Pretreatment with FK506 up-regulates insulin receptors in regenerating rat liver

Pretreatment With FK506 Up-regulates Insulin Receptors in Regenerating Rat Liver ´ Pina, ˜ Jesus ´ Fueyo, Ce´ sar Menor, Oscar Escribano, Marı´a Dolores Ferna´ ndez-Moreno, Marı´a Jesus Irene Dolores Roma´ n, and Luis G. Guijarro This report examines the effect of FK506 pretreatment on liver insulin receptor expression in partially (70%) hepatectomized rats. FK506 pretreatment led to an increased insulin receptor number 24 hours after hepatectomy, detected by means of insulin binding and crosslinking procedures. This increase was related to enhanced insulin receptor expression determined by in vitro mRNA translation and Western blot techniques. We also tested the functionality of the expressed insulin receptors by [3H] thymidine incorporation into DNA in insulin-stimulated hepatocytes. The results show that FK506 pretreatment elicits an increase in the amount of insulin receptor ␣–subunits as measured by Western blot. Maximum ␣-subunit expression recorded 24 hours after surgery was preceded by increased insulin receptor mRNA levels, which were detected 6 hours after hepatectomy. Moreover, in FK506 –pretreated rat hepatocytes, obtained from remnant livers 24 hours after partial hepatectomy (PH), the increase in insulin receptor number was associated with improved sensitivity to the hormone. However, in both experimental groups (FK506-pretreated and nonpretreated rats), the sensitivity of hepatocytes toward epidermal growth factor (EGF) showed no significant change, which suggests a specific effect of FK506 on insulin receptor expression. In conclusion, our findings suggest that FK506 pretreatment induces insulin receptor expression in regenerating rat liver and promotes liver regeneration in hepatectomized rats. (HEPATOLOGY 2002;36:555-561.)

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retreatment with immunosuppressive drugs, such as FK506 and cyclosporin A (CsA), increases the liver regenerative response after partial hepatectomy (PH) in rats.1 Several research efforts have tried to determine the factors involved in the proliferative response associated with drug treatment. FK506 or CsA could modulate the liver response by (1) increasing the expression of local mitogens such as hepatocyte growth factor2 or insulin-like growth factor I3; (2) decreasing the production of inhibitory cytokines such as transforming

Abbreviations: CsA, cyclosporin A; PH, partial hepatectomy; ALT, alanine aminotransferase; AST, aspartate aminotransferase; EGF, epidermal growth factor. From the Unidad de Toxicologı´a Molecular Hepa´tica, Departamento de Bioquı´mica y Biologı´a Molecular, Universidad de Alcala´, E-28871, Alcala´ de Henares, Spain. Received December 6, 2001; accepted June 19, 2002. Supported by a grant from the Direccio´n General de Investigacio´n Cientı´fica y Te´cnica (PM 98-0154). Address reprint requests to: Luis G. Guijarro, M.D., Unidad de Toxicologı´a Molecular Hepa´tica, Departamento de Bioquı´mica y Biologı´a Molecular, Universidad de Alcala´, E-28871, Alcala´ de Henares, Spain. E-mail: luis.gonzalez@ uah.es; fax: (34) 918854585. Copyright © 2002 by the American Association for the Study of Liver Diseases. 0270-9139/02/3603-0006$35.00/0 doi:10.1053/jhep.2002.35439

growth factor ␤21 or interleukin 24; (3) inhibiting natural killer activity4; or (4) predisposing the liver to regenerative signals. This last hypothesis has been suggested but not shown. Insulin is considered a secondary mitogen that enhances liver sensitivity to mitogens during its regeneration.5 Administering insulin to 70% PH rats increases [3H] thymidine labeling of DNA in hepatic parenchyma cells.6 Insulin induces a specific peak in [3H] thymidine incorporation 24 hours after PH, which is not observed in insulin-untreated or glucagon-treated animals.6 This 24hour period after PH also coincides with maximal effects of FK5061 or CsA2 on the liver regenerative response. Moreover, the role of insulin in liver regeneration is supported by its blunting of the immediate-early gene response after PH in diabetic animals.7 In a very recent study performed in 70% PH/50% pancreatectomized rats, it was shown that liver mass recovery in these animals was delayed with respect to that in simply hepatectomized animals.8 These data all support a potential role for insulin during hepatic regeneration. Because FK506 inhibits insulin secretion in cultured cells9 and FK506 treatment in transplant patients has been associated with low serum 555

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insulin levels in the absence of insulin resistance,10 we hypothesized that FK506 could stimulate the regenerative process that follows PH regulating the insulin signal transduction system. We thus undertook the following in FK506-pretreated and partially hepatectomized rats and in the corresponding control animals: (1) determination of plasma glucose, albumin, alanine aminotransferase(ALT), and aspartate aminotransferase (AST); (2) determination of insulin binding to liver membranes and subsequent molecular analysis of insulin receptors by cross-linking procedures; (3) quantification of insulin receptor ␣-subunits by immunoblot procedures; (4) analysis of insulin receptor mRNA levels; and (5) determination of the responsiveness of cultured hepatocytes to insulin.

Materials and Methods Animals, FK506 Treatment, and PH. Male Wistar rats (180 to 200 g) were intramuscularly administered FK506 (1 mg/kg body weight) daily for 4 days before surgery (treated group). A further group receiving an equivalent volume of saline (before surgery) served as the control. The animals were allowed free access to food and tap water. After treatment, animals were subjected to 70% PH as reported elsewhere.11 At the follow-up times 0, 1⁄4, 1, 2, and 7 days after surgery, rat livers were harvested or hepatocytes were isolated as described below. The animals were caged and handled according to the guidelines detailed in the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Institutes of Health. Materials. FK506 was purchased from Fujisawa GmbH (Munich, Germany) and stock solutions were prepared in pyrogen-free normal saline. Insulin and human epidermal growth factor (EGF) were obtained from Boehringer (Barcelona, Spain). Antibiotic-antimycotic solution, collagen, sodium dodecyl sulfate, and dithiothreitol were purchased from Sigma (Alcobendas, Spain). Collagenase (type A) was provided by Life Technologies (Madrid, Spain). Williams’ medium E and bovine fetal serum were from Gibco (Madrid, Spain). [3H] thymidine (64 Ci/mmol) was obtained from ICN Pharmaceuticals (Madrid, Spain) and rabbit reticulocyte lysate from Promega (Madrid, Spain). Insulin was labeled with Na125I by a chloramine-T method12 and purified on a Sephadex G-50 column. The antibody against insulin receptor ␣-subunit and goat antirabbit antibody conjugated to horseradish peroxidase were provided by Santa Cruz Bio-

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technologies, Inc (Santa Cruz, CA). Disuccinimidyl suberate and a chemiluminescent substrate were obtained from Pierce (Rockford, IL). Acrylamide/bisacrylamide were from Bio-Rad Laboratories (Hercules, CA). Hyperfilm ECL was provided by Amersham Pharmacia Biotech (Buckinghamshire, UK). The Ultraspec RNA Isolation System was supplied by Biotecx (Houston, TX). All other reagents were of the highest grade of purity available. Quantitative Plasma Analysis. After killing the animals, blood was collected in heparinized tubes and centrifuged at 2,000g; plasma samples were stored at ⫺80°C until use. ALT, AST, and glucose levels were determined using an Ektachem DT60 II analyzer. Albumin was identified by cellulose acetate electrophoresis and subsequent computer-assisted densitometry. Liver Membrane Isolation. A partially purified fraction of liver plasma membranes was obtained by centrifugation in a sucrose gradient as previously described.13 Plasma membranes were resuspended in Krebs Ringer phosphate buffer (0.3 mg tissue/mL, pH 7.4) and stored at ⫺80°C until use. Protein content was determined by the Bradford method.14 Binding Assay. Liver plasma membranes (150 ␮g protein) were incubated for 45 minutes at 30°C in Krebs Ringer phosphate buffer containing bovine serum albumin (1%), [125I] insulin (20 pmol/L), and increasing concentrations of unlabeled insulin (10⫺10 to 10⫺6 mol/L). Unbound insulin was separated by centrifugation and the [125I] insulin fraction bound to the membrane was tested for radioactivity. Nonspecific binding was determined in the presence of unlabeled insulin (1 ␮mol/L). Cross-linking of [125I] Insulin to Its Receptor. Chemical cross-linking of peptide-receptor complexes was achieved using disuccinimidyl suberate (100 mmol/ L). Membrane proteins (0.4 mg/mL) were allowed to bind to the tracer in the absence or presence of unlabeled insulin (1 ␮mol/L) in 5 mL of incubation medium. Labeled membranes were solubilized and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 8% gels under reducing conditions (dithiothreitol, 50 mmol/L) according to the method of Laemmli.15 The gels were dried and exposed to x-ray film. Western Blot Analysis of the Insulin Receptor ␣-Subunit. Liver membranes containing 40 ␮g of protein were loaded onto 8% SDS-PAGE gels in reducing conditions (dithiothreitol, 50 mmol/L) and transferred to nitrocellulose membranes overnight. The membranes were developed using the specific antibody for the insulin receptor ␣-subunit. Horseradish peroxidase conjugated goat antirabbit antibody was used as the secondary antibody. Enhanced chemiluminescence reagents were used

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according to the manufacturer’s instructions, and the resultant membranes were exposed to X-Omat AR film. Autoradiographs were then quantified by computer-assisted densitometry. In Vitro Translation. Total cellular RNA was isolated from frozen rat liver by the Ultraspec RNA Isolation System procedure (Biotecx, Houston, TX). To translate proteins in vitro,16 total RNA (4 ␮g) from each experimental group was added to 35 ␮L of rabbit reticulocyte lysate and to 1 ␮L of amino acid mix (50 ␮L final volume). The translation reaction was carried out for 90 minutes at 30°C, and the translated proteins were stored at – 80°C until Western blot analysis of the insulin receptor ␣-subunit, as described above. We detected a linear relationship between translated mRNA (0.5 to 4 ␮g) and the amount of insulin receptor ␣-subunit produced (data not shown). Hepatocyte Isolation. Six hours or 1 day after PH, hepatocytes from control and FK506 pretreated rats were isolated by in situ 2-step collagenase perfusion of the remnant liver.17 Cell viability, estimated by trypan-blue exclusion, was over 90%. The isolated cells were plated at a density of 105 cells/mL in 24 multiwell plastic dishes (1-mL volume) coated with rat tail collagen. They were then cultured in Williams’ medium E containing 10% fetal bovine serum and 1% antibiotic-antimycotic solution in a humidified atmosphere (5% CO2 in air) at 37°C. After incubation for 2 hours, the cells were washed twice in phosphate buffered saline and placed in serum-free Williams’ medium E. Unless otherwise indicated, at this time (time 0), insulin (0.1 ␮mol/L) or EGF (20 ng/mL) was added to the wells, and cells were cultured for 24 hours. Determination of DNA Synthesis. DNA synthesis was estimated by determining [3H] thymidine incorporation into trichloroacetic acid–precipitable material. Twenty-four hours before the end of the assay, 2 ␮Ci of [3H] thymidine were added to the wells. The cells were subsequently washed twice in phosphate buffered saline, and 0.5 mL of 5% trichloroacetic acid was added. After 20 minutes at 4°C, trichloroacetic acid was removed and 0.2 mL of KOH (2N) was added to the precipitate for 60 minutes at room temperature and neutralized with 0.25 mL of HCl (2N). The plate contents were emptied into a glass fiber filter and radioactivity determined. Statistical Analysis. Statistical analysis was performed using the GraphPad Prism package (GraphPad Software Inc., San Diego, CA) Values are reported as the mean ⫾ SEM. Unless otherwise indicated, data were compared by ANOVA and the Student’s t test. The level of significance was set at P ⬍.05.

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Fig. 1. FK506 pretreatment effect on liver and body weights 0, 1, 2, and 7 days after PH in FK506 (■)- and vehicle (䊐)-pretreated rats. Data are expressed as percent liver weight/body weight ratios for nonhepatectomized and FK506-nontreated rats. All values are mean ⫾ SEM (n ⫽ 8) (*P ⬍ .05).

Results Effect of FK506 Pretreatment on Hepatectomized Rat Liver Weight. We first evaluated the effect of FK506 pretreatment on the animals’ general status. Hepatic growth after PH in the FK506- or vehicle-treated animals is shown in Fig. 1. One day after PH, the liver weight/body weight ratio decreased in both groups. After this period, the liver weight/body weight ratio increased at a constant rate in both groups; this increase was higher in FK506-pretreated rats than in nonpretreated animals, and was significantly different 7 days after surgery. Thus, FK506 may have an effect on hepatic regeneration. Effect of FK506 Pretreatment on Hepatic Function in Hepatectomized Rats. Next we evaluated plasma variables related to liver injury. FK506 pretreatment showed no significant effect on plasma glucose levels after PH, neither in the control nor in the treated animals (Fig. 2A). However, a significant decrease in glucose levels was observed 1 day after PH in control and treated rats. Plasma albumin levels decreased 1 day after PH and reached control values 7 days after surgery (Fig. 2B). Changes in glucose and albumin levels after PH could be related to the surgical procedure. In nonhepatectomized animals (day 0), FK506 pretreatment induced a significant decrease in plasma albumin levels with respect to the control group, returning to normal levels 2 days after injury. Plasma AST and ALT levels showed significant increases 1 day after surgery (Figs. 2C and 2D), and returned to normal values 7 days after PH in both groups.

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These results indicate that FK506 pretreatment led to enhanced insulin receptor expression and that the maximum response occurred 1 day after PH. To examine whether the insulin receptor increase was associated with a corresponding increase in insulin receptor mRNA levels, total RNA was isolated from rat livers and subjected to in vitro translation. The translation products then underwent Western blot testing against the insulin receptor ␣-subunit. Six hours after PH, insulin receptor mRNA levels in FK506-pretreated animals showed a 2-fold increase over levels detected in nonpretreated rats (Fig. 6).

Discussion Fig. 2. FK506 pretreatment effect on hepatic function after PH. Plasma (A) glucose, (B) albumin, (C) AST, and (D) ALT levels at 0, 1, 2, and 7 days after PH in FK506 (■)- and vehicle (䊐)-pretreated rats. Albumin values are expressed as percentage plasma levels for nonhepatectomized and FK506-nontreated rats. All values are mean ⫾ SEM (n ⫽ 8). Data were subjected to analysis of variance with the Bonferroni test (*P ⬍ .05; **P ⬍ .01).

Effect of FK506 Pretreatment on Hepatocyte Responsiveness to Insulin or EGF. Given that insulin is known to be involved in liver regeneration, we examined the effect of insulin on hepatocyte proliferation. Hepatocytes from control or FK506-pretreated rats were obtained from remnant livers 1⁄4 or 1 day after PH. Only 1 day after PH, FK506 pretreatment significantly increased cell proliferation in the absence or presence of insulin; however, no significant changes were observed between the 2 groups in the presence of EGF (Fig. 3B). These findings suggest that FK506 could, at least partly, modify the insulin signaling pathway in this experimental model. Effect of FK506 on Insulin Receptor Expression. To evaluate insulin signaling in the liver regenerative response induced by FK506, we first explored insulin receptor binding capacity in purified liver membranes. [125I] insulin binding to liver membranes was determined 0, 1, and 7 days after PH in FK506-pretreated and control animals. A significant increase in maximal binding capacity was observed in animals pretreated with FK506 1 day after surgery. This increase was transient, because by 7 days after PH, insulin binding capacity returned to that recorded in the nonhepatectomized animals (Fig. 4). We then went on to perform a molecular study of insulin receptor cross-linking or Western immunoblotting. Cross-linking of [125I] insulin to its receptor in rat liver membranes increased 1 day after surgery in FK506-pretreated animals (Fig. 4, inset). This finding was consistent with data derived from insulin binding and immunodetection of the insulin receptor ␣-subunit in liver membranes (Fig. 5). However, 6 hours after PH, insulin receptor expression decreased slightly in both groups.

This report shows the effect of FK506 pretreatment on liver insulin receptor expression in 70% hepatectomized rats. Increased numbers of insulin receptors were detected both in [125I] insulin binding and in cross-linking experiments. These findings were found to correlate well with enhanced insulin receptor ␣-subunit expression detected

Fig. 3. Effect of FK506 pretreatment followed by PH on hepatocyte proliferation. The effect of in vivo FK506 pretreatment on mitogeninduced hepatocyte proliferation was explored. [3H] thymidine incorporation into DNA was evaluated in remnant liver hepatocytes, isolated (A) 6 hours and (B) 24 hours after 70% PH from rats pretreated with vehicle (䊐) or FK506 (■). The cells were incubated with insulin (100 nmol/L) or EGF (20 ng/mL) for 24 hours (as described in experimental procedures). Values are the mean ⫾ SEM of desintegrations per minute obtained in 7 experiments performed in duplicate. Significant differences with respect to controls were determined by ANOVA (**P ⬍ .01).

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Fig. 5. Effect of FK506 on insulin receptor ␣-subunit expression. (A) Western blot analysis of the insulin receptor ␣-subunit (129 kDa) in rat liver membranes. Rats were pretreated with vehicle (䊐) or FK506 (■) followed by PH, and killed 0, 1⁄4, 1, and 7 days after surgery. This is a representative experiment from 4 performed on 2 groups of rats. (B) Autoradiographs were subjected to computer-assisted densitometry.

generative response after PH,1 suggesting that FK506 increases the sensitivity of liver to insulin. In our in vitro translation experiments, an increased insulin receptor transcription rate preceded an increase in levels of the corresponding protein. Similarly, maximal effects of FK506 on insulin receptor transcription rate were observed 6 hours after PH. At this stage after hepatectomy, the amount of insulin receptor ␣-subunit decreased in

Fig. 4. Effect of FK506 pretreatment followed by PH on insulin receptor binding capacity. [125I] insulin binding to rat liver membranes 0, 1, and 7 days after PH, in vehicle (E)- and FK506 (●)-pretreated rats. Increasing unlabeled polypeptide concentrations were used. Values represent specific [125I] insulin binding to liver membranes as a percentage of total [125I] insulin in the assay medium. Insets show autoradiographs of [125I] insulin cross-linked to rat liver membranes obtained from 0-, 1-, and 7-day hepatectomized animals. NS corresponds to control group autoradiographs (nonhepatectomized and FK506-nontreated rats) obtained in the presence of 1 ␮mol/L unlabeled insulin; C and T correspond to the control and FK506-treated groups, respectively, in the absence of unlabeled insulin.

by in vitro mRNA translation and Western blotting. We also tested the functionality of the expressed insulin receptors through their ability to induce [3H] thymidine incorporation into DNA. FK506 pretreatment increased insulin receptor expression at all of the follow-up times established, although its greatest effect was shown 24 hours after PH. At this time, tacrolimus also shows its maximal effect on the liver re-

Fig. 6. FK506 effect on insulin receptor transcription. (A) Western blot analysis of the insulin receptor ␣-subunit (129 kDa) obtained by in vitro–translated mRNA from remnant livers 1⁄4, 1, and 7 days after PH in FK506 (■)- or vehicle (䊐)-treated animals. (B) Autoradiographs were subjected to computer-assisted densitometry. This is a representative experiment out of 6 performed.

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both experimental groups, as did hepatocyte sensitivity toward insulin or EGF. These data suggest an adaptive response to PH, a phenomenon previously described for circulating insulin levels.18 However, insulin receptor expression was more quickly recovered in liver extracts from FK506-pretreated rats compared with the control group. Moreover, 24 hours after PH, the liver of FK506-pretreated rats showed an increased number of insulin receptors associated with higher hepatocyte sensitivity toward the hormone, and the sensitivity of both groups of hepatocytes to EGF was unmodified. These findings suggest that the FK506 effect on insulin-dependent hepatocyte proliferation is caused by an increased insulin receptor transcription rate, rather than by effects on the transduction pathway shared by insulin and EGF.19 In previous studies, an inhibitory posttranslational effect of FK506 on insulin receptor expression was noted in cultured bovine adrenal gland cells.20 However, similar discrepancies in the effects of FK506 on hepatocyte proliferation have been observed when comparing in vitro21 or in vivo1 models. In the same way, treatment of cultured hepatocytes with CsA inhibits thymidine incorporation into DNA21 and increases apoptosis,22 whereas the opposite occurs after the in vivo administration of CsA before PH.23 The changes in insulin receptor expression after FK506 treatment observed here cannot be attributed to a diabetic state because neither drug pretreatment nor PH provoked a dramatic change in circulating glucose levels. A slight decrease in glucose levels was recorded in the control group 24 hours after PH, but this could be related to the fasting state of the animals after surgery. The mechanism of glucose homeostasis during the regenerative period remains to be established. Initial reports suggest that insulin levels show an abrupt decrease within the first minutes of PH18 and return to normal levels after a few hours.24 In general terms, the changes we observed in plasma metabolites (glucose, albumin, AST, and ALT) could be more related to PH than to FK506 pretreatment. The fact that no significant effects of FK506 on glucose levels were noted during liver regeneration suggests that FK506 does not significantly modify glucose metabolism in our model. In addition, the enhanced liver regeneration induced by FK506 was unassociated with a loss of specific liver functions such as albumin synthesis. The mechanisms involved in the selective effect of FK506 on liver insulin receptor expression during liver regeneration are unknown. Our results suggest that FK506 exerts its effect at the transcription level. It has been recently shown that FK506 binding protein–12 can physically associate with high-mobility group proteins25 and in turn regulate insulin receptor gene expression.26 Indeed, rat liver FK506 binding protein–12 levels have

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been shown to increase in response to PH.27 Thus, it is possible that this protein increases liver sensitivity to FK506 after PH. Modulatory factors such as these could have direct clinical applications, and their effects on insulin therapy after PH are currently being evaluated. In the rat, insulin treatment is able to enhance liver regeneration after PH impaired by either ethanol28 or CCl4.29 Moreover, intraportal insulin injection promotes the recovery of remnant liver functions in hepatectomized patients.30 The results reported here suggest that FK506 promotes liver regeneration in hepatectomized rats by increasing liver sensitivity to insulin. Acknowledgment: FK506 was provided by Dr. Murato from Fujisawa GmbH (Munich, Germany). Experimental animals were housed at the Centro de Experimentacio´ n Animal, Universidad de Alcala´ (Madrid, Spain).

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23. Khan D, Makowka L, Lai H, Eagon PK, Dindzans V, Starzl TE, Van Thiel DH. Cyclosporin augments hepatic regenerative response in rats. Dig Dis Sci 1990;35:392-398. 24. Macho L, Fickova´ M, Zorad S, Knopp J. Changes of insulin and glucagon binding to receptors in hepatocytes during liver regeneration. Physiol Res 1994;43:281-187. 25. Dolinski KJ, Heitman J. Hmo lp, a high mobility group 1/2 homolog, genetically and physically interacts with the yeast FKBP12 prolyl isomerase. Genetics 1999;151:935-944. 26. Brunetti A, Manfioletti G, Chiefari E, Goldfine ID, Foti D. Transcriptional regulation of human insulin receptor gene by the high-mobility group protein HMGI(Y). FASEB J 2001;15:492-500. 27. Francavilla A, Carr BI, Starzl TE, Azzarone A, Carrieri G, Zeng QH. Effects of rapamycin on cultured hepatocyte proliferation and gene expression. HEPATOLOGY 1992;15:871-877. 28. Imano M. Effect of glucagon and insulin administration on the inhibition of rat liver regeneration by acute ethanol treatment after partial hepatectomy. Nihon Arukoru Yakubutsu Igakkai Zasshi 1998;33: 241-251. 29. Hashimoto M, Kothary PC, Eckhauser FE, Raper SE. Treatment of cirrhotic rats with epidermal growth factor and insulin accelerates liver DNA synthesis after partial hepatectomy. J Gastroenterol Hepatol 1998;13: 1259-1265. 30. Wang HJ, Kim JH, Kim WH, Kim MW. Intraportal insulin therapy after partial hepatectomy for hepatoma patients with insulinopenia. Hepatogastroenterology 2000;47:465-467.