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Influence of high cholesterol diet and pravastatin sodium on the initiation of liver regeneration in rats after partial hepatectomy
BASIC NUTRITIONAL INVESTIGATION
Influence of High Cholesterol Diet and Pravastatin Sodium on the Initiation of Liver Regeneration in Rats After Partial Hepatectomy Helena Zivna, MD, PhD, Pavel Zivny, MD, PhD, Vladimir Palicka, MD, PhD, and Eva Simakova, MD From the Institute of Physiology, the Institute of Clinical Biochemistry and Diagnostics, and the Department of Pathology, Charles University, University Hospital, Hradec Kralove, Czech Republic OBJECTIVES: Liver regeneration is influenced by cholesterol and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA-reductase). HMG-CoA-reductase is a key enzyme for cholesterol synthesis. Recent studies have shown that inhibitors of HMG-CoA-reductase improve liver functions after 67% partial hepatectomy (PH). METHODS: Male Wistar rats (W) and Prague hereditary hypercholesterolemic rats (PHHC) were used. Aqua pro injectione (AI) or pravastatin (prava; 1 mg/kg) was administered orally once daily. Group 1: W, standard diet (SD) ⫹ AI; group 2: W, SD ⫹ prava; group 3: W, cholesterol-enriched diet (chol) ⫹ AI; group 4: PHHC, chol ⫹ AI; group 5: PHHC, chol ⫹ prava. After 27 d, PH was performed in all groups. RESULTS: Groups fed chol before PH had significantly higher liver triacylglycerol content (group 3: 25.8 ⫾ 2.6 mg/g of liver weight; group 4: 16.0 ⫾ 1.0 of liver weight; group 5: 22.0 ⫾ 1.0 of liver weight) than did the groups fed SD (group 1: 6.1 ⫾ 0.5; group 2: 5.9 ⫾ 0.7). Liver DNA synthesis after PH was significantly lower in chol-fed groups (group 3: 561 ⫾ 78; group 4: 472 ⫾ 92) than in SD-fed groups (group 1: 1645 ⫾ 574; group 2: 2935 ⫾ 1298), except the chol-fed PHHC given prava (group 5: 3230 ⫾ 527). CONCLUSIONS: In prava-treated rats, the induction of HMG-CoA activity overcame the inhibitory capability of pravastatin. The induction of HMG-CoA-reductase activity had a stimulatory effect on the initiation of liver regeneration. Nutrition 2002;18:51–55. ©Elsevier Science Inc. 2002 KEY WORDS: pravastatin, liver regeneration, rat, cholesterol, 3-hydroxy-3-methylglutaryl coenzyme A reductase, triacylglycerol
INTRODUCTION Liver regeneration after partial hepatectomy (PH) is a process in which normally quiescent hepatocytes replicate in regulated manner to restore liver mass. Liver regeneration involves the activation of multiple protooncogenes and transcription and growth factors. The sequential activation of tumor necrosis factor-␣, interleukin-6, and transcription factors nuclear factor-B and STAT3 are considered key for the initiation of liver regeneration. The complex process of liver regeneration requires large amounts of simple substrates to cover energy and structural requirements. One of these substrates is cholesterol. A recent study of Cai et al.1 showed that lovastatin, a potent inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, decreases mortality from hepatic failure after 90% PH in rats. Lovastatin increases the function of existing hepatocytes and might be used to improve liver function after extensive hepatic resection in rats. Kakkis et al.2 found that pravastatin improves survival in rats after orthotopic liver transplantation. Very important in this context are the findings of Fujioka et al.3 in rabbits, where pravastatin administration
Supported by grant MSM:J13/98:111500003 from the Ministry of Education. Correspondence to: Pavel Zivny, MD, PhD, Institute of Clinical Biochemistry and Diagnostics, Charles University and University Hospital, CZ 50005 Hradec Kralove, Czech Republic. E-mail: [email protected] Nutrition 18:51–55, 2002 ©Elsevier Science Inc., 2002. Printed in the United States. All rights reserved.
(50 mg/kg of body weight) for 14 d decreased serum and liver cholesterol by 40% and 8%, respectively. In contrast, in rats, serum cholesterol increased by 14% at 50 mg/kg and liver cholesterol by 27% at 250 mg/kg of body weight after 7 d. At 250 mg/kg, liver cholesterol increased by 11%. The investigators concluded that induced HMG-CoA-reductase activity in rats might overcome the inhibitory capability of pravastatin, resulting in an increase of net cholesterol synthesis. Cholesterol is a very important component of cell membranes and intracellular membranes in animals. HMG-CoA-reductase is the key enzyme determining the rate of cholesterol biosynthesis. Intracellular cholesterol homeostasis is controlled by low-density lipoprotein (LDL)–receptor activity. Increasing the dietary load of cholesterol suppresses LDL-receptor protein synthesis and expression in hepatocytes and decreases exogenous cholesterol uptake. The hydrophilic HMG-CoA-reductase inhibitor (pravastatin) supposedly lowers serum cholesterol by the following mechanism: hepatic cholesterol depletion by inhibition of cholesterol synthesis triggers the induction of the hepatic LDL receptor, resulting in the stimulation of LDL removal from the blood, and decreases the secretion of very low-density lipoprotein (VLDL), resulting in an decrease in serum cholesterol.4 In contrast, Mohammadi et al.5 reported that the inhibition of cholesterol synthesis in HepG2 cells by lipophilic reductase inhibitor (atorvastatin) likely occurs because of impairment of the translocation of apolipoprotein B into the lumen of the endoplasmic reticulum; apoliprotein B is degraded intracellularly and its incorporation into VLDL decreases. 0899-9007/02/$22.00 PII S0899-9007(01)00678-5
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FIG. 1. Histologic findings. (A) Group 1: Sporadic eosinophil intraplasmatic inclusions were found in Wistar rats administered AI, 18 h after PH. (H&E, 150⫻). (B) Group 2: Numerous eosinophil intraplasmatic inclusions were found in Wistar administered pravastatin, 18 h after PH (H&E, 150⫻). (C) Group 3: Sporadic eosinophil inclusions were found in Wistar rats on a cholesterol-enriched diet, 18 h after PH (H&E, 200⫻). (D) Group 4: Numerous eosinophil inclusions were found in PHHC on a cholesterol-enriched diet and administered AI, 18 h after PH (H&E, 200⫻). Group 5: Numerous eosinophil inclusions were found in PHHC on a cholesterol-enriched diet and administered AI, 18 h after PH (not shown). AI, aqua pro injectione; H&E, hematoxylin and eosin stain; PH, partial hepatectomy; PHHC, Prague hereditary hypercholesterolemic rats.
MATERIALS AND METHODS Male Wistar (W) rats and Prague hereditary hypercholesterolemic (PHHC) rats (Velaz, Prague, Czech Republic) were maintained under controlled temperatures (from 22°C to 24°C) and a constant 12-h light-and-dark cycle. Animals were housed in hanging plastic cages and fed commercial pelleted food (ST-1-TOP, Velaz; total energy content: 15.18 kJ/g, fat: 1.13 kJ, protein: 4.17 kJ, carbohydrate: 9.89 kJ) or a cholesterol-enriched diet (diet preparation: 1 kg of crystalline cholesterol obtained from Sigma-Aldrich, Diesenhofen, Germany, was diluted in 3 L of sunflower oil melted to 60°C and subsequently mixed with 30 kg of powdery ST-1-TOP, and then this mixture was pelleted), and tap water was available ad libitum from day 1 of the experiment. Pravastatin sodium or aqua pro injectione (AI) was administered once daily by gastric tube from the day 10 of the experiment. All procedures were performed under ether anesthesia. On day 24, blood samples were taken from the retroorbital sinuses of all animals. On day 27, 67% PH according to the method of Higgins and Anderson6 was performed in all groups. One hour before the rats were killed, [3H]thymidine was administered into left femoral vein. Rats were killed 18 h after PH by exsanguination from the
abdominal aorta (i.e., day 28 of the experiment). Serum samples and livers were immediately frozen and stored for further analysis. Thirty-five rats were assigned to five groups (n ⫽ 7 animals/ group). Group 1 consisted of W rats on the ST-1-TOP diet that, from the day 10 of the experiment, were administered AI (1 mL/kg of body weight) once daily by gastric tube. Group 2 consisted of W rats on the ST-1-TOP diet that, from day 10 of the experiment, were administered pravastatin sodium (1 mg/kg of body weight, dissolved in 1 mL of AI) once daily by gastric tube. Group 3 consisted of W rats on the cholesterol-enriched diet that, from day 10 of the experiment, were administered AI (1 mL/kg of body weight) once daily by gastric tube. Group 4 consisted of PHHC rats on the cholesterol-enriched diet that, from day 10 of the experiment, were administered AI (1 mL/kg of body weight) once daily by gastric tube. Group 5 consisted of PHHC rats on the cholesterol-enriched diet that, from day 10 of the experiment, were administered pravastatin sodium (1 mg/kg of body weight, dissolved in 1 mL of AI) once daily by gastric tube. Analyses Analyses were performed in liver parts removed during PH and regenerating liver remnants obtained after rats were killed.
Nutrition Volume 18, Number 1, 2002
High Cholesterol, Pravastatin Sodium, and Partial Hepatectomy
FIG. 2. Serum cholesterol concentrations in rats before and after PH (mmol/L). No significant differences were observed between Wistar-aqua and Wistar-prava and between PHHC-chol-aqua and PHHC-chol-prava groups. Rats on the cholesterol-enriched diet had higher serum cholesterol concentration than did rats on the standard diet. PHHCs before PH had significantly higher serum cholesterol concentrations than did Wistar rats (including those on the cholesterol-enriched diet). Proportional nonsignificant decreases in serum cholesterol concentrations were observed after PH. #and *Statistical significant in comparison with the Wistar-aqua group (P ⬍ 0.05). aqua, aqua pro injectione; chol, cholesterol-enriched diet; PH, partial hepatectomy; PHHC, Prague hereditary hypercholesterolemic rats; prava, pravastatin.
The initiation of liver regeneration was evaluated from DNA content in the liver and liver DNA synthesis. Liver DNA synthesis was determined by using a modified version of the method described by Short et al.7 with methyl [3H]- thymidine (Institute for Radioisotope Research, Prague, Czech Republic). One hour before the rats were killed, the isotope was administered intravenously in a dose of 1.48 Mbq/kg (specific activity: 1.48 Tbq/mmol). The radioactivity of the samples was measured with a Delta 300 (Nuclear Chicago, Chicago, IL, USA) scintillation counter. The results were converted to disintegrations per minute per milligram of DNA. The DNA content of the liver was determined with the use of diphenylamine reagent according to the method of Burton.8 Liver triacylglycerols were extracted by using a suspended mixture of methanol and chloroform, and triacylglycerol content from the suspension was estimated spectrophotometrically (570 nm) by using a colorimetric assay with chromotropic acid (expressed in milligrams per gram of wet liver tissue). Protein content in liver tissue was estimated with the method of Lowry et. al.9 and Folin
FIG. 3. Liver triacylglycerol content (mg/g of wet liver tissue). Rats on the cholesterol-enriched diet before PH had significantly higher liver triacylglycerol content than did rats on the standard diet (P ⬍ 0.05). Significant increases of liver triacylglycerol content after PH were observed in all but the Wistar-chol group (P ⬍ 0.02). aqua, aqua pro injectione; chol, cholesterol-enriched diet; PH, partial hepatectomy; PHHC, Prague hereditary hypercholesterolemic rats; prava, pravastatin.
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FIG. 4. Specific activity of liver DNA (disintegrations per minute per milligram of DNA). Specific activity of liver DNA (18 h after PH) was significantly lower in rats fed the cholesterol-enriched diet (*P ⬍ 0.05) except the PHHC-chol-prava group. The PHHC-chol-prava group had significantly higher specific activity of liver DNA in comparison with the Wistar-chol and PHHC-chol-aqua groups (#P ⬍ 0.05). aqua, aqua pro injectione; chol, cholesterol-enriched diet; PH, partial hepatectomy; PHHC, Prague hereditary hypercholesterolemic rats; prava, pravastatin.
phenol reagent. Serum concentrations of cholesterol and glucose were measured with an automatic analyzer (Modular Roche, Mannheim, Germany). Liver samples were fixed in 10% buffered formalin. Samples were embedded in parrafin and sectioned at 3 m. Sections were stained with hematoxylin and eosin. Statistics The data are presented as mean ⫾ standard error of the mean. SigmaStat software (Jandel Scientific Corporation, San Rafael, CA) was used for analysis. We used unpaired t test and one-way analysis of variance.
RESULTS Histologic Findings Eighteen hours after PH, sporadic eosinophil intraplasmatic inclusions were found in group 1 (Fig. 1A, hematoxylin and eosin, 150⫻), numerous eosinophil intraplasmatic inclusions were found in group 2 (Fig. 1B, hematoxylin and eosin, 150⫻), sporadic eosinophil inclusions were found in groups 3 (Fig. 1C, hematoxylin and eosin, 200⫻), and numerous eosinophil inclusions were found in groups 4 (Fig. 1D, hematoxylin and eosin, 200⫻) and 5 (not shown). The serum cholesterol concentrations of the different groups are shown in Figure 2. No significant differences were observed between groups 1 and 2 and between groups 4 and 5. Groups 3, 4, and 5 had higher serum cholesterol concentrations than did groups 1 and 2. PHHC rats (group 4 and 5) before PH had significantly higher serum cholesterol concentrations than the W rats (including group 3 on the cholesterol-enriched diet). Proportional not significant decreases in serum cholesterol concentrations were observed after PH. Groups 3, 4, and 5 (cholesterol-enriched diet) before PH had significantly higher liver triacylglycerol content than did groups 1 and 2 (ST-1-TOP diet). Significant increases of liver triacylglycerol content after PH were observed in all but group 3 (Fig. 3). The specific activity of liver DNA is shown in Figure 4. The specific activity of liver DNA (18 h after PH) was significantly lower in groups 3 and 4 (cholesterol-enriched diet) but not in group 5, which had a significantly higher specific activity of liver DNA in comparison with groups 3 and 4. Serum glucose concentrations are reported in Table I. No
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Nutrition Volume 18, Number 1, 2002 TABLE I. SERUM GLUCOSE CONCENTRATIONS*
mmol/L
Wistar-aqua
Wistar-prava
Wistar-chol
PHHC-chol-aqua
PHHC-chol-prava
Before PH After PH
7.92 ⫾ 0.27 6.34 ⫾ 0.49
7.66 ⫾ 0.39 6.91 ⫾ 0.64
7.63 ⫾ 0.45 7.67 ⫾ 0.41
8.31 ⫾ 0.45 7.18 ⫾ 0.36
8.67 ⫾ 0.43 5.79 ⫾ 0.15
* Values are presented as mean ⫾ standard error of the mean. aqua, aqua pro injectione; chol, cholesterol-enriched diet; PH, partial hepatectomy; PHHC, Prague hereditary hypercholesterolemic rats; prava, pravastatin.
significant differences were observed across groups. Liver DNA content and liver protein content are reported in Table II.
DISCUSSION Our experiments are based on the findings of Fujioka et al.3 in rabbits, where pravastatin administration at 50 mg/kg of body weight for 14 d decreased serum and liver cholesterol by 40% nad 8%, respectively. The hepatic LDL-receptor activity was increased 1.7-fold, and VLDL cholesterol secretion was decreased. Cholesterol 7␣-hydroxylase activity was not changed. In rats, serum cholesterol increased by 14% at 50 mg/kg and liver cholesterol by 27% at 250 mg/kg for 7 d. At 250 mg/kg, liver cholesterol was increased by 11%. Those researchers concluded that induced HMG-CoA activity in rats might overcome the inhibitory capability of pravastatin, resulting in an increase of net cholesterol synthesis. Fujioka et al.10 also studied the effect of pravastatin sodium on fatty acid synthesis in rat liver. The repeated administration of pravastatin to rats at 250 mg/kg for 7 d led to a 2.8-fold increase in fatty acid synthesis in the liver. In rat hepatocytes, the incubation with 2 g/mL of pravastatin for 24 h increased fatty acid synthase activity 1.5-fold and HMG-CoA-reductase activity 2.8fold. These results suggest that HMG-CoA-reductase inhibitors increase fatty acid synthesis in vivo by the induction of hepatic fatty acid synthase. Fatty acid oxidation is the main source for reconstution of adenosine triphosphate after PH in rats.11 Moreover, Hotta12 studied the activity of the key enzyme of cholesterol synthesis, HMG-CoA-reductase, in PH rats of different ages and found a positive correlation between HMG-CoA-reductase activity and hepatocyte mitotic activity after PH. Those findings were basis for our study. We tried to adjust the dose of pravastatin sodium to mimic that in humans. The recommended dose in humans is approximately 0.2 mg/kg of body weight, but metabolic turnover in rats is five-fold quicker than in humans, so we used the 1 mg/kg as the dose. This dose of HMG-CoA inhibitor was substantially lower than that used by Fujioka et al.4 In agreement with the recent
literature, we did not observe decreases in serum cholesterol concentrations in rats treated with pravastatin sodium, but the cholesterol concentration increased slightly. A moderate induction of HMG-CoA-reductase and fatty acid synthase might have occurred. Based on the results of Irie et al.11 and Hotta,12 we expected the liver to regenerate, and our results confirmed that expectation. We observed moderate increases in serum cholesterol concentrations and greater liver DNA synthesis 18 h after PH in rats treated with pravastatin sodium. The explanation for these changes is not known. Cholesterol is a very important component of cell membranes and intracellular organelle membranes in animals. Cholesterol is the precursor of steroid hormones and bile acids and essential for cell proliferation and plasma membrane fluidity maintenance. The results showed that cholesterol administration (with the cholesterol-enriched diet) had no beneficial effect on the initiation of liver regeneration after PH, but administration of pravastatin followed by induction of HMGCoA-reductase in rats led to greater liver DNA synthesis after PH. Free fatty acids seem to be essential nutritional substrates for liver remnants after PH. The results obtained by Sabugal et al.13 showed a marked increase in the levels of lipoprotein lipase mRNA in the regenerating liver, coincident with high levels of lipoprotein lipase activity in the liver. The presence of high quantities of active lipoprotein lipase in a regenerating liver that is actively growing could enable it to take up fatty acids from the circulating triacylglycerols in the VLDL and the chylomicrons. These fatty acids are necessary for the generation of membranes. PH in rats increased the metabolic load of the liver remnants. After consumption of liver glycogen, resulting in a transient decrease of glycemia, intensive gluconeogenesis promptly normalized blood glucose levels. We found nonsignificant changes in serum glucose levels before and after PH. Eosinophil inclusions were found in histologic sections from liver samples 18 h after PH. Those inclusions were more numerous in pravastatin-treated rats and their source probably was lipoprotein particles.
TABLE II. LIVER DNA CONTENT AND LIVER PROTEIN CONTENT* mg/g
Wistar-aqua
Wistar-prava
Wistar-chol
PHHC-chol-aqua
PHHC-chol-prava
Before PH (DNA) After PH (DNA) Before PH (protein) After PH (protein)
2.03 ⫾ 0.05 2.25 ⫾ 0.15 153.3 ⫾ 3.6 153.5 ⫾ 6.7
1.98 ⫾ 0.10 2.13 ⫾ 0.07 157.0 ⫾ 2.7 166.1 ⫾ 5.9
1.48 ⫾ 0.06 1.62 ⫾ 0.10 149.9 ⫾ 4.9 174.7 ⫾ 6.5
2.14 ⫾ 0.08 1.94 ⫾ 0.08 150.7 ⫾ 5.6 147.3 ⫾ 3.5
2.00 ⫾ 0.09 1.76 ⫾ 0.06 142.9 ⫾ 5.8 157.9 ⫾ 6.9
* Values are presented as mean ⫾ standard error of the mean. aqua, aqua pro injectione; chol, cholesterol-enriched diet; PH, partial hepatectomy; PHHC, Prague hereditary hypercholesterolemic rats; prava, pravastatin.
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High Cholesterol, Pravastatin Sodium, and Partial Hepatectomy
Potential Clinical Implication for Humans This study demonstrates that pravastatin can improve the function of remnant hepatocytes after PH in rats. We suggest that HMGCoA-reductase was the key in that improvement. The increase of HMG-CoA activity in rats correlated with the increase of hepatic mitotic activities.12 In constrast, in humans, pravastatin decreases HMG-CoA activity. However, Kakkis et al.2 suggested that pravastatin has a cholesterol-independent effect on immune function (and possibly liver regeneration) in humans and rats. The aim of future studies might be dose optimization and understanding the different actions of pravastatin in rats and humans.
4. 5.
6. 7. 8. 9. 10.
REFERENCES 1. Cai SR, Motoyama K, Shen KJ, et al. Lovastatin decreases mortality and improves liver functions in fulminant hepatic failure from 90% partial hepatectomy in rats. J Hepatol 2000;32:67 2. Kakkis JL, Ke B, Dawson S, et al. Pravastatin increases survival and inhibits natural killer cell enhancement factor in liver transplanted rats. J Surg Res 1997;69:393 3. Fujioka T, Nara F, Tsujita Y, et al. The mechanism of lack of hypocholester-
11.
12.
13.
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olemic effects of pravastatin sodium, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, in rats. Biochim Biophys Acta 1995;1254:7 Fujioka T, Tsujita Y. Effects of single administration of pravastatin sodium on hepatic cholesterol metabolism in rats. Eur J Pharm 1997;323:223 Mohammadi A, Macri J, Newton R, et al. Effects of Atorvastatin on the intracellular stability and secretion of apolipoprotein B in HepG2 cells. Arterioscler Thromb Vasc Biol 1998;18:783 Higgins GM, Andersson RM. Experimental pathology of the liver. I. Restoration of the liver following partial surgical removal. Arch Pathol 1931;272:186 Short J, Zemel R, Kanta J, Lieberman I. Stimulation of deoxyribonucleic acid synthesis in the liver parenchymal cells of the intact rats. Nature 1969;223:956 Burton K. A study of the condition and mechanism of the colorimetric estimation of deoxyribonucleic acid. Biochem J 1956;62:315 Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265 Fujioka T, Tsujita Y, Shimotsu H. Induction of fatty acid synthesis by pravastatin sodium in rat liver and primary hepatocytes. Eur J Pharmacol 1997;328:235 Irie R, Kono Y, Aoyama H, et al. Impaired glucose tolerance related to changes in the energy metabolism of the remnant liver after major hepatic resection. J Lab Clin Med 1983;101:692 Hotta SS. The influence of age on the increased activity of 3-hydroxy-3methylglutaryl-CoA reductase in regenerating livers of partially hepatectomized rats. Exp Gerontol 1981;16:495 Sabugal R, Robert MQ, Julve J, et al. Hepatic regeneration induces changes in lipoprotein lipase activity in several tissues and its re-expression in the liver. Biochem J 1966;318:597
Influence of High Cholesterol Diet and Pravastatin Sodium on the Initiation of Liver Regeneration in Rats After Partial Hepatectomy Helena Zivna, MD, PhD, Pavel Zivny, MD, PhD, Vladimir Palicka, MD, PhD, and Eva Simakova, MD From the Institute of Physiology, the Institute of Clinical Biochemistry and Diagnostics, and the Department of Pathology, Charles University, University Hospital, Hradec Kralove, Czech Republic OBJECTIVES: Liver regeneration is influenced by cholesterol and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA-reductase). HMG-CoA-reductase is a key enzyme for cholesterol synthesis. Recent studies have shown that inhibitors of HMG-CoA-reductase improve liver functions after 67% partial hepatectomy (PH). METHODS: Male Wistar rats (W) and Prague hereditary hypercholesterolemic rats (PHHC) were used. Aqua pro injectione (AI) or pravastatin (prava; 1 mg/kg) was administered orally once daily. Group 1: W, standard diet (SD) ⫹ AI; group 2: W, SD ⫹ prava; group 3: W, cholesterol-enriched diet (chol) ⫹ AI; group 4: PHHC, chol ⫹ AI; group 5: PHHC, chol ⫹ prava. After 27 d, PH was performed in all groups. RESULTS: Groups fed chol before PH had significantly higher liver triacylglycerol content (group 3: 25.8 ⫾ 2.6 mg/g of liver weight; group 4: 16.0 ⫾ 1.0 of liver weight; group 5: 22.0 ⫾ 1.0 of liver weight) than did the groups fed SD (group 1: 6.1 ⫾ 0.5; group 2: 5.9 ⫾ 0.7). Liver DNA synthesis after PH was significantly lower in chol-fed groups (group 3: 561 ⫾ 78; group 4: 472 ⫾ 92) than in SD-fed groups (group 1: 1645 ⫾ 574; group 2: 2935 ⫾ 1298), except the chol-fed PHHC given prava (group 5: 3230 ⫾ 527). CONCLUSIONS: In prava-treated rats, the induction of HMG-CoA activity overcame the inhibitory capability of pravastatin. The induction of HMG-CoA-reductase activity had a stimulatory effect on the initiation of liver regeneration. Nutrition 2002;18:51–55. ©Elsevier Science Inc. 2002 KEY WORDS: pravastatin, liver regeneration, rat, cholesterol, 3-hydroxy-3-methylglutaryl coenzyme A reductase, triacylglycerol
INTRODUCTION Liver regeneration after partial hepatectomy (PH) is a process in which normally quiescent hepatocytes replicate in regulated manner to restore liver mass. Liver regeneration involves the activation of multiple protooncogenes and transcription and growth factors. The sequential activation of tumor necrosis factor-␣, interleukin-6, and transcription factors nuclear factor-B and STAT3 are considered key for the initiation of liver regeneration. The complex process of liver regeneration requires large amounts of simple substrates to cover energy and structural requirements. One of these substrates is cholesterol. A recent study of Cai et al.1 showed that lovastatin, a potent inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, decreases mortality from hepatic failure after 90% PH in rats. Lovastatin increases the function of existing hepatocytes and might be used to improve liver function after extensive hepatic resection in rats. Kakkis et al.2 found that pravastatin improves survival in rats after orthotopic liver transplantation. Very important in this context are the findings of Fujioka et al.3 in rabbits, where pravastatin administration
Supported by grant MSM:J13/98:111500003 from the Ministry of Education. Correspondence to: Pavel Zivny, MD, PhD, Institute of Clinical Biochemistry and Diagnostics, Charles University and University Hospital, CZ 50005 Hradec Kralove, Czech Republic. E-mail: [email protected] Nutrition 18:51–55, 2002 ©Elsevier Science Inc., 2002. Printed in the United States. All rights reserved.
(50 mg/kg of body weight) for 14 d decreased serum and liver cholesterol by 40% and 8%, respectively. In contrast, in rats, serum cholesterol increased by 14% at 50 mg/kg and liver cholesterol by 27% at 250 mg/kg of body weight after 7 d. At 250 mg/kg, liver cholesterol increased by 11%. The investigators concluded that induced HMG-CoA-reductase activity in rats might overcome the inhibitory capability of pravastatin, resulting in an increase of net cholesterol synthesis. Cholesterol is a very important component of cell membranes and intracellular membranes in animals. HMG-CoA-reductase is the key enzyme determining the rate of cholesterol biosynthesis. Intracellular cholesterol homeostasis is controlled by low-density lipoprotein (LDL)–receptor activity. Increasing the dietary load of cholesterol suppresses LDL-receptor protein synthesis and expression in hepatocytes and decreases exogenous cholesterol uptake. The hydrophilic HMG-CoA-reductase inhibitor (pravastatin) supposedly lowers serum cholesterol by the following mechanism: hepatic cholesterol depletion by inhibition of cholesterol synthesis triggers the induction of the hepatic LDL receptor, resulting in the stimulation of LDL removal from the blood, and decreases the secretion of very low-density lipoprotein (VLDL), resulting in an decrease in serum cholesterol.4 In contrast, Mohammadi et al.5 reported that the inhibition of cholesterol synthesis in HepG2 cells by lipophilic reductase inhibitor (atorvastatin) likely occurs because of impairment of the translocation of apolipoprotein B into the lumen of the endoplasmic reticulum; apoliprotein B is degraded intracellularly and its incorporation into VLDL decreases. 0899-9007/02/$22.00 PII S0899-9007(01)00678-5
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FIG. 1. Histologic findings. (A) Group 1: Sporadic eosinophil intraplasmatic inclusions were found in Wistar rats administered AI, 18 h after PH. (H&E, 150⫻). (B) Group 2: Numerous eosinophil intraplasmatic inclusions were found in Wistar administered pravastatin, 18 h after PH (H&E, 150⫻). (C) Group 3: Sporadic eosinophil inclusions were found in Wistar rats on a cholesterol-enriched diet, 18 h after PH (H&E, 200⫻). (D) Group 4: Numerous eosinophil inclusions were found in PHHC on a cholesterol-enriched diet and administered AI, 18 h after PH (H&E, 200⫻). Group 5: Numerous eosinophil inclusions were found in PHHC on a cholesterol-enriched diet and administered AI, 18 h after PH (not shown). AI, aqua pro injectione; H&E, hematoxylin and eosin stain; PH, partial hepatectomy; PHHC, Prague hereditary hypercholesterolemic rats.
MATERIALS AND METHODS Male Wistar (W) rats and Prague hereditary hypercholesterolemic (PHHC) rats (Velaz, Prague, Czech Republic) were maintained under controlled temperatures (from 22°C to 24°C) and a constant 12-h light-and-dark cycle. Animals were housed in hanging plastic cages and fed commercial pelleted food (ST-1-TOP, Velaz; total energy content: 15.18 kJ/g, fat: 1.13 kJ, protein: 4.17 kJ, carbohydrate: 9.89 kJ) or a cholesterol-enriched diet (diet preparation: 1 kg of crystalline cholesterol obtained from Sigma-Aldrich, Diesenhofen, Germany, was diluted in 3 L of sunflower oil melted to 60°C and subsequently mixed with 30 kg of powdery ST-1-TOP, and then this mixture was pelleted), and tap water was available ad libitum from day 1 of the experiment. Pravastatin sodium or aqua pro injectione (AI) was administered once daily by gastric tube from the day 10 of the experiment. All procedures were performed under ether anesthesia. On day 24, blood samples were taken from the retroorbital sinuses of all animals. On day 27, 67% PH according to the method of Higgins and Anderson6 was performed in all groups. One hour before the rats were killed, [3H]thymidine was administered into left femoral vein. Rats were killed 18 h after PH by exsanguination from the
abdominal aorta (i.e., day 28 of the experiment). Serum samples and livers were immediately frozen and stored for further analysis. Thirty-five rats were assigned to five groups (n ⫽ 7 animals/ group). Group 1 consisted of W rats on the ST-1-TOP diet that, from the day 10 of the experiment, were administered AI (1 mL/kg of body weight) once daily by gastric tube. Group 2 consisted of W rats on the ST-1-TOP diet that, from day 10 of the experiment, were administered pravastatin sodium (1 mg/kg of body weight, dissolved in 1 mL of AI) once daily by gastric tube. Group 3 consisted of W rats on the cholesterol-enriched diet that, from day 10 of the experiment, were administered AI (1 mL/kg of body weight) once daily by gastric tube. Group 4 consisted of PHHC rats on the cholesterol-enriched diet that, from day 10 of the experiment, were administered AI (1 mL/kg of body weight) once daily by gastric tube. Group 5 consisted of PHHC rats on the cholesterol-enriched diet that, from day 10 of the experiment, were administered pravastatin sodium (1 mg/kg of body weight, dissolved in 1 mL of AI) once daily by gastric tube. Analyses Analyses were performed in liver parts removed during PH and regenerating liver remnants obtained after rats were killed.
Nutrition Volume 18, Number 1, 2002
High Cholesterol, Pravastatin Sodium, and Partial Hepatectomy
FIG. 2. Serum cholesterol concentrations in rats before and after PH (mmol/L). No significant differences were observed between Wistar-aqua and Wistar-prava and between PHHC-chol-aqua and PHHC-chol-prava groups. Rats on the cholesterol-enriched diet had higher serum cholesterol concentration than did rats on the standard diet. PHHCs before PH had significantly higher serum cholesterol concentrations than did Wistar rats (including those on the cholesterol-enriched diet). Proportional nonsignificant decreases in serum cholesterol concentrations were observed after PH. #and *Statistical significant in comparison with the Wistar-aqua group (P ⬍ 0.05). aqua, aqua pro injectione; chol, cholesterol-enriched diet; PH, partial hepatectomy; PHHC, Prague hereditary hypercholesterolemic rats; prava, pravastatin.
The initiation of liver regeneration was evaluated from DNA content in the liver and liver DNA synthesis. Liver DNA synthesis was determined by using a modified version of the method described by Short et al.7 with methyl [3H]- thymidine (Institute for Radioisotope Research, Prague, Czech Republic). One hour before the rats were killed, the isotope was administered intravenously in a dose of 1.48 Mbq/kg (specific activity: 1.48 Tbq/mmol). The radioactivity of the samples was measured with a Delta 300 (Nuclear Chicago, Chicago, IL, USA) scintillation counter. The results were converted to disintegrations per minute per milligram of DNA. The DNA content of the liver was determined with the use of diphenylamine reagent according to the method of Burton.8 Liver triacylglycerols were extracted by using a suspended mixture of methanol and chloroform, and triacylglycerol content from the suspension was estimated spectrophotometrically (570 nm) by using a colorimetric assay with chromotropic acid (expressed in milligrams per gram of wet liver tissue). Protein content in liver tissue was estimated with the method of Lowry et. al.9 and Folin
FIG. 3. Liver triacylglycerol content (mg/g of wet liver tissue). Rats on the cholesterol-enriched diet before PH had significantly higher liver triacylglycerol content than did rats on the standard diet (P ⬍ 0.05). Significant increases of liver triacylglycerol content after PH were observed in all but the Wistar-chol group (P ⬍ 0.02). aqua, aqua pro injectione; chol, cholesterol-enriched diet; PH, partial hepatectomy; PHHC, Prague hereditary hypercholesterolemic rats; prava, pravastatin.
53
FIG. 4. Specific activity of liver DNA (disintegrations per minute per milligram of DNA). Specific activity of liver DNA (18 h after PH) was significantly lower in rats fed the cholesterol-enriched diet (*P ⬍ 0.05) except the PHHC-chol-prava group. The PHHC-chol-prava group had significantly higher specific activity of liver DNA in comparison with the Wistar-chol and PHHC-chol-aqua groups (#P ⬍ 0.05). aqua, aqua pro injectione; chol, cholesterol-enriched diet; PH, partial hepatectomy; PHHC, Prague hereditary hypercholesterolemic rats; prava, pravastatin.
phenol reagent. Serum concentrations of cholesterol and glucose were measured with an automatic analyzer (Modular Roche, Mannheim, Germany). Liver samples were fixed in 10% buffered formalin. Samples were embedded in parrafin and sectioned at 3 m. Sections were stained with hematoxylin and eosin. Statistics The data are presented as mean ⫾ standard error of the mean. SigmaStat software (Jandel Scientific Corporation, San Rafael, CA) was used for analysis. We used unpaired t test and one-way analysis of variance.
RESULTS Histologic Findings Eighteen hours after PH, sporadic eosinophil intraplasmatic inclusions were found in group 1 (Fig. 1A, hematoxylin and eosin, 150⫻), numerous eosinophil intraplasmatic inclusions were found in group 2 (Fig. 1B, hematoxylin and eosin, 150⫻), sporadic eosinophil inclusions were found in groups 3 (Fig. 1C, hematoxylin and eosin, 200⫻), and numerous eosinophil inclusions were found in groups 4 (Fig. 1D, hematoxylin and eosin, 200⫻) and 5 (not shown). The serum cholesterol concentrations of the different groups are shown in Figure 2. No significant differences were observed between groups 1 and 2 and between groups 4 and 5. Groups 3, 4, and 5 had higher serum cholesterol concentrations than did groups 1 and 2. PHHC rats (group 4 and 5) before PH had significantly higher serum cholesterol concentrations than the W rats (including group 3 on the cholesterol-enriched diet). Proportional not significant decreases in serum cholesterol concentrations were observed after PH. Groups 3, 4, and 5 (cholesterol-enriched diet) before PH had significantly higher liver triacylglycerol content than did groups 1 and 2 (ST-1-TOP diet). Significant increases of liver triacylglycerol content after PH were observed in all but group 3 (Fig. 3). The specific activity of liver DNA is shown in Figure 4. The specific activity of liver DNA (18 h after PH) was significantly lower in groups 3 and 4 (cholesterol-enriched diet) but not in group 5, which had a significantly higher specific activity of liver DNA in comparison with groups 3 and 4. Serum glucose concentrations are reported in Table I. No
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Zivna et al.
Nutrition Volume 18, Number 1, 2002 TABLE I. SERUM GLUCOSE CONCENTRATIONS*
mmol/L
Wistar-aqua
Wistar-prava
Wistar-chol
PHHC-chol-aqua
PHHC-chol-prava
Before PH After PH
7.92 ⫾ 0.27 6.34 ⫾ 0.49
7.66 ⫾ 0.39 6.91 ⫾ 0.64
7.63 ⫾ 0.45 7.67 ⫾ 0.41
8.31 ⫾ 0.45 7.18 ⫾ 0.36
8.67 ⫾ 0.43 5.79 ⫾ 0.15
* Values are presented as mean ⫾ standard error of the mean. aqua, aqua pro injectione; chol, cholesterol-enriched diet; PH, partial hepatectomy; PHHC, Prague hereditary hypercholesterolemic rats; prava, pravastatin.
significant differences were observed across groups. Liver DNA content and liver protein content are reported in Table II.
DISCUSSION Our experiments are based on the findings of Fujioka et al.3 in rabbits, where pravastatin administration at 50 mg/kg of body weight for 14 d decreased serum and liver cholesterol by 40% nad 8%, respectively. The hepatic LDL-receptor activity was increased 1.7-fold, and VLDL cholesterol secretion was decreased. Cholesterol 7␣-hydroxylase activity was not changed. In rats, serum cholesterol increased by 14% at 50 mg/kg and liver cholesterol by 27% at 250 mg/kg for 7 d. At 250 mg/kg, liver cholesterol was increased by 11%. Those researchers concluded that induced HMG-CoA activity in rats might overcome the inhibitory capability of pravastatin, resulting in an increase of net cholesterol synthesis. Fujioka et al.10 also studied the effect of pravastatin sodium on fatty acid synthesis in rat liver. The repeated administration of pravastatin to rats at 250 mg/kg for 7 d led to a 2.8-fold increase in fatty acid synthesis in the liver. In rat hepatocytes, the incubation with 2 g/mL of pravastatin for 24 h increased fatty acid synthase activity 1.5-fold and HMG-CoA-reductase activity 2.8fold. These results suggest that HMG-CoA-reductase inhibitors increase fatty acid synthesis in vivo by the induction of hepatic fatty acid synthase. Fatty acid oxidation is the main source for reconstution of adenosine triphosphate after PH in rats.11 Moreover, Hotta12 studied the activity of the key enzyme of cholesterol synthesis, HMG-CoA-reductase, in PH rats of different ages and found a positive correlation between HMG-CoA-reductase activity and hepatocyte mitotic activity after PH. Those findings were basis for our study. We tried to adjust the dose of pravastatin sodium to mimic that in humans. The recommended dose in humans is approximately 0.2 mg/kg of body weight, but metabolic turnover in rats is five-fold quicker than in humans, so we used the 1 mg/kg as the dose. This dose of HMG-CoA inhibitor was substantially lower than that used by Fujioka et al.4 In agreement with the recent
literature, we did not observe decreases in serum cholesterol concentrations in rats treated with pravastatin sodium, but the cholesterol concentration increased slightly. A moderate induction of HMG-CoA-reductase and fatty acid synthase might have occurred. Based on the results of Irie et al.11 and Hotta,12 we expected the liver to regenerate, and our results confirmed that expectation. We observed moderate increases in serum cholesterol concentrations and greater liver DNA synthesis 18 h after PH in rats treated with pravastatin sodium. The explanation for these changes is not known. Cholesterol is a very important component of cell membranes and intracellular organelle membranes in animals. Cholesterol is the precursor of steroid hormones and bile acids and essential for cell proliferation and plasma membrane fluidity maintenance. The results showed that cholesterol administration (with the cholesterol-enriched diet) had no beneficial effect on the initiation of liver regeneration after PH, but administration of pravastatin followed by induction of HMGCoA-reductase in rats led to greater liver DNA synthesis after PH. Free fatty acids seem to be essential nutritional substrates for liver remnants after PH. The results obtained by Sabugal et al.13 showed a marked increase in the levels of lipoprotein lipase mRNA in the regenerating liver, coincident with high levels of lipoprotein lipase activity in the liver. The presence of high quantities of active lipoprotein lipase in a regenerating liver that is actively growing could enable it to take up fatty acids from the circulating triacylglycerols in the VLDL and the chylomicrons. These fatty acids are necessary for the generation of membranes. PH in rats increased the metabolic load of the liver remnants. After consumption of liver glycogen, resulting in a transient decrease of glycemia, intensive gluconeogenesis promptly normalized blood glucose levels. We found nonsignificant changes in serum glucose levels before and after PH. Eosinophil inclusions were found in histologic sections from liver samples 18 h after PH. Those inclusions were more numerous in pravastatin-treated rats and their source probably was lipoprotein particles.
TABLE II. LIVER DNA CONTENT AND LIVER PROTEIN CONTENT* mg/g
Wistar-aqua
Wistar-prava
Wistar-chol
PHHC-chol-aqua
PHHC-chol-prava
Before PH (DNA) After PH (DNA) Before PH (protein) After PH (protein)
2.03 ⫾ 0.05 2.25 ⫾ 0.15 153.3 ⫾ 3.6 153.5 ⫾ 6.7
1.98 ⫾ 0.10 2.13 ⫾ 0.07 157.0 ⫾ 2.7 166.1 ⫾ 5.9
1.48 ⫾ 0.06 1.62 ⫾ 0.10 149.9 ⫾ 4.9 174.7 ⫾ 6.5
2.14 ⫾ 0.08 1.94 ⫾ 0.08 150.7 ⫾ 5.6 147.3 ⫾ 3.5
2.00 ⫾ 0.09 1.76 ⫾ 0.06 142.9 ⫾ 5.8 157.9 ⫾ 6.9
* Values are presented as mean ⫾ standard error of the mean. aqua, aqua pro injectione; chol, cholesterol-enriched diet; PH, partial hepatectomy; PHHC, Prague hereditary hypercholesterolemic rats; prava, pravastatin.
Nutrition Volume 18, Number 1, 2002
High Cholesterol, Pravastatin Sodium, and Partial Hepatectomy
Potential Clinical Implication for Humans This study demonstrates that pravastatin can improve the function of remnant hepatocytes after PH in rats. We suggest that HMGCoA-reductase was the key in that improvement. The increase of HMG-CoA activity in rats correlated with the increase of hepatic mitotic activities.12 In constrast, in humans, pravastatin decreases HMG-CoA activity. However, Kakkis et al.2 suggested that pravastatin has a cholesterol-independent effect on immune function (and possibly liver regeneration) in humans and rats. The aim of future studies might be dose optimization and understanding the different actions of pravastatin in rats and humans.
4. 5.
6. 7. 8. 9. 10.
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olemic effects of pravastatin sodium, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, in rats. Biochim Biophys Acta 1995;1254:7 Fujioka T, Tsujita Y. Effects of single administration of pravastatin sodium on hepatic cholesterol metabolism in rats. Eur J Pharm 1997;323:223 Mohammadi A, Macri J, Newton R, et al. Effects of Atorvastatin on the intracellular stability and secretion of apolipoprotein B in HepG2 cells. Arterioscler Thromb Vasc Biol 1998;18:783 Higgins GM, Andersson RM. Experimental pathology of the liver. I. Restoration of the liver following partial surgical removal. Arch Pathol 1931;272:186 Short J, Zemel R, Kanta J, Lieberman I. Stimulation of deoxyribonucleic acid synthesis in the liver parenchymal cells of the intact rats. Nature 1969;223:956 Burton K. A study of the condition and mechanism of the colorimetric estimation of deoxyribonucleic acid. Biochem J 1956;62:315 Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265 Fujioka T, Tsujita Y, Shimotsu H. Induction of fatty acid synthesis by pravastatin sodium in rat liver and primary hepatocytes. Eur J Pharmacol 1997;328:235 Irie R, Kono Y, Aoyama H, et al. Impaired glucose tolerance related to changes in the energy metabolism of the remnant liver after major hepatic resection. J Lab Clin Med 1983;101:692 Hotta SS. The influence of age on the increased activity of 3-hydroxy-3methylglutaryl-CoA reductase in regenerating livers of partially hepatectomized rats. Exp Gerontol 1981;16:495 Sabugal R, Robert MQ, Julve J, et al. Hepatic regeneration induces changes in lipoprotein lipase activity in several tissues and its re-expression in the liver. Biochem J 1966;318:597