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Inhibition of microsomal triglyceride transfer protein (MTP) and hepatic lipoprotein secretion: Another important mechanism for drug-induced steatosis
30
Tuesday, April 1, 2003
Parallel Session 9: Experimental
I83
ACTIVATION CAUSES
OF PPAR ALPHA-DEPENDENT
RAPID REGRESSION
STEATOHEPATITIS
Hepatology
2
PATHWAYS
OF FIBROSING
IN MCD DIET-FED
MICE
l? Ha112, R. Kirsch2, I. Leclercq’. G. Farrell’, G. Robertson’, ‘Starr Liver Unit, Westmead Millennium Institute, University Of Sydney, Sydney, NSK Australia; 2Department Of Anatomical Pathology, Faculty Of Health Sciences, University Of Cape Town, Cape Town, South Africa
E.Ip’,
Pathogenesis of steatohepatitis involves interplay between accumulated hepatic lipid and oxidative stress that generates pro-inflammatory lipoperoxides. We have shown that activation of PPARalpha-dependent pathways clears hepatic lipid, reduces oxidative stress and prevents development of steatohepatitis induced by a methionine and choline deficient (MCD). We have now tested the hypothesis that PPARalpha activation could ameliorate established steatohepatitis. Male C57BL6 mice were fed MCD diet for 8 weeks. During the last 5 days, Wy-14, 643 (0.1% w/w), a potent PPARalpha agonist, was added to the diet; another group (controls) were maintained on the MCD diet. Liver sections were scored for steatosis and necroinflammation; Sirius red was used to stain collagen and PPARalpha-responsive gene expression was determined (northern blots or RNase protection assay). In MCD-fed mice, ALT levels were elevated and livers exhibited widespread macrovesicular steatosis, necroinflammation and extensive pericellular fibrosis. In mice given Wy-14,643 for 5 days, ALT levels were lower and livers showed minimal inflammation and less steatosis. Consistent with reduced steatosis, Wy-14, 643 increased expression of genes involved with fatty acid turnover. Wy-14, 643 treatment produced impressive regression of hepatic fibrosis, demonstrated by unweighting of extracellular collagen networks leaving only scattered fib&. In conclusion, treatment with Wy-14, 643 reverses established fibrosing steatohepatitis in mice. This PPARalpha agonist rapidly depletes the liver of lipid, decreases inflammation and appears to promote collagen degradation caused by the MCD diet.
I
84
PEROXISOME
PROLIFERATOR
(PPAR-ALPHA)
EXPRESSION
ENZYME
RESPONSE
ACTIVATED IS INDUCED
TO CLOFIBRATE
RECEPTOR-ALPHA BUT PEROXISOMAL
IS BLUNTED
IN A RAT
MODEL OF FAlTY LIVER
F. Akbiyik’, K. Cinar2, T. Ozsullu’, E. Demirpence’, R. Tunca3, R. Haziroglu3, 0. 0nder4, S. Civris4, C. Yurdaydin2, 0. Uzunalimoglu2, H. Bozkaya2. ‘Biochemistry, Hacettepe University, Faculty Of Medicine; 2Gastroenterology, Ankara University; ‘Pathology, Veterinary Faculty Of Ankara University; 41nternal Medicine, Ankara University, Ankara, Turkey Background and Aims: In this study we aimed to asses the PPAR-alpha expression pattern and mitocondrial/peroxisomal enzyme activities in response to clofibrate in a rat model of fatty liver(n). Methods: Four-week-old/male/star-Albino rats(48) were grouped (12 each group) as: 1: normal diet(ND) (6. weeks) (10 kcal% fat). 2: ND(6weeks) +clofibrate(last 2-weeks) (0.25%wt/wt). 3: high fat diet(HFD) (6. weeks) (60 kcal% fat). 4: HFD(6-weeks) +clofibrate(last 2-weeks). After 6 weeks, animals were sacrificed. Peroxisomal-acyl-CoA-oxidase(AOX), mitocondrial-acyl-CoA-dehydrogenase(ACD), catalase activities and malonaldehyde(MDA) and glutathion(GSH) levels were measured in the liver tissues by spectrophotometrically. Liver steatosis was graded by Oil-red staining. PPAR-alpha expression was immunohistochemically determined. Results: All animals fed HFD developed FL while only 2/12 animals fed HFD+clofibrate developed FL. PPAR-alpha expression was increased in
rats fed HID. Clofibrate further increased PPAR-alpha expression (labeling index for l/2/3/4: 2.5/50/10/49, p
I 85
INHIBITION
OF MICROSOMAL
TRIGLYCERIDE
PROTEIN (MTP) AND HEPATIC LIPOPROTEIN ANOTHER
IMPORTANT
MECHANISM
TRANSFER SECRETION:
FOR DRUG-INDUCED
STEATOSIS F! Letteron,
A. Sutton, A. Mansomi, B. Fromenty, U481, Hospital Beaujon, Clichy, France
D. Pessayre.
‘ZNSERM
Although several steatogenic drugs have been shown to inhibit mitochondrial fatty acid beta-oxidation, little is known on their effects on hepatic lipoprotein secretion. MTP lipidates apolipoprotein B (apo B) in the microsomal lumen to form triglyceride (TG) -rich very low density lipoproteins (VLDL), which are secreted, whereas incompletely lipidated apo B is degraded. We studied MTP activity, the lipoproteins present in the microsomal lumen and hepatic lipoprotein secretion 4 hours after a single dose of amineptine (1 mmol/kg), amiodarone (1 mmol/kg), doxycycline (0.25 mmol/kg), tetracycline (0.25 mmol/kg), tianeptine (0.5 mmol/kg) or pirprofen (2 mmol/kg) in mice. These doses acutely inhibit lipid oxidation and were also shown to cause steatosis after repeated doses (doxycycline) or a single dose (other compounds). We found that doxycycline had no effect on MTP or lipoprotein secretion. In contrast, amineptine, amiodarone, pirprofen, tetracycline and tianeptine inhibited MTP in vitro, decreased ex viva MTP activity in the hepatic homogenate (by 46, 48, 24, 70 and 60%), decreased TG in the luminal VLDL fraction of microsomes (by 89, 83, 33, 83 and 59%) and decreased the in viva hepatic secretion of TG (by 57, 51, 24, 36 and 51%) and that of Apo B (by 53, 59, 36, 45 and 85%). These results indicate that several steatogenic drugs inhibit not only mitochondrial beta-oxidation, but also MTP activity, which decreases Apo B lipidation and hepatic lipoprotein secretion. Drugs inhibiting both betaoxidation and MTP may be more steatogenic than drugs acting only on beta-oxidation or only MTl?
I 86
IRON AND INSULIN RESISTANCE: MANIPULATION
EFFECT
ON INSULIN RECEPTOR
OF IRON
IN HEPGS
HEPATOCYTES F! Dongiovanni,
L. Valenti, G. Occhino, A.L. Fracanzani, M. Mattioli, S. Fargion. Dipartimento Di Medicina Interna, Universit De&i Studi, Ospedale Policlinico IRCCS, Milano, Italy
Background: Mild hepatic iron overload is a frequent clinical feature in patients with NAFLD, characterized by hepatic insulin resistance. Iron depletion improves insulin sensitivity in patients with diabetes, but the mechanisms are unclear. Aim: To define the interaction between iron and insulin sensitivity, we determined the effect of iron manipulation on insulin receptor expression in a hepatocyte cell line. Methods: HepG2 cells were cultured in RPM1 complete medium. 1125. insulin binding was evaluated 48 hours after plating in basal condition and in cells treated for 24 hours with iron (FeAmCit/holo-transfenin),
Tuesday, April 1, 2003
Parallel Session 9: Experimental
I83
ACTIVATION CAUSES
OF PPAR ALPHA-DEPENDENT
RAPID REGRESSION
STEATOHEPATITIS
Hepatology
2
PATHWAYS
OF FIBROSING
IN MCD DIET-FED
MICE
l? Ha112, R. Kirsch2, I. Leclercq’. G. Farrell’, G. Robertson’, ‘Starr Liver Unit, Westmead Millennium Institute, University Of Sydney, Sydney, NSK Australia; 2Department Of Anatomical Pathology, Faculty Of Health Sciences, University Of Cape Town, Cape Town, South Africa
E.Ip’,
Pathogenesis of steatohepatitis involves interplay between accumulated hepatic lipid and oxidative stress that generates pro-inflammatory lipoperoxides. We have shown that activation of PPARalpha-dependent pathways clears hepatic lipid, reduces oxidative stress and prevents development of steatohepatitis induced by a methionine and choline deficient (MCD). We have now tested the hypothesis that PPARalpha activation could ameliorate established steatohepatitis. Male C57BL6 mice were fed MCD diet for 8 weeks. During the last 5 days, Wy-14, 643 (0.1% w/w), a potent PPARalpha agonist, was added to the diet; another group (controls) were maintained on the MCD diet. Liver sections were scored for steatosis and necroinflammation; Sirius red was used to stain collagen and PPARalpha-responsive gene expression was determined (northern blots or RNase protection assay). In MCD-fed mice, ALT levels were elevated and livers exhibited widespread macrovesicular steatosis, necroinflammation and extensive pericellular fibrosis. In mice given Wy-14,643 for 5 days, ALT levels were lower and livers showed minimal inflammation and less steatosis. Consistent with reduced steatosis, Wy-14, 643 increased expression of genes involved with fatty acid turnover. Wy-14, 643 treatment produced impressive regression of hepatic fibrosis, demonstrated by unweighting of extracellular collagen networks leaving only scattered fib&. In conclusion, treatment with Wy-14, 643 reverses established fibrosing steatohepatitis in mice. This PPARalpha agonist rapidly depletes the liver of lipid, decreases inflammation and appears to promote collagen degradation caused by the MCD diet.
I
84
PEROXISOME
PROLIFERATOR
(PPAR-ALPHA)
EXPRESSION
ENZYME
RESPONSE
ACTIVATED IS INDUCED
TO CLOFIBRATE
RECEPTOR-ALPHA BUT PEROXISOMAL
IS BLUNTED
IN A RAT
MODEL OF FAlTY LIVER
F. Akbiyik’, K. Cinar2, T. Ozsullu’, E. Demirpence’, R. Tunca3, R. Haziroglu3, 0. 0nder4, S. Civris4, C. Yurdaydin2, 0. Uzunalimoglu2, H. Bozkaya2. ‘Biochemistry, Hacettepe University, Faculty Of Medicine; 2Gastroenterology, Ankara University; ‘Pathology, Veterinary Faculty Of Ankara University; 41nternal Medicine, Ankara University, Ankara, Turkey Background and Aims: In this study we aimed to asses the PPAR-alpha expression pattern and mitocondrial/peroxisomal enzyme activities in response to clofibrate in a rat model of fatty liver(n). Methods: Four-week-old/male/star-Albino rats(48) were grouped (12 each group) as: 1: normal diet(ND) (6. weeks) (10 kcal% fat). 2: ND(6weeks) +clofibrate(last 2-weeks) (0.25%wt/wt). 3: high fat diet(HFD) (6. weeks) (60 kcal% fat). 4: HFD(6-weeks) +clofibrate(last 2-weeks). After 6 weeks, animals were sacrificed. Peroxisomal-acyl-CoA-oxidase(AOX), mitocondrial-acyl-CoA-dehydrogenase(ACD), catalase activities and malonaldehyde(MDA) and glutathion(GSH) levels were measured in the liver tissues by spectrophotometrically. Liver steatosis was graded by Oil-red staining. PPAR-alpha expression was immunohistochemically determined. Results: All animals fed HFD developed FL while only 2/12 animals fed HFD+clofibrate developed FL. PPAR-alpha expression was increased in
rats fed HID. Clofibrate further increased PPAR-alpha expression (labeling index for l/2/3/4: 2.5/50/10/49, p
I 85
INHIBITION
OF MICROSOMAL
TRIGLYCERIDE
PROTEIN (MTP) AND HEPATIC LIPOPROTEIN ANOTHER
IMPORTANT
MECHANISM
TRANSFER SECRETION:
FOR DRUG-INDUCED
STEATOSIS F! Letteron,
A. Sutton, A. Mansomi, B. Fromenty, U481, Hospital Beaujon, Clichy, France
D. Pessayre.
‘ZNSERM
Although several steatogenic drugs have been shown to inhibit mitochondrial fatty acid beta-oxidation, little is known on their effects on hepatic lipoprotein secretion. MTP lipidates apolipoprotein B (apo B) in the microsomal lumen to form triglyceride (TG) -rich very low density lipoproteins (VLDL), which are secreted, whereas incompletely lipidated apo B is degraded. We studied MTP activity, the lipoproteins present in the microsomal lumen and hepatic lipoprotein secretion 4 hours after a single dose of amineptine (1 mmol/kg), amiodarone (1 mmol/kg), doxycycline (0.25 mmol/kg), tetracycline (0.25 mmol/kg), tianeptine (0.5 mmol/kg) or pirprofen (2 mmol/kg) in mice. These doses acutely inhibit lipid oxidation and were also shown to cause steatosis after repeated doses (doxycycline) or a single dose (other compounds). We found that doxycycline had no effect on MTP or lipoprotein secretion. In contrast, amineptine, amiodarone, pirprofen, tetracycline and tianeptine inhibited MTP in vitro, decreased ex viva MTP activity in the hepatic homogenate (by 46, 48, 24, 70 and 60%), decreased TG in the luminal VLDL fraction of microsomes (by 89, 83, 33, 83 and 59%) and decreased the in viva hepatic secretion of TG (by 57, 51, 24, 36 and 51%) and that of Apo B (by 53, 59, 36, 45 and 85%). These results indicate that several steatogenic drugs inhibit not only mitochondrial beta-oxidation, but also MTP activity, which decreases Apo B lipidation and hepatic lipoprotein secretion. Drugs inhibiting both betaoxidation and MTP may be more steatogenic than drugs acting only on beta-oxidation or only MTl?
I 86
IRON AND INSULIN RESISTANCE: MANIPULATION
EFFECT
ON INSULIN RECEPTOR
OF IRON
IN HEPGS
HEPATOCYTES F! Dongiovanni,
L. Valenti, G. Occhino, A.L. Fracanzani, M. Mattioli, S. Fargion. Dipartimento Di Medicina Interna, Universit De&i Studi, Ospedale Policlinico IRCCS, Milano, Italy
Background: Mild hepatic iron overload is a frequent clinical feature in patients with NAFLD, characterized by hepatic insulin resistance. Iron depletion improves insulin sensitivity in patients with diabetes, but the mechanisms are unclear. Aim: To define the interaction between iron and insulin sensitivity, we determined the effect of iron manipulation on insulin receptor expression in a hepatocyte cell line. Methods: HepG2 cells were cultured in RPM1 complete medium. 1125. insulin binding was evaluated 48 hours after plating in basal condition and in cells treated for 24 hours with iron (FeAmCit/holo-transfenin),