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Intravenous administration of follistatin: Delivery to the liver and effect on liver regeneration after partial hepatectomy
Intravenous Administration of Follistatin: Delivery to the Liver and Effect on Liver Regeneration After Partial Hepatectomy KIMITAKA KOGURE,1 YOU-QING ZHANG,2 MAKOTO KANZAKI,2 WAKA OMATA,2 TETSUYA MINE,3
When 1 mg 125I-follistatin was administered into a rat intravenously, radioactivity levels in serum decreased rapidly. Analysis with a biexponential equation showed that the initial half-life and the terminal half-life were 4.0 and 130.8 minutes, respectively. After 2 hours of infusion, approximately 9% of the follistatin infused remained in the liver, which was much more than that in kidney, spleen, pancreas, intestine, or lung. Autoradiography of the liver obtained at 24 hours of infusion revealed that numerous grains were located in parenchymal cells. Radioactivity of 125I-follistatin in the liver remained elevated until 72 hours and declined markedly thereafter. When a booster shot of 125I-follistatin was administered at 72 hours, radioactivity in the liver at 120 hours was markedly increased compared with that in rats that received a single shot of 125I-follistatin. We then examined the effect of intravenous infusion of follistatin on liver regeneration after hepatectomy of 70%. Immediately after the hepatectomy, either 1 mg follistatin or saline was infused intravenously. In some rats, a booster shot was infused at 72 hours. After 120 hours of hepatectomy of 70%, remnant liver weight, liver regeneration rate, and DNA content were significantly (P õ .05) higher in rats that received a booster shot of follistatin at 72 hours than those in control rats. These results indicate that follistatin administered intravenously accumulates in the liver and promotes liver regeneration after partial hepatectomy. (HEPATOLOGY 1996;24:361-366.) Augmentation of liver regeneration, with an artificial support to maintain liver function necessary for survival, would be beneficial for the treatment of fulminant hepatic failure caused by viral infection and hepatic toxins.1 As well, massive hepatectomy to resect liver tumors can be performed safely if the growth of the remnant liver is promoted effectively. Various attempts have been made to promote liver regeneration. Farivar et al.2 have reported that the infusion of insulin and glucagon decreased mortality in murine hepatic failure by stimulating liver regeneration. However, the effectiveness of the infusion of insulin and glucagon has not been confirmed in humans.3-5 The roles of various growth factors in the regulation of hepatocyte growth have been elucidated.1,6 The administration of growth factors should therefore promote liver regeneration. Hepatocyte growth factor increases murine
Abbreviation: PCNA, proliferating cell nuclear antigen. From the 1First Department of Surgery, Gunma University School of Medicine, 2Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, and 3Fourth Department of Internal Medicine, University of Tokyo School of Medicine, Tokyo, Japan. Received July 24, 1995; accepted April 1, 1996. The present study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan and grants from the Kato Memorial Foundation for Nanbyo Research and the Japanese Viral Hepatitis Foundation. Address reprint requests to: Itaru Kojima, M.D., Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371, Japan. Copyright q 1996 by the American Association for the Study of Liver Diseases. 0270-9139/96/2402-0012$3.00/0
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survival after hepatic injury induced by carbon tetrachloride.7 In addition, hepatocyte growth factor augmented liver regeneration after partial hepatectomy.7 Nevertheless, the efficacy of hepatocyte growth factor is modest. Activin A is a dimeric protein with a variety of biological actions in many types of cells.8,9 It belongs to the transforming growth factor b supergene family and it modulates growth and differentiation in many tissues. Studies in our laboratory have provided evidence that activin A is an autocrine growth inhibitor in hepatocytes.10 Activin A is produced in cultured rat parenchymal hepatocytes stimulated by epidermal growth factor or hepatocyte growth factor. Messenger RNA expression and the release of mature protein are markedly increased in mid- to late-G1 phase. The growth inhibition caused by activin A is significant because a blockade of the action of endogenous activin A leads to enhanced DNA synthesis.11 Activin A is also induced in vivo after partial hepatectomy.10 Low levels of the messenger RNA for the bA -subunit of activin have been detected in the liver; these disappeared 3 hours after partial hepatectomy and are markedly increased 20 hours after hepatectomy. When follistatin, which binds activin and blocks its action,11,12 is infused into the portal vein immediately after hepatectomy of 70%, DNA is synthesized several hours earlier than in control rats and liver regeneration is significantly augmented.13 A single administration of follistatin is much more effective than several administrations of hepatocyte growth factor in promoting liver regeneration.7,13 Follistatin provides a potential therapeutic approach to promoting liver regeneration because it specifically blocks autocrine growth inhibitor. In the present study, we investigated whether the intravenous administration of follistatin effectively promoted liver regeneration after partial hepatectomy. We studied the tissue distribution of follistatin infused intravenously and determined an effective means of maintaining the follistatin content in the liver. The results indicate that intravenous follistatin is effective in promoting liver regeneration after hepatectomy of 70%. MATERIALS AND METHODS Experimental Animals. All studies were performed according to the American Association for the Accreditation of Laboratory Animal Care guidelines. Male Wistar rats obtained from Imai Animal Company (Saitama, Japan) were fed with regular laboratory chow (Oriental Mouse Food, Tokyo, Japan) and kept in a temperature-controlled (24 { 17C) and artificially illuminated room (light on from 7 AM to 7 PM). Food and water were withdrawn from the animals for 3 hours before surgery. After the experiments, animals were allowed food and water ad libitum. Iodination of Follistatin. Recombinant human follistatin produced in Chinese hamster ovary cells was provided by Dr. Y. Eto of Ajinomoto Inc. (Kawasaki, Japan). Follistatin was radioiodinated using lactoperoxidase as described.13 The specific activity of the [125I]follistatin was 20 to 40 mCi/mg. Biological activity of the labeled follistatin was confirmed by activin-binding.13 An autoradiogram was obtained after dissolving 125I-follistatin by sodium dodecyl sulfate– polyacrylamide gel electrophoresis.
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FIG. 1. Autoradiogram of 125I-follistatin. I-follistatin was dissolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and an autoradiogram was taken. 125
Pharmacokinetical Analysis of 125I-Follistatin. Animals were not given sodium iodide before the follistatin infusion. Animals were weighed and anesthetized with sodium pentobarbital (50 mg/kg). Cannulae were implanted into the right jugular vein for blood sampling and femoral vein for the administration of the iodinated follistatin. All blood samples were drawn via the juguler venous catheter at 1, 5, 15, 30, 45, 60, 90, and 120 minutes after a single bolus injection (0.5 mL; 1 mg) of labeled follistatin, which was followed by the administration of 0.5 mL saline. Blood was transferred to a serum separator tube and allowed to clot for 45 minutes at room temperature. Radioactivity levels in the blood samples were measured in a g-counter and then the serum was separated by centrifugation at 10,000g for 5 minutes. The total radioactivity in serum was measured using a g-counter. The serum was then diluted with phosphate-buffered saline containing 1% bovine serum albumin to 500 mL just before the addition of 500 mL of chilled 20% trichloroacetic acid. After vortex mixing and incubation on ice for 30 minutes, the samples were centrifuged at 12,000g for 5 minutes. The supernatant was removed and radioactivity in the trichloroacetic acid–precipitable materials was counted. The serum concentration of [125I]follistatin was analyzed by fitting a biexponential equation to the trichloroacetic acid–precipitable counts versus time using a nonlinear leastsquares regression analysis. The disposition of [125I]follistatin was characterized by calculating the clearance, initial, and steady-state volume of distribution, and mean residence time in the body using noncompartment methods. Tissue Distribution of 125I-Follistatin. At 120 minutes, and 24, 48, 72, and 120 hours after the infusion, the liver was removed and flushed with 10 mL of normal saline through the inferior vena cava and the hepatic vein, using the portal vein as the outflow. The spleen, pancreas, kidney, intestine, and lung were simultaneously removed, weighed, and specimens were taken. Total radioactivity in each specimen was measured using a Packard Radioisotope Calibrator (COBRA TM 5010, Packard Instrument Company, Meriden, CT). 125 I-Follistatin Autoradiography. At 24 hours after the administration of 125I-follistatin, the liver was obtained as described above. Specimens were taken from each hepatic lobe and fixed immediately in 4% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4), and after 120 minutes, they were immersed in 20% saccharose. After 12 hours of fixation, the specimens embedded in OCT compound 4583 (Miles, Inc., Elkhart) were slowly frozen in liquid nitrogen. After drying, the frozen sections were cut at a thickness of 3 mm and mounted on glass slides covered with 1% gelatin and stretched on a heat board. The slides were dipped with Kodak NTB-3 emulsion at
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457C for 10 seconds and packed in a dark box. Following exposure for 2 weeks, the slides were developed in a Kodak developer for 20 minutes. After drying, the slides were stained with hematoxylin for 2 minutes. Partial Hepatectomy. Partial hepatectomy of 70% was performed according to Higgins and Anderson.14 Under diethylether anesthesia, the peritoneal cavity was entered through a median incision. After the dissection of the falciform ligament, the left lateral and left median lobes were excised.14 After the surgery, coagulated blood was removed with gauze and 100 mg of cefazol sodium was administered intraperitoneally. The wound was closed by layers with running 5– 0 silk. Administration of Follistatin. After hepatectomy of 70%, the intestine was drawn out from the peritoneal cavity and the inferior vena cava was exposed. One microgram of follistatin dissolved in 0.5 mL of physiological saline was infused into the inferior vena cava via the bifurcation of the common iliac vein through peritoneum. In control animals, the same volume of saline was infused into the inferior vena cava. In some experiments, the same doses of follistatin or saline were infused via the right jugular vein 72 hours after hepatectomy. Measurement of Liver Regeneration. Changes in postsurgical body weight were measured at indicated time points. At 120 hours of hepatectomy, rats were decapitated and the remnant liver weight was measured. The liver regeneration rate described by Fishback15 was calculated. Statistical significance was assessed by multiple comparisons using modified Student’s t test. For measurement of expression of proliferating cell nuclear antigen (PCNA), mouse antiPCNA antibody (Seikagaku Kogyo, Tokyo, Japan) and an indirect immunoperoxidase technique were used. Deparaffinized tissue sections were preincubated with normal goat serum (Chemicon International, Inc., Temecula, CA) for 4 hours at room temperature and then incubated overnight at 47C with the primary antibody. Antigen-antibody complex was visualized after reacting with peroxidase-conjugated, affinity-purified goat anti-mouse immunoglobulin G–peroxidase serum (Sigma), followed by colar development with 33*-diaminobenzidine tetrahydrochloride medium. The 3-3*-diaminobenzidine tetrahydrochloride reaction was stopped in phosphate-buffered saline solution for 10 minutes. Negative controls comprised test tissue without the addition of the primary antibody. For each of the tissues studied, sections of tissue from fixative were immunostained at the same time. To determine DNA content, liver DNA was extracted using the method by Byvoet.16 Briefly, a small piece of liver (0.5 g) was homoge-
FIG. 2. Time course of changes in serum 125I-follistatin after intravenous infusion. 125I-follistatin (1 mg) was infused at time 0 and blood samples were drawn at 1, 5, 15, 30, 45, 60, 90, and 120 minutes. Radioactivity levels in serum (●) and in trichloroacetic acid precipitate (s) were counted. Values are the means { SE of six independent experiments.
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FIG. 3. Tissue distribution of 125I-follistatin 24 hours after intravenous infusion. 125I-follistatin (1 mg) was infused intravenously. Various organs were obtained after 24 hours and radioactivity was measured. Values are the means { SE of six independent experiments.
nized in ice-cold 0.25 mol/L sucrose, and 6 mol/L HCl was added to a final concentration of 0.5 mol/L. The homogenized liver was centrifuged at 1,500g for another 15 minutes at 47C. The pellet was homogenized again in 5% trichloroacetic acid solution and centrifuged at 1,500g for another 15 minutes at 47C. The pellet was washed with 95% ethanol, 100% ethanol, chloroform:methanol (2:1, vol/vol), and ether, each for 5 to 10 minutes. Then the DNA pellet was measured by using the method by Hinegardner.17 RESULTS 125
Distribution of I-Follistatin Administered Intravenously. To determine the tissue distribution of follistatin after
the intravenous infusion, we infused 1 mg of 125I-follistatin into the femoral vein. Figure 1 shows an autoradiogram of 125 I-follistatin used in this study. A single band with a molecular weight of 40 kd was observed. Figure 2 demonstrates the time course of changes in the serum 125I-follistatin levels after intravenous infusion. Data are described by a biexponential equation. After intravenous infusion of 125I-follistatin, the initial half life (t1/2a ) and the terminal half life (t1/2b ) were 4.0 and 130.8 minutes, respectively. The weight-normalized clearance was 31.1 { 6.1 mL/min/kg (mean { SE, n Å 6). 125Ifollistatin had a rapid clearance and a large initial volume
FIG. 4. Autoradiography of 125I-follistatin in the liver. 125I-follistatin was infused intravenously and the liver was obtained at 24 hours. The specimens were prepared as described in Materials and Methods and autoradiographed.
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FIG. 5. Time course of changes in 125I-follistatin in the liver after an intravenous infusion. 125I-follistatin was infused intravenously and the amounts of radioactivity remaining in the liver were measured at 2, 24, 48, 72, and 120 hours. Values are the means { SE of four independent determinations.
of distribution (4.3 I/kg; mean, 6), suggesting the extensive binding to tissue component. Tissue Distribution of 125I-Follistatin After Intravenous Administration. Figure 3 shows the tissue distribution of 125I-
follistatin 24 hours after the intravenous infusion. Among several organs examined, the liver was the predominant site of accumulation of 125I-follistatin. Approximately 4% of the infused radioactive follistatin remained in the liver at 24 hours. Considerable radioactivity was also detected in the kidney. Accumulation of 125I-Follistatin in the Liver. Figure 4 demonstrates an autoradiography of 125I-follistatin in the liver 24 hours after the intravenous infusion. Numerous grains were distributed diffusely. In some experiments, we infused 1 mg
FIG. 6. Tissue distribution of 125I-follistatin 120 hours after intravenous infusion. 125I-follistatin (1 mg) was infused intravenously at time 0. In one group of rats, an additional 1 mg of 125I-follistatin was infused at 72 hours. Rats were killed at 120 hours and the tissue distribution of 125I-follistatin was measured. Values are the means { SE of four independent experiments.
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FIG. 7. Effect of follistatin on remnant liver weight, liver regeneration rate, and DNA content after 120 hours of hepatectomy. Follistatin (1 mg) was infused intravenously immediately after hepatectomy of 70%. Some rats received an additional 1 mg of follistatin, which was infused at 72 hours. The rats were killed at 120 hours and remnant liver weight (A), liver regeneration rate (B), and DNA content (C) were measured. Values are the means { SE of five to six experiments. N.S., not significant.
125 I-follistatin and 100-fold excess unlabeled follistatin. Nevertheless, accumulation of 125I-follistatin in the liver was not displaced by unlabeled excess follistatin (data not shown). We then measured time course of radioactivity of 125I-follistatin remaining in the liver after intravenous infusion. As shown in Fig. 5, the peak value was obtained at 2 hours of infusion. Approximately 9% of 125I-follistatin infused remained in the liver at 2 hours. 125I-follistatin level in the liver at 24 hours was approximately 50% of the peak value observed at 2 hours, and it remained elevated for 72 hours; the radioactivity declined thereafter. At 120 hours after the intravenous infusion, the amount of radioactivity remaining in the liver was less than 10% of the peak value. Considerable radioactivity was found in feces (data not shown). These results indicate that a significant amount of 125I-follistatin remains in the liver for at least 72 hours. To analyze the molecular form of 125Ifollistatin remaining in the liver at 72 hours, we homogenized the liver tissue, and the homogenate was dissolved in sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by an autoradiogram. However, the radioactivity of 125I-follistatin was not detected, probably because of the low radioactivity remaining in the tissue. Effect of a Second Administration of 125I-Follistatin. The
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above results suggested that a second administration of follistatin would be necessary to sustain elevated intrahepatic follistatin levels for 120 hours. In the next set of experiments, we infused 1 mg 125I-follistatin at the start of the experiment, and a booster shot was given after 72 hours. Figure 6 demonstrates the tissue distribution of 125I-follistatin after 120 hours. When the second administration of 125I-follistatin was given at 72 hours, the radioactivity level remaining in the liver at 120 hours was much greater (P õ .01) than that in rats receiving a single intravenous infusion at 0 minutes. In addition, the radioactivity remaining in the kidney was remarkably increased. Effect of Follistatin on Liver Regeneration After Partial Hepatectomy. An intraportal administration of follistatin
augments liver regeneration after hepatectomy of 70%.13 The effects of follistatin on the remnant liver weight and liver regeneration rate are significant after 120 hours of hepatectomy.13 We therefore examined the effect of intravenous follistatin on remnant liver weight 120 hours after hepatectomy of 70%. One microgram of follistatin was infused intravenously immediately after hepatectomy. Some rats received a booster infusion of the same amount of follistatin at 72 hours. As shown in Fig. 7, remnant liver weight was not changed
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ing was observed 120 hours after hepatectomy, whereas PCNA labeling was detected in rats receiving one or two shots of follistatin (Fig. 8). The number of PCNA-positive cell was, however, small. Histologically, morphology of hepatocytes in rats receiving follistatin was not changed (data not shown). Table 1 shows biochemical parameters in serum in control and follistatin-treated rats. No significant changes were observed in follistatin-treated rats. DISCUSSION
As we reported previously, an administration of follistatin accelerates liver regeneration in 70% hepatectomized rats.13 We infused 1 mg of follistatin into the portal vein immediately after hepatectomy of 70%. DNA synthesis was initiated several hours earlier than in untreated rats. The liver regeneration rate and the remnant liver weight were significantly greater at 120 hours of hepatectomy. In the present study, we examined whether an intravenous administration of the same amount of follistatin promoted liver regeneration. As shown in Fig. 3, follistatin infused intravenously accumulated predominantly in the liver. Approximately 4% of the infused radioactive follistatin remained in the liver at 24 hours. Our unpublished observations indicated that the serum concentration of follistatin in the normal rat was approximately 5 ng/mL (Kanzaki M, Kojima I, Unpublished observations, September 1994). Because we infused a relatively large amount of labeled follistatin, its specific activity may not have changed greatly. The amount of labeled follistatin found in the liver probably reflected the mass of follistatin delivered. Autoradiography of the liver indicated that infused follistatin accumulated in the liver and grains were found in parenchymal cells. We showed previously that follistatin infused into the portal vein remained in the liver and was released after perfusion with 2 mol/L NaCl.13 These results, together with the fact that follistatin binds to the heparan sulfate chain of proteoglycan in granulosa cells,18 suggest that follistatin binds to the heparan sulfate chain located in the glycocalix of hepatocytes. The fact that follistatin remained in the liver for as long as 72 hours may result from the binding of follistatin to the extracellular matrix. Consistent with this notion, follistatin remained in the liver for 72 hours, and radioactive follistatin levels in the liver declined rather quickly thereafter. Because considerable radioactivity was found in feces (data not shown), it is likely that follistatin was taken up by parenchymal cells, then was excreted into the bile duct. To maintain the hepatic content of follistatin for over 72 hours, an additional infusion would be necessary. A booster shot achieved a high hepatic content of follistatin. We studied the delivery of follistatin in intact rats, but it is possible that both distribution of infused follistatin and
FIG. 8. Effect of follistatin on expression of PCNA. Follistatin (1 mg) was infused intravenously immediately after hepatectomy of 70%. Some rats received an additional 1 mg of follistatin at 72 hours. The rats were killed at 120 hours and PCNA labeling was determined. (A) Control rat. (B) Rat receiving follistatin at 0 hours. (C) Rat receiving follistatin at 0 and 72 hours.
significantly in rats receiving a single intravenous infusion of follistatin. In contrast, the remnant liver weight was significantly (P õ .05) increased in rats receiving a booster shot of follistatin at 72 hours compared with control rats. Likewise, the liver regeneration rate was significantly (P õ .05) elevated in rats that received two shots of follistatin, whereas it was not changed significantly in rats that were given a single intravenous infusion at the time of hepatectomy (Fig. 7B). Similarly, the DNA content of the remnant liver was significantly greater in rats receiving an additional shot of follistatin (Fig. 7C). In control rats, virtually no PCNA label-
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TABLE 1. Changes in Biochemical Parameters in Serum Administration of Follistatin None
Albumin (g/dL) Aspartate transaminase (U/L) Lactate dehydrogenase (U/L) g-Glutamyl transpeptidase (U/L) Cholesterol (mg/dL) Glucose (mg/dL)
0h
0 and 72 h
4.57 { 0.25
4.65 { 0.20
4.68 { 0.31
233.0 { 24.7
272.0 { 23.1
281.6 { 60
1,595 { 244
1,922 { 148
2,440 { 725
2.5 { 0.28 67 { 3.5 159.2 { 10.0
2.16 { 0.16 76.3 { 7.6 141.3 { 10.3
1.8 { 0.37 65.8 { 5.8 137.2 { 3.7
NOTE. Follistatin (1 mg) or saline was administered intravenously immediately after hepatectomy of 70%. In some rats an additional 1 mg of follistatin was infused at 72 hours. Various serum parameters were determined after 120 hours of hepatectomy. Values are the means { SE (n Å 6).
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metabolism of the protein in the liver are altered in hepatectomized rats. In partially hepatectomized rats, however, a booster shot of follistatin was effective in promoting liver regeneration. Therefore, the information as to the fate of infused follistatin obtained in intact rats may be useful to achieve effective follistatin content in the liver. When 1 mg of follistatin was infused intravenously immediately after hepatectomy of 70%, the remnant liver weight or liver regeneration rate measured at 120 hours was not changed compared with that in control rats. The amount of follistatin remaining in the liver after 2 hours was approximately 9% of that infused. When the same amount was infused intraportally, about 40% of the follistatin remained in the liver, and 10% was released in response to the subsequent infusion of high salt.13 Hence, the amount of follistatin delivered to the liver after an intravenous administration may have been much less compared with the intraportal administration. Despite the limited delivery of intravenous follistatin to the liver, a combination of initial and booster shots increased the remnant liver weight and liver regeneration rate. Both parameters were significantly greater than those in untreated rats. Although we did not perform morphometry of the hepatocytes, the increases in these two parameters were not simply caused by the increase in cell volume, because DNA content of the remnant liver was significantly greater in rats receiving two shots of follistatin. These results indicate the efficacy of follistatin and suggest that endogenous activin A exerts tonic inhibition on liver regeneration not only in the initial round of DNA synthesis, but also in subsequent replications. Furthermore, the results also raise the possibility that a more extensive administration of follistatin, especially in the later phase, would be more effective in promoting regeneration. In conclusion, follistatin infused intravenously accumulates significantly in the liver and, when administered appropriately, effectively promotes liver regeneration. Acknowledgment: The authors thank Dr. Shigeyasu Tanaka of the Institute for Molecular and Cellular Regulation,
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Gunma University, for helpful discussion, and Kiyomi Ohgi for her secretarial assistance. REFERENCES 1. LaBrecque D. Liver regeneration: a picture emerges from the puzzle. Am J Gastroenterol 1994;89:S86-S96. 2. Farivar M, Wands JR, Isselbacher KJ. Effect of insulin and glucagon on fulminant murine hepatitis. N Engl J Med 1976;295:1517-1519. 3. Baker AI, Jaspan JB, Haines NW. A randomized clinical trial of insulin and glucagon infusion for treatment of alcoholic hepatitis. Gastroenterology 1981;80:1410-1414. 4. Jaspan JB, Landan RL, Schneider J. Insulin and glucagon infusion in the treatment of liver failure. Arch Intern Med 1984;144:2075-2078. 5. Harrison PM, Hughes RD, Forbes A. Failure of insulin and glucagon infusion to stimulate liver regeneration in fulminant hepatic failure. J Hepatol 1990;10:332-336. 6. Michalopoulos GK. Liver regeneration: molecular mechanisms of growth control. FASEB J 1990;4:176-187. 7. Ishiki Y, Ohnishi H, Muto Y, Matsumoto N, Nakamura T. Direct evidence that hepatocyte growth factor is a hepatotropic factor for liver regeneration and has a potent antihepatitis effect in vivo. HEPATOLOGY 1992;16:12271235. 8. Ying SY. Inhibins, activins, and follistatins: gonadal proteins modulating the secretion of follicle-stimulating hormone. Endocr Rev 1988;9:267-293. 9. DePaolo LV, Bicsak TA, Ericson GF, Shimasaki S, Ling N. Follistatin and activin: a potential intrinsic regulatory system within diverse tissues. Proc Soc Exp Biol Med 1991;198:500-512. 10. Yasuda H, Mine T, Shibata H, Eto Y, Hasegawa Y, Takeuchi T, Asano S, et al. Activin A: an autocrine inhibitor of initiation of DNA synthesis in rat hepatocytes. J Clin Invest 1993;92:1491-1496. 11. Nakamura T, Takio K, Eto Y, Shibai H, Titani K, Sugino H. Activinbinding protein from ovary is follistatin. Science 1990;247:836-838. 12. Farnworth UM, Findlay JK. Follistatin: more than follicle-stimulating hormone suppressing proteins. Mol Cell Endocrinol 1993;91:1-11. 13. Kogure K, Omata W, Kanzaki M, Zhang YQ, Yasuda H, Mine T, Kojima I. A single intraportal administration of follistatin accelerates liver regeneration in partially hepatectomized rats. Gastroenterology 1995;108:11361142. 14. Higgins GM, Anderson RM. Experimental pathology of the liver. Arch Pathol 1931;12:186-202. 15. Fishback FC. A morphologic study of regeneration of the liver after partial removal. Arch Pathol 1929;7:956-977. 16. Byvoet P. Determination of nucleic acids with concentrated H2SO4 . Anal Biochem 1966;15:31-39. 17. Hinegardner TT. An improved fluorometric assay for DNA. Anal Biochem 1971;39:197-201. 18. Nakamura T, Sugino K, Titani K, Sugino H. Follistatin, an activin-binding protein, associates with heparan sulfate chains of proteoglycans on follicular granulosa cells. J Biol Chem 1991;266:19432-19437.
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When 1 mg 125I-follistatin was administered into a rat intravenously, radioactivity levels in serum decreased rapidly. Analysis with a biexponential equation showed that the initial half-life and the terminal half-life were 4.0 and 130.8 minutes, respectively. After 2 hours of infusion, approximately 9% of the follistatin infused remained in the liver, which was much more than that in kidney, spleen, pancreas, intestine, or lung. Autoradiography of the liver obtained at 24 hours of infusion revealed that numerous grains were located in parenchymal cells. Radioactivity of 125I-follistatin in the liver remained elevated until 72 hours and declined markedly thereafter. When a booster shot of 125I-follistatin was administered at 72 hours, radioactivity in the liver at 120 hours was markedly increased compared with that in rats that received a single shot of 125I-follistatin. We then examined the effect of intravenous infusion of follistatin on liver regeneration after hepatectomy of 70%. Immediately after the hepatectomy, either 1 mg follistatin or saline was infused intravenously. In some rats, a booster shot was infused at 72 hours. After 120 hours of hepatectomy of 70%, remnant liver weight, liver regeneration rate, and DNA content were significantly (P õ .05) higher in rats that received a booster shot of follistatin at 72 hours than those in control rats. These results indicate that follistatin administered intravenously accumulates in the liver and promotes liver regeneration after partial hepatectomy. (HEPATOLOGY 1996;24:361-366.) Augmentation of liver regeneration, with an artificial support to maintain liver function necessary for survival, would be beneficial for the treatment of fulminant hepatic failure caused by viral infection and hepatic toxins.1 As well, massive hepatectomy to resect liver tumors can be performed safely if the growth of the remnant liver is promoted effectively. Various attempts have been made to promote liver regeneration. Farivar et al.2 have reported that the infusion of insulin and glucagon decreased mortality in murine hepatic failure by stimulating liver regeneration. However, the effectiveness of the infusion of insulin and glucagon has not been confirmed in humans.3-5 The roles of various growth factors in the regulation of hepatocyte growth have been elucidated.1,6 The administration of growth factors should therefore promote liver regeneration. Hepatocyte growth factor increases murine
Abbreviation: PCNA, proliferating cell nuclear antigen. From the 1First Department of Surgery, Gunma University School of Medicine, 2Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, and 3Fourth Department of Internal Medicine, University of Tokyo School of Medicine, Tokyo, Japan. Received July 24, 1995; accepted April 1, 1996. The present study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan and grants from the Kato Memorial Foundation for Nanbyo Research and the Japanese Viral Hepatitis Foundation. Address reprint requests to: Itaru Kojima, M.D., Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371, Japan. Copyright q 1996 by the American Association for the Study of Liver Diseases. 0270-9139/96/2402-0012$3.00/0
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survival after hepatic injury induced by carbon tetrachloride.7 In addition, hepatocyte growth factor augmented liver regeneration after partial hepatectomy.7 Nevertheless, the efficacy of hepatocyte growth factor is modest. Activin A is a dimeric protein with a variety of biological actions in many types of cells.8,9 It belongs to the transforming growth factor b supergene family and it modulates growth and differentiation in many tissues. Studies in our laboratory have provided evidence that activin A is an autocrine growth inhibitor in hepatocytes.10 Activin A is produced in cultured rat parenchymal hepatocytes stimulated by epidermal growth factor or hepatocyte growth factor. Messenger RNA expression and the release of mature protein are markedly increased in mid- to late-G1 phase. The growth inhibition caused by activin A is significant because a blockade of the action of endogenous activin A leads to enhanced DNA synthesis.11 Activin A is also induced in vivo after partial hepatectomy.10 Low levels of the messenger RNA for the bA -subunit of activin have been detected in the liver; these disappeared 3 hours after partial hepatectomy and are markedly increased 20 hours after hepatectomy. When follistatin, which binds activin and blocks its action,11,12 is infused into the portal vein immediately after hepatectomy of 70%, DNA is synthesized several hours earlier than in control rats and liver regeneration is significantly augmented.13 A single administration of follistatin is much more effective than several administrations of hepatocyte growth factor in promoting liver regeneration.7,13 Follistatin provides a potential therapeutic approach to promoting liver regeneration because it specifically blocks autocrine growth inhibitor. In the present study, we investigated whether the intravenous administration of follistatin effectively promoted liver regeneration after partial hepatectomy. We studied the tissue distribution of follistatin infused intravenously and determined an effective means of maintaining the follistatin content in the liver. The results indicate that intravenous follistatin is effective in promoting liver regeneration after hepatectomy of 70%. MATERIALS AND METHODS Experimental Animals. All studies were performed according to the American Association for the Accreditation of Laboratory Animal Care guidelines. Male Wistar rats obtained from Imai Animal Company (Saitama, Japan) were fed with regular laboratory chow (Oriental Mouse Food, Tokyo, Japan) and kept in a temperature-controlled (24 { 17C) and artificially illuminated room (light on from 7 AM to 7 PM). Food and water were withdrawn from the animals for 3 hours before surgery. After the experiments, animals were allowed food and water ad libitum. Iodination of Follistatin. Recombinant human follistatin produced in Chinese hamster ovary cells was provided by Dr. Y. Eto of Ajinomoto Inc. (Kawasaki, Japan). Follistatin was radioiodinated using lactoperoxidase as described.13 The specific activity of the [125I]follistatin was 20 to 40 mCi/mg. Biological activity of the labeled follistatin was confirmed by activin-binding.13 An autoradiogram was obtained after dissolving 125I-follistatin by sodium dodecyl sulfate– polyacrylamide gel electrophoresis.
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FIG. 1. Autoradiogram of 125I-follistatin. I-follistatin was dissolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and an autoradiogram was taken. 125
Pharmacokinetical Analysis of 125I-Follistatin. Animals were not given sodium iodide before the follistatin infusion. Animals were weighed and anesthetized with sodium pentobarbital (50 mg/kg). Cannulae were implanted into the right jugular vein for blood sampling and femoral vein for the administration of the iodinated follistatin. All blood samples were drawn via the juguler venous catheter at 1, 5, 15, 30, 45, 60, 90, and 120 minutes after a single bolus injection (0.5 mL; 1 mg) of labeled follistatin, which was followed by the administration of 0.5 mL saline. Blood was transferred to a serum separator tube and allowed to clot for 45 minutes at room temperature. Radioactivity levels in the blood samples were measured in a g-counter and then the serum was separated by centrifugation at 10,000g for 5 minutes. The total radioactivity in serum was measured using a g-counter. The serum was then diluted with phosphate-buffered saline containing 1% bovine serum albumin to 500 mL just before the addition of 500 mL of chilled 20% trichloroacetic acid. After vortex mixing and incubation on ice for 30 minutes, the samples were centrifuged at 12,000g for 5 minutes. The supernatant was removed and radioactivity in the trichloroacetic acid–precipitable materials was counted. The serum concentration of [125I]follistatin was analyzed by fitting a biexponential equation to the trichloroacetic acid–precipitable counts versus time using a nonlinear leastsquares regression analysis. The disposition of [125I]follistatin was characterized by calculating the clearance, initial, and steady-state volume of distribution, and mean residence time in the body using noncompartment methods. Tissue Distribution of 125I-Follistatin. At 120 minutes, and 24, 48, 72, and 120 hours after the infusion, the liver was removed and flushed with 10 mL of normal saline through the inferior vena cava and the hepatic vein, using the portal vein as the outflow. The spleen, pancreas, kidney, intestine, and lung were simultaneously removed, weighed, and specimens were taken. Total radioactivity in each specimen was measured using a Packard Radioisotope Calibrator (COBRA TM 5010, Packard Instrument Company, Meriden, CT). 125 I-Follistatin Autoradiography. At 24 hours after the administration of 125I-follistatin, the liver was obtained as described above. Specimens were taken from each hepatic lobe and fixed immediately in 4% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4), and after 120 minutes, they were immersed in 20% saccharose. After 12 hours of fixation, the specimens embedded in OCT compound 4583 (Miles, Inc., Elkhart) were slowly frozen in liquid nitrogen. After drying, the frozen sections were cut at a thickness of 3 mm and mounted on glass slides covered with 1% gelatin and stretched on a heat board. The slides were dipped with Kodak NTB-3 emulsion at
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457C for 10 seconds and packed in a dark box. Following exposure for 2 weeks, the slides were developed in a Kodak developer for 20 minutes. After drying, the slides were stained with hematoxylin for 2 minutes. Partial Hepatectomy. Partial hepatectomy of 70% was performed according to Higgins and Anderson.14 Under diethylether anesthesia, the peritoneal cavity was entered through a median incision. After the dissection of the falciform ligament, the left lateral and left median lobes were excised.14 After the surgery, coagulated blood was removed with gauze and 100 mg of cefazol sodium was administered intraperitoneally. The wound was closed by layers with running 5– 0 silk. Administration of Follistatin. After hepatectomy of 70%, the intestine was drawn out from the peritoneal cavity and the inferior vena cava was exposed. One microgram of follistatin dissolved in 0.5 mL of physiological saline was infused into the inferior vena cava via the bifurcation of the common iliac vein through peritoneum. In control animals, the same volume of saline was infused into the inferior vena cava. In some experiments, the same doses of follistatin or saline were infused via the right jugular vein 72 hours after hepatectomy. Measurement of Liver Regeneration. Changes in postsurgical body weight were measured at indicated time points. At 120 hours of hepatectomy, rats were decapitated and the remnant liver weight was measured. The liver regeneration rate described by Fishback15 was calculated. Statistical significance was assessed by multiple comparisons using modified Student’s t test. For measurement of expression of proliferating cell nuclear antigen (PCNA), mouse antiPCNA antibody (Seikagaku Kogyo, Tokyo, Japan) and an indirect immunoperoxidase technique were used. Deparaffinized tissue sections were preincubated with normal goat serum (Chemicon International, Inc., Temecula, CA) for 4 hours at room temperature and then incubated overnight at 47C with the primary antibody. Antigen-antibody complex was visualized after reacting with peroxidase-conjugated, affinity-purified goat anti-mouse immunoglobulin G–peroxidase serum (Sigma), followed by colar development with 33*-diaminobenzidine tetrahydrochloride medium. The 3-3*-diaminobenzidine tetrahydrochloride reaction was stopped in phosphate-buffered saline solution for 10 minutes. Negative controls comprised test tissue without the addition of the primary antibody. For each of the tissues studied, sections of tissue from fixative were immunostained at the same time. To determine DNA content, liver DNA was extracted using the method by Byvoet.16 Briefly, a small piece of liver (0.5 g) was homoge-
FIG. 2. Time course of changes in serum 125I-follistatin after intravenous infusion. 125I-follistatin (1 mg) was infused at time 0 and blood samples were drawn at 1, 5, 15, 30, 45, 60, 90, and 120 minutes. Radioactivity levels in serum (●) and in trichloroacetic acid precipitate (s) were counted. Values are the means { SE of six independent experiments.
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FIG. 3. Tissue distribution of 125I-follistatin 24 hours after intravenous infusion. 125I-follistatin (1 mg) was infused intravenously. Various organs were obtained after 24 hours and radioactivity was measured. Values are the means { SE of six independent experiments.
nized in ice-cold 0.25 mol/L sucrose, and 6 mol/L HCl was added to a final concentration of 0.5 mol/L. The homogenized liver was centrifuged at 1,500g for another 15 minutes at 47C. The pellet was homogenized again in 5% trichloroacetic acid solution and centrifuged at 1,500g for another 15 minutes at 47C. The pellet was washed with 95% ethanol, 100% ethanol, chloroform:methanol (2:1, vol/vol), and ether, each for 5 to 10 minutes. Then the DNA pellet was measured by using the method by Hinegardner.17 RESULTS 125
Distribution of I-Follistatin Administered Intravenously. To determine the tissue distribution of follistatin after
the intravenous infusion, we infused 1 mg of 125I-follistatin into the femoral vein. Figure 1 shows an autoradiogram of 125 I-follistatin used in this study. A single band with a molecular weight of 40 kd was observed. Figure 2 demonstrates the time course of changes in the serum 125I-follistatin levels after intravenous infusion. Data are described by a biexponential equation. After intravenous infusion of 125I-follistatin, the initial half life (t1/2a ) and the terminal half life (t1/2b ) were 4.0 and 130.8 minutes, respectively. The weight-normalized clearance was 31.1 { 6.1 mL/min/kg (mean { SE, n Å 6). 125Ifollistatin had a rapid clearance and a large initial volume
FIG. 4. Autoradiography of 125I-follistatin in the liver. 125I-follistatin was infused intravenously and the liver was obtained at 24 hours. The specimens were prepared as described in Materials and Methods and autoradiographed.
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FIG. 5. Time course of changes in 125I-follistatin in the liver after an intravenous infusion. 125I-follistatin was infused intravenously and the amounts of radioactivity remaining in the liver were measured at 2, 24, 48, 72, and 120 hours. Values are the means { SE of four independent determinations.
of distribution (4.3 I/kg; mean, 6), suggesting the extensive binding to tissue component. Tissue Distribution of 125I-Follistatin After Intravenous Administration. Figure 3 shows the tissue distribution of 125I-
follistatin 24 hours after the intravenous infusion. Among several organs examined, the liver was the predominant site of accumulation of 125I-follistatin. Approximately 4% of the infused radioactive follistatin remained in the liver at 24 hours. Considerable radioactivity was also detected in the kidney. Accumulation of 125I-Follistatin in the Liver. Figure 4 demonstrates an autoradiography of 125I-follistatin in the liver 24 hours after the intravenous infusion. Numerous grains were distributed diffusely. In some experiments, we infused 1 mg
FIG. 6. Tissue distribution of 125I-follistatin 120 hours after intravenous infusion. 125I-follistatin (1 mg) was infused intravenously at time 0. In one group of rats, an additional 1 mg of 125I-follistatin was infused at 72 hours. Rats were killed at 120 hours and the tissue distribution of 125I-follistatin was measured. Values are the means { SE of four independent experiments.
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FIG. 7. Effect of follistatin on remnant liver weight, liver regeneration rate, and DNA content after 120 hours of hepatectomy. Follistatin (1 mg) was infused intravenously immediately after hepatectomy of 70%. Some rats received an additional 1 mg of follistatin, which was infused at 72 hours. The rats were killed at 120 hours and remnant liver weight (A), liver regeneration rate (B), and DNA content (C) were measured. Values are the means { SE of five to six experiments. N.S., not significant.
125 I-follistatin and 100-fold excess unlabeled follistatin. Nevertheless, accumulation of 125I-follistatin in the liver was not displaced by unlabeled excess follistatin (data not shown). We then measured time course of radioactivity of 125I-follistatin remaining in the liver after intravenous infusion. As shown in Fig. 5, the peak value was obtained at 2 hours of infusion. Approximately 9% of 125I-follistatin infused remained in the liver at 2 hours. 125I-follistatin level in the liver at 24 hours was approximately 50% of the peak value observed at 2 hours, and it remained elevated for 72 hours; the radioactivity declined thereafter. At 120 hours after the intravenous infusion, the amount of radioactivity remaining in the liver was less than 10% of the peak value. Considerable radioactivity was found in feces (data not shown). These results indicate that a significant amount of 125I-follistatin remains in the liver for at least 72 hours. To analyze the molecular form of 125Ifollistatin remaining in the liver at 72 hours, we homogenized the liver tissue, and the homogenate was dissolved in sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by an autoradiogram. However, the radioactivity of 125I-follistatin was not detected, probably because of the low radioactivity remaining in the tissue. Effect of a Second Administration of 125I-Follistatin. The
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above results suggested that a second administration of follistatin would be necessary to sustain elevated intrahepatic follistatin levels for 120 hours. In the next set of experiments, we infused 1 mg 125I-follistatin at the start of the experiment, and a booster shot was given after 72 hours. Figure 6 demonstrates the tissue distribution of 125I-follistatin after 120 hours. When the second administration of 125I-follistatin was given at 72 hours, the radioactivity level remaining in the liver at 120 hours was much greater (P õ .01) than that in rats receiving a single intravenous infusion at 0 minutes. In addition, the radioactivity remaining in the kidney was remarkably increased. Effect of Follistatin on Liver Regeneration After Partial Hepatectomy. An intraportal administration of follistatin
augments liver regeneration after hepatectomy of 70%.13 The effects of follistatin on the remnant liver weight and liver regeneration rate are significant after 120 hours of hepatectomy.13 We therefore examined the effect of intravenous follistatin on remnant liver weight 120 hours after hepatectomy of 70%. One microgram of follistatin was infused intravenously immediately after hepatectomy. Some rats received a booster infusion of the same amount of follistatin at 72 hours. As shown in Fig. 7, remnant liver weight was not changed
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ing was observed 120 hours after hepatectomy, whereas PCNA labeling was detected in rats receiving one or two shots of follistatin (Fig. 8). The number of PCNA-positive cell was, however, small. Histologically, morphology of hepatocytes in rats receiving follistatin was not changed (data not shown). Table 1 shows biochemical parameters in serum in control and follistatin-treated rats. No significant changes were observed in follistatin-treated rats. DISCUSSION
As we reported previously, an administration of follistatin accelerates liver regeneration in 70% hepatectomized rats.13 We infused 1 mg of follistatin into the portal vein immediately after hepatectomy of 70%. DNA synthesis was initiated several hours earlier than in untreated rats. The liver regeneration rate and the remnant liver weight were significantly greater at 120 hours of hepatectomy. In the present study, we examined whether an intravenous administration of the same amount of follistatin promoted liver regeneration. As shown in Fig. 3, follistatin infused intravenously accumulated predominantly in the liver. Approximately 4% of the infused radioactive follistatin remained in the liver at 24 hours. Our unpublished observations indicated that the serum concentration of follistatin in the normal rat was approximately 5 ng/mL (Kanzaki M, Kojima I, Unpublished observations, September 1994). Because we infused a relatively large amount of labeled follistatin, its specific activity may not have changed greatly. The amount of labeled follistatin found in the liver probably reflected the mass of follistatin delivered. Autoradiography of the liver indicated that infused follistatin accumulated in the liver and grains were found in parenchymal cells. We showed previously that follistatin infused into the portal vein remained in the liver and was released after perfusion with 2 mol/L NaCl.13 These results, together with the fact that follistatin binds to the heparan sulfate chain of proteoglycan in granulosa cells,18 suggest that follistatin binds to the heparan sulfate chain located in the glycocalix of hepatocytes. The fact that follistatin remained in the liver for as long as 72 hours may result from the binding of follistatin to the extracellular matrix. Consistent with this notion, follistatin remained in the liver for 72 hours, and radioactive follistatin levels in the liver declined rather quickly thereafter. Because considerable radioactivity was found in feces (data not shown), it is likely that follistatin was taken up by parenchymal cells, then was excreted into the bile duct. To maintain the hepatic content of follistatin for over 72 hours, an additional infusion would be necessary. A booster shot achieved a high hepatic content of follistatin. We studied the delivery of follistatin in intact rats, but it is possible that both distribution of infused follistatin and
FIG. 8. Effect of follistatin on expression of PCNA. Follistatin (1 mg) was infused intravenously immediately after hepatectomy of 70%. Some rats received an additional 1 mg of follistatin at 72 hours. The rats were killed at 120 hours and PCNA labeling was determined. (A) Control rat. (B) Rat receiving follistatin at 0 hours. (C) Rat receiving follistatin at 0 and 72 hours.
significantly in rats receiving a single intravenous infusion of follistatin. In contrast, the remnant liver weight was significantly (P õ .05) increased in rats receiving a booster shot of follistatin at 72 hours compared with control rats. Likewise, the liver regeneration rate was significantly (P õ .05) elevated in rats that received two shots of follistatin, whereas it was not changed significantly in rats that were given a single intravenous infusion at the time of hepatectomy (Fig. 7B). Similarly, the DNA content of the remnant liver was significantly greater in rats receiving an additional shot of follistatin (Fig. 7C). In control rats, virtually no PCNA label-
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TABLE 1. Changes in Biochemical Parameters in Serum Administration of Follistatin None
Albumin (g/dL) Aspartate transaminase (U/L) Lactate dehydrogenase (U/L) g-Glutamyl transpeptidase (U/L) Cholesterol (mg/dL) Glucose (mg/dL)
0h
0 and 72 h
4.57 { 0.25
4.65 { 0.20
4.68 { 0.31
233.0 { 24.7
272.0 { 23.1
281.6 { 60
1,595 { 244
1,922 { 148
2,440 { 725
2.5 { 0.28 67 { 3.5 159.2 { 10.0
2.16 { 0.16 76.3 { 7.6 141.3 { 10.3
1.8 { 0.37 65.8 { 5.8 137.2 { 3.7
NOTE. Follistatin (1 mg) or saline was administered intravenously immediately after hepatectomy of 70%. In some rats an additional 1 mg of follistatin was infused at 72 hours. Various serum parameters were determined after 120 hours of hepatectomy. Values are the means { SE (n Å 6).
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metabolism of the protein in the liver are altered in hepatectomized rats. In partially hepatectomized rats, however, a booster shot of follistatin was effective in promoting liver regeneration. Therefore, the information as to the fate of infused follistatin obtained in intact rats may be useful to achieve effective follistatin content in the liver. When 1 mg of follistatin was infused intravenously immediately after hepatectomy of 70%, the remnant liver weight or liver regeneration rate measured at 120 hours was not changed compared with that in control rats. The amount of follistatin remaining in the liver after 2 hours was approximately 9% of that infused. When the same amount was infused intraportally, about 40% of the follistatin remained in the liver, and 10% was released in response to the subsequent infusion of high salt.13 Hence, the amount of follistatin delivered to the liver after an intravenous administration may have been much less compared with the intraportal administration. Despite the limited delivery of intravenous follistatin to the liver, a combination of initial and booster shots increased the remnant liver weight and liver regeneration rate. Both parameters were significantly greater than those in untreated rats. Although we did not perform morphometry of the hepatocytes, the increases in these two parameters were not simply caused by the increase in cell volume, because DNA content of the remnant liver was significantly greater in rats receiving two shots of follistatin. These results indicate the efficacy of follistatin and suggest that endogenous activin A exerts tonic inhibition on liver regeneration not only in the initial round of DNA synthesis, but also in subsequent replications. Furthermore, the results also raise the possibility that a more extensive administration of follistatin, especially in the later phase, would be more effective in promoting regeneration. In conclusion, follistatin infused intravenously accumulates significantly in the liver and, when administered appropriately, effectively promotes liver regeneration. Acknowledgment: The authors thank Dr. Shigeyasu Tanaka of the Institute for Molecular and Cellular Regulation,
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Gunma University, for helpful discussion, and Kiyomi Ohgi for her secretarial assistance. REFERENCES 1. LaBrecque D. Liver regeneration: a picture emerges from the puzzle. Am J Gastroenterol 1994;89:S86-S96. 2. Farivar M, Wands JR, Isselbacher KJ. Effect of insulin and glucagon on fulminant murine hepatitis. N Engl J Med 1976;295:1517-1519. 3. Baker AI, Jaspan JB, Haines NW. A randomized clinical trial of insulin and glucagon infusion for treatment of alcoholic hepatitis. Gastroenterology 1981;80:1410-1414. 4. Jaspan JB, Landan RL, Schneider J. Insulin and glucagon infusion in the treatment of liver failure. Arch Intern Med 1984;144:2075-2078. 5. Harrison PM, Hughes RD, Forbes A. Failure of insulin and glucagon infusion to stimulate liver regeneration in fulminant hepatic failure. J Hepatol 1990;10:332-336. 6. Michalopoulos GK. Liver regeneration: molecular mechanisms of growth control. FASEB J 1990;4:176-187. 7. Ishiki Y, Ohnishi H, Muto Y, Matsumoto N, Nakamura T. Direct evidence that hepatocyte growth factor is a hepatotropic factor for liver regeneration and has a potent antihepatitis effect in vivo. HEPATOLOGY 1992;16:12271235. 8. Ying SY. Inhibins, activins, and follistatins: gonadal proteins modulating the secretion of follicle-stimulating hormone. Endocr Rev 1988;9:267-293. 9. DePaolo LV, Bicsak TA, Ericson GF, Shimasaki S, Ling N. Follistatin and activin: a potential intrinsic regulatory system within diverse tissues. Proc Soc Exp Biol Med 1991;198:500-512. 10. Yasuda H, Mine T, Shibata H, Eto Y, Hasegawa Y, Takeuchi T, Asano S, et al. Activin A: an autocrine inhibitor of initiation of DNA synthesis in rat hepatocytes. J Clin Invest 1993;92:1491-1496. 11. Nakamura T, Takio K, Eto Y, Shibai H, Titani K, Sugino H. Activinbinding protein from ovary is follistatin. Science 1990;247:836-838. 12. Farnworth UM, Findlay JK. Follistatin: more than follicle-stimulating hormone suppressing proteins. Mol Cell Endocrinol 1993;91:1-11. 13. Kogure K, Omata W, Kanzaki M, Zhang YQ, Yasuda H, Mine T, Kojima I. A single intraportal administration of follistatin accelerates liver regeneration in partially hepatectomized rats. Gastroenterology 1995;108:11361142. 14. Higgins GM, Anderson RM. Experimental pathology of the liver. Arch Pathol 1931;12:186-202. 15. Fishback FC. A morphologic study of regeneration of the liver after partial removal. Arch Pathol 1929;7:956-977. 16. Byvoet P. Determination of nucleic acids with concentrated H2SO4 . Anal Biochem 1966;15:31-39. 17. Hinegardner TT. An improved fluorometric assay for DNA. Anal Biochem 1971;39:197-201. 18. Nakamura T, Sugino K, Titani K, Sugino H. Follistatin, an activin-binding protein, associates with heparan sulfate chains of proteoglycans on follicular granulosa cells. J Biol Chem 1991;266:19432-19437.
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