Hidekazu Tsukamoto

Relationship between fatty liver and subsequent development of necrosis, inflammation and fibrosis in experimental alcoholic liver disease

Rats fed a diet varying in the amount of fat, infused with ethanol, were studied to determine the relationship among diet, degree of fatty liver, and development of necrosis, inflammation, and fibrosis. Three groups of experimental animals, male Wistar rats, were fed diets containing 25% fat, 35% fat, and 32% fat with low protein. Morphologic assessment of liver injury was performed monthly by obtaining liver biopsies. The greatest degree of fatty infiltration at 1 month was seen in the high fat-low protein group, the mean fat score (3.8 +/- 0.37) was significantly higher than in the other two groups (P less than 0.05 and P less than 0.01). When the subsequent development of necrosis, inflammation, and fibrosis was related to the degree of fatty infiltration at 1 month, a significant relationship was seen between the number of animals developing these pathologic lesions and the severity of fatty liver. Our results show that the degree of fatty infiltration of the liver, influenced by the dietary intake of both fat and protein, is related to the subsequent development of necrosis, inflammation, and fibrosis in our intragastric feeding model for alcoholic liver disease.

Aortic compliance in hypercholesterolemic Watanabe rabbits compared to normal New Zealand controls.

This study was designed to test the hypothesis that severe atherosclerosis changes aortic compliance. Compliance of a vessel is defined as change in volume per unit change in pressure and is a measure of the stiffness or distensibility of the vascular wall. Part of the energy delivered by the left ventricle in systole is used to propel the blood forward into the aorta and part of it to distend the aorta and major vessels. During diastole, the arterial walls recoil and provide energy for propulsion of blood, thereby making blood flow continuous. It is known that Watanabe hereditary hyperlipidemic rabbits develop severe atherosclerosis beginning at 6 months of age. Compliance of the ascending thoracic aorta was studied angiographically in eight Watanabe hereditary hyperlipidemic rabbits of ages greater than 6 months and six normal lipidemic New Zealand white rabbits of ages greater than 6 months, used as controls. The normal New Zealand white rabbits had an average blood cholesterol of 27.4 mg/dL, SD = 13.8, and a regional compliance in the ascending aorta of 0.004 mL/mm Hg, SD = 0.002, compared to the Watanabe hereditary hyperlipidemic rabbits with a cholesterol of 583.1 mg/dL, SD = 162.7, and a compliance of 0.0022 mL/mm Hg, SD = 0.0015. These are significant differences (p less than .05). In addition, the histopathology of the aorta of the Watanabe hyperlipidemic rabbit compared to that of the controls showed a significant decrease in the number of medial lamellar elastin units, an indicator of the decreased elasticity of the blood vessel wall.

Expression of TGFβ1 by Rat Kupffer Cells: Implication for a Pathogenetic Role in Alcoholic Liver Fibrosisa

The importance of cytokines in regulation of collagen metabolism has recently been suggested!,2 In particular, TGFP has been shown to have a specific stimulatory effect on collagen formation by Ito cells? a cell type believed to be a major source of extracellular matrices in the liver: Since monocytes/macrophages are the known source of this cytokine? Kupffer cells, resident macrophages in the liver may also release TGFP and act as effector cells to enhance Ito cell collagen production during active liver fibrogenesis. The present study has tested this hypothetical mechanism of liver fibrogenesis using Ito cells and Kupffer cells isolated from the rat model of alcoholic liver fibrosis: Kupffer cell-conditioned medium derived from this model (KCCM) but not that from pair-fed control animals significantly enhanced collagen synthesis of Ito cells in culture established from autologous livers and those of pair-fed control and chowfed rats, as assessed by the incorporation of [3H]proline into the intracellular collagen. Acidification of KCCM potentiated this effect by three to fourfold indicating the presence of the latent form (FIG. 1). Simultaneous addition of anti-TGFD1 IgG (25 Bg/ml) to the Ito cell culture with either unacidified (K,) or acidified (KA) KCCM inhibited their stimulatory activities by 100% or 60%, respectively (FIG. 1). Preincubation of K, with anti-TGFPl IgG and subsequent addition of the preincubated K, to the culture following the complete removal of free IgG with protein A, resulted in neutralization of the stimulation that was as effective as that seen with the simultaneous addition. This indicated that Kupffer cells rather than Ito cells were the primary source of the TGFPlike activity responsible for the augmentation of Ito cell collagen production. Fractionation of K, by HPLC gel filtration revealed the major peak of the stimulatory activity corresponding to the molecular weight of 20 to 30 K, which was also completely neutralized with anti-TGFpl IgG. Furthermore, Northern blotting and hybridization of Kupffer cell mRNA from alcohol-fed rats with 32P-labeled TGFPl cDNA

Superior Mesenteric Artery Vasoactivity in Hyperlipidemic Watanabe Rabbits versus Normal Lipidemic New Zealand Controls

Recent in vitro studies on isolated coronary and mesenteric arteries have shown that hyperlipidemia appears to hypersensitize the vascular arterial smooth muscle to drugs such as ergonovine and that this increased contractility seems to be mediated by a serotinergic mechanism. This results in vasospasm with exposure to certain vasoactive drugs such as serotonin or norepinephrine. However, in vivo quantification of this observed phenomenon has not been done. In the present study we used Watanabe hereditary hyperlipidemic (WHHL) rabbits (cholesterol level 459 +/- 216 mg/dL) and the normal lipidemic New Zealand white (NZW) rabbit (cholesterol level 35 +/- 19) as a control in the study of hyperlipidemia and blood flow changes in response to various vasoactive drugs. Blood flow measurements were made by the video dilution technique (VDT) following catheterization of the superior mesenteric artery. The serotinergic vasoactive drug ergonovine maleate was injected into the superior mesenteric artery at low dose (0.002) mg/kg) and high dose (0.004 mg/kg). A significant decrease (p less than .05) in blood flow was observed in response to high-dose ergonovine maleate in WHHL rabbits compared to the NZW rabbits. This in vivo experiment confirms the in vitro studies showing that hyperlipidemia sensitizes mesenteric arteries in the presence of serotinergic stimuli. The vasodilators verapamil hydrochloride and calcitonin gene-related peptide (CGRP) injected into the superior mesenteric artery caused a marked increase in flow in both the WHHL and the normal lipidemic NZW rabbits. This model can be used in the assessment of superior mesenteric artery ischemia and its reversal.

Effects of Cholecystokinin, Food Intake and Cephalic Stimuli on Plasma Levels of Amylase, Lipase, and Immunoreactive Cationic Trypsinogen in Rats

Plasma levels of amylase, lipase, and immunoreactive cationic trypsinogen (ICT) were monitored in conscious rats to study the effects of cholecystokinin octapeptide (CCK-8) plus secretin administration, cephalic stimuli, and food intake. Stepwise increasing doses of CCK-8 (1, 5, 15, 30 Ivy dog units: IDU/kg/h) caused significant dose-related increases in plasma levels of each enzyme in a similar manner as those previously observed for response patterns of CCK-induced exocrine protein secretion in the same species. ICT showed the greatest response to CCK-8 with a maximal concentration 30 times above basal levels resulting in the steepest slope of the dose-response curve. Plasma lipase showed a maximal response that was 5 times above the basal level, while the plasma amylase level was increased only by 50% at the maximal response. Computed ED50 of CCK-8 for each enzyme confirmed this relative sensitivity of the response: 3.0, 8.7, 11.0 IDU/kg/h for ICT, lipase, and amylase, respectively. Plasma levels of amylase and lipase did not change significantly in response to the intake of either a liquid diet or fiber pellets containing no caloric value. Plasma ICT levels, however, were elevated significantly by 23 and 53% at the time when the liquid diet or fiber pellets were given and when cephalic stimulation appeared maximally induced. The increased levels declined thereafter and were no longer significantly different from the basal levels for postprandial 2 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Watanabe Hyperlipidemic Rabbit as a Model of Aortic Degeneration of the Medial Lamellar Elastin Unit

At age 3 years, WHHL rabbits are near the end of their lifespan, frequently dying from the progression of their hyperlipidemic disease from events such as myocardial infarction. Out of a colony of 20 three-year-old WHHL rabbits raised as part of a NIH breeding project, 2 rabbits actually died of a ruptured thoracic aortic aneurysm. The need for a model to study abdominal aortic aneurysm formation led us to explore further the abdominal aortic pathology in aged WHHL rabbits. Six rabbit abdominal aortas from 3-year-old WHHL rabbits were preserved in formalin, sectioned, and stained for elastin. These were compared to the same sections of six normolipidemic age matched New Zealand white (NZW) rabbits. There was significant (P less than or equal to .001) destruction of the medial lamellar elastin unit in the aorta of the WHHL rabbits compared with the control NZW rabbits. Severe cholesterol deposits appeared to destroy the medial lamellae from the inside out. No definite aneurysm formation was seen in the abdominal aorta despite the significant changes in the medial lamellar elastin units. Thus, this model could be used to study the elastin degeneration of the media, but not necessarily abdominal aortic aneurysm formation.

Effects of sustained ethanol intoxication and optimal nutrition on free amino acid levels in plasma, liver and muscle of the rat

Abstract The increased plasma levels of αamino-n-butyric acid (AANB) and branched chain amino acids (BCA) are commonly observed after chronic ethanol consumption. These changes are respectively attributed to enhanced turnover of hepatic glutathione (GSH) and increased muscle protein catabolism. On the other hand, the ethanol-induced decrease in plasma level of alanine (ALA) is thought to be caused by enhanced hepatic production of lactate. The objective of this study was to investigate how these alterations can be exhibited in rats subjected to sustained ethanol intoxication and concomitant optimal nutrition. Plasma lactate levels and content of free amino acids in the liver and gastroenemius muscle were also determined to facilitate better understanding of factors involved in the aforementioned changes. Male Wistar rats were intragastrically infused with liquid diet plus ethanol or isocaloric glucose for 30 days. This regimen resulted in sustained blood alcohol levels and optimal nutrition as indicated by similar weight gains of ethanol or pair-fed animals to that of chow-fed rats. Hepatic content and plasma concentrations of ALA were not decreased in the ethanol-fed group, and the plasma lactate levels in these animals were not different from those in the controls. Levels of BCA (valine, isoleucine, leucine) were increased by 42–44% in plasma and 15–23% in the muscle of the ethanol-fed rats. AANB concentrations were significantly increased by 5-fold in the muscle and by 4-fold in plasma with only a 2-fold increase in the liver. These data indicate that the reported ethanol-induced depression in the plasma level and liver content of ALA can be prevented by optimal nutrition. Furthermore, the increased plasma levels of AANB seem to be associated more closely with increased protein turnover in the muscle than with the enhanced GSH metabolism in the liver. The elevated BCA levels in plasma are also likely to be specific effects of ethanol stimulating the muscle protein turnover.