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Inhibition of vesicle transport and lipid secretion into bile by calpain inhibitor-1 in isolated perfused rat liver (IPRL)
April 1995
INHIBITION O16 VESICLE TRANSPORT AND LIPID SECRETION INTO BILE BY CALPAIN INHIBITOR-1 IN ISOLATED PERFUSED RAT LIVER (IPRL). K.Mizuno, T.Hayakawa, Y.Kamiya, H.Ohara, T.Yamada, H.Yamada, T.Nakazawa, 'T.Inagaki, A,Uchida, M.Hoshino, M.Miyaji, T.Takeuchi 1st Dept. of Internal Medicine, School of Medicine, Nagoya City University. Nagoya, JAPAN Ca~-dependent neutral proteases (calpains) have been reported to exist in the cytosol of rat liver, however, the physiological role of calpains in fiver have yet to be elucidated. Calpain inhibitor-1 (CI-1;N-acetyMeucyl-leucylnorleueinal) is a cysteine protease inhibitor which strongly inhibits the activities of ealpains. We investigated the effects of CI-I on the biliary excretion of lipids and horseradish peroxidase (HRP), a conventional marker of transcytotic vesicle transport, in the single-pass IPRL study. During constant infusion of tanroeholate (251,tM), a 15-minute infusion of CI-1 (10p.M) increased bile flow (l.tl/mirgg.liver) from 2,21_+0.05 to 3.49-+0.23, and increased slightly bile acid secretion (nmol/min/g.liver) from 107.6-+3.98 to 125.4:£-6.34. However, CI-1 reduced significantly the biliary excretion of phospholipids (nmol/min/g.liver)from 6.57+0.40 to 3.99-+0.47. The phospholipid/bile acid molar ratio declined from 0.062+0.005 to 0.033-+0.004. It has been shown that in vitro CI-1 inhibits lysosomal enzymes, including eathepsin B, cathepsin L. However, the lysosomotropic agent chloroquine did not affect hiliary lipid excretion, suggesting that cathepsins are not the site of action of this inhibitor. When HRP (25rag) was infused for 1 min as a pulse load in the portal vein during a continuous infusion of taurocholate, HRP appeared in bile in an early and late peak. CI-1 (10p,M) reduced HRP excretion in the late peak, from 0.466+_0.092 to 0.192+0.050 (nmol/min/g.liver), We also found that the Ca 2÷ -agonist vasopressin erthanced HRP excretion at a low concentration (330pmol/l) without causing uneven perfusion secondary to vasoconstriction. Further, this vasopressin-stimulated HRP excretion was reduced by CI-1 (10p.M). Thus, CI-1, a cell-permeable potent inhibitor of calpains, causes both the uncoupling of biliary lipid secretion from that of bile acids and the inhibition of transcytotie vesicle transport labelled by HRP. These results suggest that eytosolie Ca~-dependent neutral proteases (calpains) may be involved in lipid secretion and vesicle transport in bile secretion.
• EFFECT OF U R S O D E S O X Y C H O L I C ACID ON LIVER ENZYMES AND TITER OF ANTI-HCV-IGM AND SEMIQUANTITATIVE HCV RNA IN PATIENTS WITH SUBTYPED CHRONIC HEPATITIS C. M~ M6hler, S. Seipp, U. Tfx, B. Kallinovsky, W. Stremmel, L. Theilmann and T. Goeser, Dept. of Int. Medicine, UniversiW of Heidelberg, Germany Ursodenxycholic acid (UDCA) admimsta'aaon has been associated with the reduction of serm~ fiver enzymes in patients with cbrome hepatitis C and with improvement of liver histology. Whether this effect of UDCA is induced by a cholore'tie effect or a tmmunomodnintory way of actaon is at present unclear. Therefore we thought to investigate the effect of UDCA in patients with chronic HCV infection by monitoring serum liver enzymes, HCV RNA titers and, in addition, anti-HCV IgM titars. Anti-HCV IgM have been found in acute and chronic active hepatitis C virus (HCV) infections, while resolved or chronic persistent hepatitis C are less commonly associated with IgM antibodies indicating that antiHCV IgM might be a marker for active hepatitis C virus replication. Methods: Sera of 19 chronic HCV-infected patients (pat.) were sampled before, dunng and affzr UDCA (500 mg q.i.d., 3 months). All sera were tested for liver eazzymes, senuquantitative HCV RNA (RT-PCR) and anti-HCV IgG and lgM Titer (Abbott). Aider extraction of RNA, RNA was reverse transcribed and amplified with a nested PCR using primers of the noncoding- 5'-region and adjacent HCV core region. Quantiiieation was done semiquantitatively by diluting HCV RNA prior to a single step RT-PCR. Amplified eDNA was detected by Southern blot. Genotyping was done by a reverse-hybridization line probe assay (Innolipa). Results: Before treamaent, all sera were anti-HCV IgG and HCV RNA positive. Most prevalent was the HCV genotype lb (67%), followed by la, 2a and 3a 11% each. Although no changes of levels of ALT were observed in 3 pat. 16/19 pat. (84%) had sigmficant decrease of ALT under UDCA (mean ALT before UDCA 90.7 U/1 vs. during UDCA 40.7 LIB;p<0.001). ;tGT significantly decreased in all pat. (mean "fGT before UDCA 75.5 U/I, during UDCA 25.1 U/I ;p<0.001). During and after UDCA~ 50% of patients had positive anti-HCV IgM status and their titer remained unchanged as did HCV ~'LNA.Semiquantitauve HCV P,aNA did not change sigmficunfly during or alter UDCA. Conclusions: 1. A three months treatment with UDCA has sigmficuntly improved transaminases and cholestatic enzymes in patients with chronic hepatitis C (mostly HCV lb). 2. UDCA has no positive impact on titer ofHCV RNA. 3. AntiHCV IgM arc found in ordy 50% of patients with chronic hepatitis C infection with no change of its titer as irrmaunresponseto UDCA. 4. Thus the mayor improvement of liver euzymes seems to be induced by the choloretie effect of UDCA.
AASLD
Al125
THE UNCOUPLING OF BILIARY LIPID FROM BILE ACID SECRETION BY FORMYL-METHIONYL-LEUCYL-PHENYLALANINE (FMLP) IN THE ISOLATED PERFUSED RAT LIVER (IPRL). K.Mizuno, T.Hayakawa, YKamiya, H.Ohara, T.Yamada, H.Yamada, T.Nakazawa, T.Inagaki, A.Uchida, M.Hoshino, M.Miyaji, T.Takeuchi. 1st Dept. of Internal Medicine, School of Medicine, Nagoya City University. Nagoya, JAPAN Hepatobiliary diseases such as pericholangitis and sclemsing cholangitis am known to be associated with some patients with inflammatory bowel disease (IBD). In experimental colitis, neutrophil chemotactic factor FMLP, is reported to circulate enterohepatically, but its effect on bile secretion is unclear. We investigated the effect of FMLP on bile secretion including biliary excretion of horseradish peroxidase(HRP), a conventional marker of transcytotic vesicle transport pathway, in a single-pass IPRL model. When FMLP was infused at different concentrations (2gM, 10pM, 20p.M) into the portal vein, bile flow increased and FMLP was excreted into Nle in its native form and in a dose-dependent manner. Approximately 12% of the infused dose was excreted into the bile independently of the presence of sodium taurocholate. During constant infusion of sodium tattrocholate (25gM), a 15 minute infusion of FMLP (20gM) resulted in a significant and reversible decrease in biliary excretion of phospholipids in a dose dependent manner, whereas bile acid secretion increased slightly. Phospholipidfoile acid molar ratios declined from 0.07+0.01 to 0.04+0.01, indicating that FMLP induces the uncoupling of biliary excretion of phospholipid with that of bile acids. A one-minute load of HRP (25mg) under a continuous infusion of taurocholate produced an early and late peak in biliary excretion of HRP. FMLP (101.tM) reduced HRP excretion in the late peak, from 0.489-+0.052 to 0.269-+0.040 (nmol/min/g.liver). When bile samples collected during the infusion of FMLP (101.tM) was applied to gel-permeation chromatography using Sepharose CL4B column, elution profiles of bile demonstrated a specific association of FMLP with bile acids. These findings suggest that FMLP causes the uncoupling phenomenon as a result of an inhibition of transcellular vesicle transport and the interaction between FMLP and bile acid micelles in the bile canaliculus. Enterohepatic circulation of FMLP might be one of the factors important in mediating the hepatobiliary diseases associated with IBD.
THE METABOLIC CHARACTERISTICS DIFFERENTIATING NONALCOHOLIC STEATOHEPATITIS, DIABETES MELLITUS AND OBESITY, S Mokshagundam, A Peiris, J Stagner, CH Tamburro Liver Research Center, Dept of Medicine, Univ of Louisville, VA Medical Center, Louisville, KY and East Tennessee State Univ, Johnson City, TN Non-alcoholic steatohepatitis (NASH) is a clinical entity defined by persistent abnormal liver enzymes (ALE) and significant fatty infiltration in the absence of other causes of hepatic disease. Diabetes mellitus and obesity are frequently associated with NASH. The metabolic characteristics of 23 NASH patients, 15 obese and 9 lean individuals were studied and also clinically compared to 50 diabetics. The comparison parameters included biochemical liver tests, blood glucose, ferritin level, anthropometric measurement, lipid profile, insulin and c-peptide levels. Of the 23 NASI4 patients 9 had evidence of diabetes mellitus; 5 bad evidence of glucose intolerance; 13/23 were women with the ages ranging 3566, women being slightly older than the men; 56.5 yrs vs 48.7 yrs. Percent ideal body weights for women were 120-146%, for men 120-164%. Over 70% had waist/hip ratios of >0,88. CT scan evidence of fatty infiltration were present in all and histologically estimated at 30-80% of biopsy tissue. Over 80% had fasting dyslipidemia and persistently elevated basal insulin levels. All had elevated ferritin levels and 65% increased fibrosis or cirrhosis. In contrast, only 6% (3/50) of the diabetic group had ALE and 50% dyslipidemia. The body fat, lipid and insulin characteristics of NASH and obese groups are below. Table I
NASH/n= 16
OBESE/n= 15
LEAN/n=9
%IBW BMI WHR Triglyceride Cholesterol HDL-C LDL-C Basal Insulin Basal C-peptide
141.5+5.5 30.2+2.2 0.93+0.02 248+31.48 198.8+9.6 31.9+2.8 111+7.9 44.5+8.5 2.2+0.4
140.93+3.4 3I .4+0.7 0.94+0.02 138:4+ 19.9 207+ I2.8 39.7+2.6 139.5+11.2 12.2+3.4 2.1+0.3
104+3.3 22.7+ 1.7 0.85+0.01 100.7+ 16.8 168+ 12.2 45.6+3.8 98.1+11.3 7.7+0.8 1.35+0.6
NASH is a metabolic disorder characterized by significant fatty infihrat on, steatonecrosis, pets stent ALE, hypertriglyceridemia, elevated ferritin and markedly elevated basal insulin levels. These metabolic characteristics distinguish NASH from individuals with obesity and diabetes mellitus. Steatonecrosis may be related to insulin nonresponsiveness of hepatic cells leading to enzyme leakage and fibrogenesis.
INHIBITION O16 VESICLE TRANSPORT AND LIPID SECRETION INTO BILE BY CALPAIN INHIBITOR-1 IN ISOLATED PERFUSED RAT LIVER (IPRL). K.Mizuno, T.Hayakawa, Y.Kamiya, H.Ohara, T.Yamada, H.Yamada, T.Nakazawa, 'T.Inagaki, A,Uchida, M.Hoshino, M.Miyaji, T.Takeuchi 1st Dept. of Internal Medicine, School of Medicine, Nagoya City University. Nagoya, JAPAN Ca~-dependent neutral proteases (calpains) have been reported to exist in the cytosol of rat liver, however, the physiological role of calpains in fiver have yet to be elucidated. Calpain inhibitor-1 (CI-1;N-acetyMeucyl-leucylnorleueinal) is a cysteine protease inhibitor which strongly inhibits the activities of ealpains. We investigated the effects of CI-I on the biliary excretion of lipids and horseradish peroxidase (HRP), a conventional marker of transcytotic vesicle transport, in the single-pass IPRL study. During constant infusion of tanroeholate (251,tM), a 15-minute infusion of CI-1 (10p.M) increased bile flow (l.tl/mirgg.liver) from 2,21_+0.05 to 3.49-+0.23, and increased slightly bile acid secretion (nmol/min/g.liver) from 107.6-+3.98 to 125.4:£-6.34. However, CI-1 reduced significantly the biliary excretion of phospholipids (nmol/min/g.liver)from 6.57+0.40 to 3.99-+0.47. The phospholipid/bile acid molar ratio declined from 0.062+0.005 to 0.033-+0.004. It has been shown that in vitro CI-1 inhibits lysosomal enzymes, including eathepsin B, cathepsin L. However, the lysosomotropic agent chloroquine did not affect hiliary lipid excretion, suggesting that cathepsins are not the site of action of this inhibitor. When HRP (25rag) was infused for 1 min as a pulse load in the portal vein during a continuous infusion of taurocholate, HRP appeared in bile in an early and late peak. CI-1 (10p,M) reduced HRP excretion in the late peak, from 0.466+_0.092 to 0.192+0.050 (nmol/min/g.liver), We also found that the Ca 2÷ -agonist vasopressin erthanced HRP excretion at a low concentration (330pmol/l) without causing uneven perfusion secondary to vasoconstriction. Further, this vasopressin-stimulated HRP excretion was reduced by CI-1 (10p.M). Thus, CI-1, a cell-permeable potent inhibitor of calpains, causes both the uncoupling of biliary lipid secretion from that of bile acids and the inhibition of transcytotie vesicle transport labelled by HRP. These results suggest that eytosolie Ca~-dependent neutral proteases (calpains) may be involved in lipid secretion and vesicle transport in bile secretion.
• EFFECT OF U R S O D E S O X Y C H O L I C ACID ON LIVER ENZYMES AND TITER OF ANTI-HCV-IGM AND SEMIQUANTITATIVE HCV RNA IN PATIENTS WITH SUBTYPED CHRONIC HEPATITIS C. M~ M6hler, S. Seipp, U. Tfx, B. Kallinovsky, W. Stremmel, L. Theilmann and T. Goeser, Dept. of Int. Medicine, UniversiW of Heidelberg, Germany Ursodenxycholic acid (UDCA) admimsta'aaon has been associated with the reduction of serm~ fiver enzymes in patients with cbrome hepatitis C and with improvement of liver histology. Whether this effect of UDCA is induced by a cholore'tie effect or a tmmunomodnintory way of actaon is at present unclear. Therefore we thought to investigate the effect of UDCA in patients with chronic HCV infection by monitoring serum liver enzymes, HCV RNA titers and, in addition, anti-HCV IgM titars. Anti-HCV IgM have been found in acute and chronic active hepatitis C virus (HCV) infections, while resolved or chronic persistent hepatitis C are less commonly associated with IgM antibodies indicating that antiHCV IgM might be a marker for active hepatitis C virus replication. Methods: Sera of 19 chronic HCV-infected patients (pat.) were sampled before, dunng and affzr UDCA (500 mg q.i.d., 3 months). All sera were tested for liver eazzymes, senuquantitative HCV RNA (RT-PCR) and anti-HCV IgG and lgM Titer (Abbott). Aider extraction of RNA, RNA was reverse transcribed and amplified with a nested PCR using primers of the noncoding- 5'-region and adjacent HCV core region. Quantiiieation was done semiquantitatively by diluting HCV RNA prior to a single step RT-PCR. Amplified eDNA was detected by Southern blot. Genotyping was done by a reverse-hybridization line probe assay (Innolipa). Results: Before treamaent, all sera were anti-HCV IgG and HCV RNA positive. Most prevalent was the HCV genotype lb (67%), followed by la, 2a and 3a 11% each. Although no changes of levels of ALT were observed in 3 pat. 16/19 pat. (84%) had sigmficant decrease of ALT under UDCA (mean ALT before UDCA 90.7 U/1 vs. during UDCA 40.7 LIB;p<0.001). ;tGT significantly decreased in all pat. (mean "fGT before UDCA 75.5 U/I, during UDCA 25.1 U/I ;p<0.001). During and after UDCA~ 50% of patients had positive anti-HCV IgM status and their titer remained unchanged as did HCV ~'LNA.Semiquantitauve HCV P,aNA did not change sigmficunfly during or alter UDCA. Conclusions: 1. A three months treatment with UDCA has sigmficuntly improved transaminases and cholestatic enzymes in patients with chronic hepatitis C (mostly HCV lb). 2. UDCA has no positive impact on titer ofHCV RNA. 3. AntiHCV IgM arc found in ordy 50% of patients with chronic hepatitis C infection with no change of its titer as irrmaunresponseto UDCA. 4. Thus the mayor improvement of liver euzymes seems to be induced by the choloretie effect of UDCA.
AASLD
Al125
THE UNCOUPLING OF BILIARY LIPID FROM BILE ACID SECRETION BY FORMYL-METHIONYL-LEUCYL-PHENYLALANINE (FMLP) IN THE ISOLATED PERFUSED RAT LIVER (IPRL). K.Mizuno, T.Hayakawa, YKamiya, H.Ohara, T.Yamada, H.Yamada, T.Nakazawa, T.Inagaki, A.Uchida, M.Hoshino, M.Miyaji, T.Takeuchi. 1st Dept. of Internal Medicine, School of Medicine, Nagoya City University. Nagoya, JAPAN Hepatobiliary diseases such as pericholangitis and sclemsing cholangitis am known to be associated with some patients with inflammatory bowel disease (IBD). In experimental colitis, neutrophil chemotactic factor FMLP, is reported to circulate enterohepatically, but its effect on bile secretion is unclear. We investigated the effect of FMLP on bile secretion including biliary excretion of horseradish peroxidase(HRP), a conventional marker of transcytotic vesicle transport pathway, in a single-pass IPRL model. When FMLP was infused at different concentrations (2gM, 10pM, 20p.M) into the portal vein, bile flow increased and FMLP was excreted into Nle in its native form and in a dose-dependent manner. Approximately 12% of the infused dose was excreted into the bile independently of the presence of sodium taurocholate. During constant infusion of sodium tattrocholate (25gM), a 15 minute infusion of FMLP (20gM) resulted in a significant and reversible decrease in biliary excretion of phospholipids in a dose dependent manner, whereas bile acid secretion increased slightly. Phospholipidfoile acid molar ratios declined from 0.07+0.01 to 0.04+0.01, indicating that FMLP induces the uncoupling of biliary excretion of phospholipid with that of bile acids. A one-minute load of HRP (25mg) under a continuous infusion of taurocholate produced an early and late peak in biliary excretion of HRP. FMLP (101.tM) reduced HRP excretion in the late peak, from 0.489-+0.052 to 0.269-+0.040 (nmol/min/g.liver). When bile samples collected during the infusion of FMLP (101.tM) was applied to gel-permeation chromatography using Sepharose CL4B column, elution profiles of bile demonstrated a specific association of FMLP with bile acids. These findings suggest that FMLP causes the uncoupling phenomenon as a result of an inhibition of transcellular vesicle transport and the interaction between FMLP and bile acid micelles in the bile canaliculus. Enterohepatic circulation of FMLP might be one of the factors important in mediating the hepatobiliary diseases associated with IBD.
THE METABOLIC CHARACTERISTICS DIFFERENTIATING NONALCOHOLIC STEATOHEPATITIS, DIABETES MELLITUS AND OBESITY, S Mokshagundam, A Peiris, J Stagner, CH Tamburro Liver Research Center, Dept of Medicine, Univ of Louisville, VA Medical Center, Louisville, KY and East Tennessee State Univ, Johnson City, TN Non-alcoholic steatohepatitis (NASH) is a clinical entity defined by persistent abnormal liver enzymes (ALE) and significant fatty infiltration in the absence of other causes of hepatic disease. Diabetes mellitus and obesity are frequently associated with NASH. The metabolic characteristics of 23 NASH patients, 15 obese and 9 lean individuals were studied and also clinically compared to 50 diabetics. The comparison parameters included biochemical liver tests, blood glucose, ferritin level, anthropometric measurement, lipid profile, insulin and c-peptide levels. Of the 23 NASI4 patients 9 had evidence of diabetes mellitus; 5 bad evidence of glucose intolerance; 13/23 were women with the ages ranging 3566, women being slightly older than the men; 56.5 yrs vs 48.7 yrs. Percent ideal body weights for women were 120-146%, for men 120-164%. Over 70% had waist/hip ratios of >0,88. CT scan evidence of fatty infiltration were present in all and histologically estimated at 30-80% of biopsy tissue. Over 80% had fasting dyslipidemia and persistently elevated basal insulin levels. All had elevated ferritin levels and 65% increased fibrosis or cirrhosis. In contrast, only 6% (3/50) of the diabetic group had ALE and 50% dyslipidemia. The body fat, lipid and insulin characteristics of NASH and obese groups are below. Table I
NASH/n= 16
OBESE/n= 15
LEAN/n=9
%IBW BMI WHR Triglyceride Cholesterol HDL-C LDL-C Basal Insulin Basal C-peptide
141.5+5.5 30.2+2.2 0.93+0.02 248+31.48 198.8+9.6 31.9+2.8 111+7.9 44.5+8.5 2.2+0.4
140.93+3.4 3I .4+0.7 0.94+0.02 138:4+ 19.9 207+ I2.8 39.7+2.6 139.5+11.2 12.2+3.4 2.1+0.3
104+3.3 22.7+ 1.7 0.85+0.01 100.7+ 16.8 168+ 12.2 45.6+3.8 98.1+11.3 7.7+0.8 1.35+0.6
NASH is a metabolic disorder characterized by significant fatty infihrat on, steatonecrosis, pets stent ALE, hypertriglyceridemia, elevated ferritin and markedly elevated basal insulin levels. These metabolic characteristics distinguish NASH from individuals with obesity and diabetes mellitus. Steatonecrosis may be related to insulin nonresponsiveness of hepatic cells leading to enzyme leakage and fibrogenesis.