16 pmol/min/mg protein) and at 6-72 hours after partial hepatectomy (p

In the control rats, the number of apoptotic hepatocytes increased significantly at 6 hours and then recovered gradually at 24 hours after partial hepatectomy (Table III). The number was significantly higher in the HF group (60 ± 11) than in the control group (15 ± 3) before hepatectomy (p

The changes in BrdU incorporation in hepatocytes in the remnant liver are shown in Table IV. The numbers were scanty in all groups preoperatively but increased markedly to 10 ± 2, 30 ± 5, 20 ± 4, and 10 ± 2 at 6, 24, 48, and 72 hours after partial hepatectomy in the control group. The number of BrdU-incorporated hepatocytes also increased after partial hepatectomy in the HF, FN, and Ff rats. However, the increases were significantly lower in the HF and FN groups at 24, 48, and 72 hours after partial hepatectomy than in the control group (p

The changes in ODC contents in the remnant liver are shown in Table V. In the control group, ODC contents increased significantly at 6, 24, 48, and 72 hours after partial hepatectomy. The value at 6 hours was almost 2 and a half times the preoperative value (656 ± 46 µg/g liver us 255 ± 26 µg/g liver). ODC contents in the HF, FN, and Ff groups also increased after partial hepatectomy. However, the increments were significantly less at 6 and 24 hours in the HF and FN rats, and at 48 hours in the HF rats, than in the control rats. ODC contents in the Ff group were not significantly different from those in the control group but were significantly higher than those in the HF group at 6 hours (650 ± 60 µg/g liver vs 444 ± 45 µg/g liver) and 24 hours (534 ± 49 µg/g liver vs 390 ± 42 µg/g liver) after partial hepatectomy.

DISCUSSION

Much is known about the mechanisms that mediate the pathogenesis of alcoholic fatty liver disease. However, research on the pathogenesis of nonalcoholic fatty liver disease is still in its infancy.21 In humans, nonalcoholic fatty liver disease is mainly associated with unbalanced diets, obesity,22 diabetes mellitus,10 and less commonly with drug effects or malabsorption-malnutrition syndrome.23 Fatty liver can be induced in rats fed a high-protein, high-energy diet24 or a high-fat diet.21 In the present study, the livers of rats fed a high-fat diet were greasy with a more rounded edge and a higher ratio of liver weight to body weight. Diffuse hepatic steatosis was also proved by his'topathologic observation.

Lipid accumulation in the liver can be caused by the development of insulin resistance.25,26 Increasing evidence suggests that central adiposity27 and fatty liver28 are important features of insulin resistance syndrome. Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors acting as transcription factors whose agonists can improve insulin action in peripheral tissue, attenuate hyperinsulinemia, and lower circulating lipid concentrations.26'29 Additionally, PPAR α/γ agonists can counteract fatty liver, hepatic insulin resistance, and visceral adiposity. The possible mechanism by which a high-fat diet induces the accumulation of triglyceride in the liver and leads to fatty liver may be through the PPAR α/γ pathway and its effects on insulin resistance.26 It is important to investigate how to prevent or reverse fatty liver because of the evidence that surgical procedures cause intestinal stasis and secondary bacterial overgrowth, which accelerate the progression of fatty liver into steatohepatitis.21 Steatohepatitis is believed to be an intermediate stage in the progression from fatty liver to cirrhosis because the probability of developing an advanced hepatic fibrosis is significantly greater in individuals with steatohepatitis than in those with simple fatty livers.30

Although gene regulation, such as overexpression of tumor necrosis factor α and its mRNA p55 receptor, has been proven to be involved in the pathogenesis of nonalcoholic fatty liver,31 it is still considered possible to reverse fatty liver. Petersen et al32 reported that leptin may play an important role in reversing severe hepatic and muscle insulin resistance and the associated hepatic steatosis. Barber et al15 studied rats that were fed a defined-formula diet which provided sufficient calories for growth primarily as simple sugars and small peptides, and found that the defined diet resulted in a significant loss of small-bowel mass and jejunal mucosal thickness and hepatomegaly compared with animals pair-fed a standard rat feed. Supplementation of the defined formula diet with fiber could prevent this loss of bowel mass and bacterial translocation. We found that hepatic steatosis, which can be induced by a high-fat diet, can be markedly reversed by a high-fiber diet for 4 weeks. This may be because the high-fiber diet can prevent bowel atrophy and thus bacterial translocation and then prevent hepatic steatosis. Another pathway through insulin responsiveness may also be considered. Gerloff2 studied the feeding management of dairy cows and suggested that a high-fiber diet can prevent subclinical rumen acidosis and ketosis and a decrease in serum nonesterified fatty acid concentrations, thus preventing fatty liver through the insulin responsiveness pathway. However, the true mechanism by which a high-fiber diet improves fatty liver is still unclear.

Apoptosis and cell proliferation are complementary and account for the maintenance, growth, or involution of a tissue.12 Baroni et al33 reported that apoptosis and mitosis were increased 4- and 5-fold, respectively, in chronic ethanol-treated rats, which suggests a physiologic equilibrium between the 2 phenomena after exposure to ethanol. In the present study, we proved that apoptosis coexists with liver regeneration during the early period after partial hepatectomy. Furthermore, we found that the apoptosis of hepatocytes increased and liver regeneration was impaired more in a fatty liver than in a normal liver after partial hepatectomy.

The mechanism for the increase in apoptosis and impairment of regeneration in fatty liver is still unclear. Immunologie response may play a role.34 Fatty liver frequently occurs in obese patients undergoing jejunoileal bypass surgery,36 a procedure that leads to portal blood endotoxemia with enhanced hepatic tumor necrosis factor α (TNFα) and polymorphonuclear cell production. TNFα can induce the hepatic synthesis of other cytokines, such as interleukin 8, a neutrophil chemokine factor. Polymorphonuclear cells can cause cell membrane peroxidation damage and possible cell apoptosis.36 Susca et al37 showed that patients with fatty liver have higher serum triglyceride concentrations and lower γ-globulins. Additionally, T lymphocytes outnumber polymorphonuclear cells in steatohepatitis, with a larger number of CD 8 lymphocytes but a comparable number of granulocytes. This phenomenon results in a changed granulocyte/Tcell ratio, which may make these cells more easily enhanced. Apoptosis and piecemeal necrosis may then be increased. Cell-mediated immunity, including γ-interferon, natural killer cells and macrophages, and several lymphokines and extrathymic T-cells, has been found to play an important role during liver regeneration.6,38,39 The immunologic response may regulate or even inhibit liver regeneration after partial hepatectomy. The immunologic response occurring in fatty liver may also be the factor that impairs regenerative capacity. We assume that whenever the steatosis of hepatocytes is reversed, immunologie response will be recovered and so too the apoptosis of hepatocytes and regeneration capacity of the posthepatectomized remnant liver.

ODC is the rate-limiting enzyme in polyamine synthesis. Increased polyamine synthesis is thought to play a critical role in stimulating hepatic regeneration after partial hepatectomy.19 On the other hand, the regeneration of hepatocytes involves an increase in DNA synthesis for mitosis, which requires DNA replication and an induction of hepatic BrdU incorporation activity in a manner analogous to the use of thymidine.40 The mechanism and timing by which ODC and BrdU affect liver regeneration differ. McGowan and Fausto40 found that ODC activity starts to increase 412 hours after partial hepatectomy. This is much earlier than the incorporation of thymidine kinase and BrdU into DNA because a cellular depletion of polyamines occurs during the early posthepatectomy stage and basal concentrations of polyamines are essential for DNA synthesis. BrdU measurement can be a sensitive method of detecting DNA replication in a manner analogous to the use of thymidine and shows maximum changes 36-40 hours after partial hepatectomy because of the direct correlation with proliferative capacity.41 Thus, these 2 factors are used as indices of liver regeneration: ODC during the early phase and BrdU measurement during the regenerating stage after partial hepatectomy.

Data concerning the effects of fatty liver on regeneration capacity and hepatocyte apoptoeis are still sparse. Yang et al42 showed that liver mass is maintained at a relatively constant value in 0b/0b C57BL-6J mice with fatty livers because the rates of hepatocyte proliferation and apoptosis increase similarly. This difference between their findings and ours may be because of the difference in animal models: Wistar rats fed a high-fat diet and with partial hepatectomy us genetically obese, leptin-deficient 0b/0b mice without partial hepatectomy. Further study of the immunologic changes and the role of leptin and insulin resistance is needed to clarify the mechanism of the effects of a high-fiber diet on hepatocyte apoptosis and liver regeneration in fatty liver.

ACKNOWLEDGMENT

The study was supported by a National Science Council grant (NSC-90-2314-B-002-321).

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Hong-Shiee Lai, MD, PhD*; Wen-His Lin, MD*; Pey-Rong Chen, RD, PhD[dagger]; Hsiu-Chuan Wu, BS*; Po-Huang Lee, MD, PhD*; and Wei-Jao Chen, MD, MPH, DMSc*

From the * Department of Surgery and the [dagger] Department of Dietetics, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan

Received for publication June 17, 2004.

Accepted for publication June 14, 2005.

Correspondence: Hong-Shiee Lai, MD, PhD, Department of Surgery, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei, Taiwan. Electronic mail may be sent to hslai@ha.mc.ntu. edu.tw.

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