Al142 AASLD ABSTRACTS GASTROENTEROLOGY, Vol. 108, No. 4

• REGULATION OF HEPATIC CHOLESTEROL AND BILE ACID BIOSYNTHESIS BY GLUCAGON AND ENTEROGLUCAGON. W.M. Pandak, D.M. Heuman, G. Compton, P.B. Hylemon and Z.R. VlahceWc. Depts. of Medicine and Microbiology, McGuire Veterans Affairs Medical Center and Medical College of Virginia, Richmond, Viriginia 23249.

Background: We have previously shown that addition of glucagon (G) to primary rat hepatocytes results in a marked concentration dependent decrease in cholesterol 7c~-hydroxylase (C7~H) mRNA levels and transcritional activity. Enteroglucagon (E), a full agonist for the hepatic glucagon receptor which is released from the ileum in response to bile acid infusion, could represent an intestinally derived progluagon involved in the regulation of C7~xH and bile acid synthesis. Therefore, it is important to determine the potential role of these polypeptides in the regulation of C7c~H in an in vivo model. Specific aim: To determine the effect of (G) or (E) administration on three key enzymes which participate in the maintenance of cholesterol homeostasis in the liver: HMG- CoA reductase (HMGR; rate-determining enzyme of the cholesterol biosynthetic pathway), C7c~H (rate-determining enzyme in the "neutral" bile acid biosynthetic pathway), and sterol 27-hydroxylase ($27OH; initial enzyme of the "acidic" bile acid biosyntheti c pathway). Methods: G or E was administered as a continuous intravenous infusion (15 to 300 ng/kg-min for 24h) to chronic biliary diverted rats. Bile was collected continuously for determination of rates of bile acid synthesis. At the study conclusion, livers were harvested and the specific activities of HMGR, C7c~H and $27OH were determined. In addition, C7cxH steady-state mRNA levels were quantitated. Results: G infusion (75, 150 and 300 ng/kg-min rat) decreased HMGR and C7c~H specific activities to 0_ 15 %, 36+ 19 % and 604-11%, and 42 4-3 %, 444-13% and 814-6% of paired control values, respectively. The specific activity of $27OH was decreased 45% with a 300ng/kg-min infusion. G infusion (300 ng/kg-min) also resulted in an approximate 50% decrease in C7c~H steady-state mRNA levels, and an 85% decrease in bile acid synthesis with no significant change in bile salt composition. In contrast infusion Of E (15 and 150 ng/kg-min) failed to reduce C7c~H specific activity or bile acid synthesis. Conclusion: Glucagon appears to be a potent down-regulat0r of cholesterol and bile acid biosynthesis of potential physiologic importance. Enteroglucagon does not appear to be involved in the regulation of C7cxH and bile acid biosynthesis.

IN VIVO ICAM-I EXPRESSION IN PORTAL HYPERTENSION. EFFECTS OF ENDOTOXIN. J Pan~, MA Perry#, DC Anderson*, DN Granger. Dept Physiology. LSU Medical Center, Shreveport, LA 7111L #Universitty of Soutn Wales, Sidney, Australia. *Upjohn Laboratories, Kalamazoo, M149001. Increased circulating levels of ICAM-1 have recently been reported in various types of liver disease. However, the origin of this ICAM-lin plasma remains obscure since measurements of ICAM- 1 expression in different organs have no t been reported to date. The objectives of the present study were to characterize and compare the constitutive expression of ICAM-1 in portal hypertensive and control rats, and the changes in ICAM-1 expression elicited by endotoxin. Methods: Portal hypertension was induced by partial portal vein ligation (PPVL). ICAM-1 expression was measured using t25I labeled anti-rat ICAM-1 mAb (1A29), and an isotype matched control mAb (~3tl-P-23, anti- dog ICAM-1) to correct for nonspecific accumulation of the binding mAb. ICAM- 1 expression was studied in PPVL and control rats and in animals treated (for 5 hours) with ahigh dose (5 mg/Kg) or low doses (1 and 10 ~tg/Kg) of endotoxin. Results: In basal conditions ICAM-1 mAb binding was observed in all organs from both PPVL and control rats. However, the magnitude of accumulation of ICAM-1 mAb varied widely in the different organs, with the highest content (per g tissue) detected in the lung, followed by liver, spleen and kidneys (1/2 lung activity) heart (1/20), splanchnic organs (1/50), thymus (1/100), testes and brain (1/300), and skeletal muscle (1/500). ICAM-1 content (per g tissue) in the different organs paralleled endothelial surface area (per g tissue). No differences were observed in the levels of constitutive ICAM- 1 expression between PPVL and control rats in any organ. Endotoxin dose-dependently increased ICAM-1 expression in all organs studied, except in the liver and spleen, in both PPVL and control animals. However, low doses of endotoxin that were without effect on ICAM-1 expression in the lung, heart, and kidney of control animals, significantly increased ICAM-1 expression in these organs in PPVL rats. The response to 10w dose endotoxin in splanchnic organs was similar in PPVL and control rats. High-dose endotoxin had the same effect in increasing ICAM-1 expression in PPVL and in control rats. Conclusions: Portal hypertension is associated with an increased upregulation of ICAM- 1 in response to low doses of endotoxin in the lung, heart, and kidneys. This may be related to a higher bioavailability of endotoxin due the existence of porto-systemic shunting. (Supported by DK 43785)

• EXPRESSION OF EARLY RESPONSE GENES AFTER MASSIVE HEPATECTOMY IN RATS

Y. Panis, N. Lomri,* J.C. Emond Liver Transplant Program and the Liver Center

*Department of Medicine and UCSF Liver Center University of California, San Francisco

INTRODUCTION The relationship between hepatic regeneration and the expression of early response genes has been widely characterized in nonqethal 70% hepatectomy (70% H) in rats. This model represents "normal" regeneration resulting in uniform survival. It is not known whether these early signals occur normally following 90% lethal hepatectomy (90% H). The finding of a altered profile of gene expression in 90% H could be used predictively to obtain an early diagnosis of tiver failure after hepatectomy or partial liver transplantation. The aim of this study was to compare proto-oncogene expression after 70% H and 90% H in rats.

MATERIAL & METHODS 110 Wistar rats underwent sham (n=30), 70% H (n=40), or 90% H (n=40).

Animals were sacrificed at intervals. Livers were excised and divided into 4 equal specimens, snap frozen, and stored at -70~C. RNA was extracted by standard methods and preparations probed for proto-oncogenes, c-myc, c-foe, and for hepatocyte growth factor HGF and it receptor c-MET. After Overnight exposure of autoradiographs, quantification was accomplished by densitometry of RNA slot blots.

RESULTS After 70% H, peaks of maximal expression for both c-myc and c-MET were

observed for t hour (5- and 10- fold, respectively) and 12 hours (10-fold for both); for c-foe, peak of maximal expression was observed at 6 hours (g-fold); for HGF, peak was observed between 12 hours and Day 2 (10-fold).

After 90% H, rats demonstrated similar patterns including peak expression of c- myc at I hour (5-fold), but altered peak at 12 hours (6-fold); for c-MET, the same pattern was observed between 1 and 12 hours (5-fold). For HGF, two peaks were noted: A first peak at 1 hour (10-fold), and a peak similar to the peak observed after 70% H at 12 hours.

CONCLUSION These results suggest that early molecular events which are part of the

regenerative response are !argely intact after massive hepatectomy. In this study, 90% H demonstrated eady response patterns similar to those following 70% H. Our results suggest that ttver dysfunction and the failure of regeneration following 90% H is not due to alteration of proto-oncogenes expression. It is possible that progressive injury to the small hepatic remnant prevents recovery despite intact early signaling.

CYTOKINES AND PROGRESSION OF LIVER DAMAGE IN EXPERIMENTAL BILE DUCT LIGATION. M.P. Panozzo, M. De Paoli, D. Basso, M.L. Valente* e M. Plebani. Departments of Laboratory Medlclne and *Pathology, University of Pedova, Italy

Cytokines are multipotential molecules produced and activated in noz~nal and/or pathological conditions. Some of them can modulate several aspects of hepatic damage either of acute or chronic origin. Aims of thls study were to verify: i. the variations in hepatic levels of transforming growth factor beta I (TGF B1), epidermal growth factor (EGF), tumor necrosis factor (TNF) and interleukin 6 (IL-6) In extrehepatic cholestasis induced by bile duct ligatlon (BDL); 2. the role of these cytoklnes in influencing the cholestatic picture. 38 male sprague Dawley rats (300 g), among which 4 sham operated, were bile duct llgated for 1 (n=Sl, 2 (n=5), 5 In=4), 8 (n=4), 10 (n=4), 14 (n=6), 22 (n=3) and 28 (n=3) days. At the end of each follow up time, TGF B1 (Genzyme), EGF (DSL), TNF and IL-6 (T Cell Diagnostic) were assayed in hepatic homogenates (1/20 phosphate buffer 0.05 M pH 7.4). Liver histology considered: ductular proliferation (DP), inflammation lII), cell necrosis (CN) after R&E and perlportal (PF), perivenular (VF) and perlneoductular (NF) liver fibrosis after Gomorl staining. Results: both EGF and TGF B1 progressively increased in relation to BDL times, although variations were significant only for EGP (F=4.81, p<0.001). TNF decreased early after BDL and thereafter increased showing two peaks on days 8 and 14 (F=5.95, p<0.001). IL-6 progressively decreased reaching 0 levels after 21 days of BDL (F=7.97, p<0.S01). EGF inversely correlated with IL-6 (r=-0.33, p<0.05) while TGF B1 with TNF lr--0.34, p<0.05). All cytokines but TGF B1 correlated with total billrubin (r=0.51, p<0.005 for EGF, r=0.66, p<0.001 for TNF and r=-0.39, p<0.05 for IL-6). The variations of DP, II and CN were consensual with those of EGF; on the other hand, PF, NF and VF were directly correlated with TOP B1 and inversely with TNF and IL-6. Conclusions: During extrahepatic cholestasis, the hepatic levels of TGF BI, EGF, TNP and IL-6 vary differently. In particular: i. EGF seems mainly associated with inflammatory and necrotic, other than proliferative phenomena. 2. The correlations found between hepatic TGF B1 and the degree of fibrosis, seem to support the pivotal role of this cytoklne in modulating the onset and the progression of extracellular matrix deposition. 3. The waving pattern of TNF indicates that this substance is probablyccunterregolated by several events not strictly associated to the cholestatlc process. 4. The severe reduction of IL-6 after long term BDL suggests that this condition inhibits the hepatic production of this cytokine while favours that of other cytokines.