Pancreatic acinar cell function and morphology in rats chronically fed an ethanol diet

The aim of the present study was to determine the effect of prolonged ethanol intake on the morphology and protein metabolism in the rat pancreatic acinar cells. Weight-matched triplets of Sprague-Dawley rats were fed Lieber-DeCarli diet containing 5% (wt/vol) concentration of ethanol, isocaloric amounts of Lieber-DeCarli diet, or rat chow ad libitum for 6, 12, and 18 mo. In the ethanol-fed group, histologic studies by light microscopy showed absence of protein plugs in the pancreatic ducts and/or pancreatitis, but electron-microscopic evaluation revealed progressive accumulation of lipid droplets in acinar and ductal cells. No definite changes in the mitochondria and endoplasmic reticulum were noticed. Biochemical studies revealed increased specific activity of trypsinogen, chymotrypsinogen, and lipase, and decreased specific activity of amylase. Trypsin-inhibiting capacity was decreased in the tissue and in the medium in a progressive fashion. There was no increase in the secretion of total protein. These data show a complex and a nonparallel alteration of specific digestive enzymes and trypsin inhibitor in this model of chronic ethanol intoxication that may be of relevance to occurrence of pancreatitis.     

Role of endothelin in endotoxin-induced hepatic microvascular dysfunction in rats fed chronically with ethanol

BACKGROUND: We examined the role of endothelin in endotoxin-induced hepatic microcirculatory disturbance in pair-fed rats given a liquid diet containing ethanol or isocaloric control. METHODS AND RESULTS: One lobe of the liver was observed with the use of an intravital microscope. Erythrocytes (RBCs) labeled with fluorescein isothiocyanate (FITC) were injected, and the flow velocity of the FITC-RBCs in the sinusoids was measured with an off-line velocimeter. The flow velocity decreased 30 min after 1 mg/kg of lipopolysaccharide (LPS) was administered to the controls, and portal pressure (PP) was increased at 60 min. In ethanol-fed rats, however, both the flow velocity and PP increased in the early phase (at 10 min), and in the late phase, flow velocity decreased and PP increased more than in the controls. The LPS-induced decrease in flow velocity was blunted, when BQ-123, an antagonist of endothelin receptor subtype A, was infused into ethanol-fed rats, and BQ-123 also attenuated the change in PP. The plasma endothelin levels in both systemic and portal blood of the ethanol-fed rats were higher than in the controls. CONCLUSIONS: These results suggest that endothelin plays a role in the LPS-induced hepatic microcirculatory disturbance, especially in alcohol-fed animals.     

Role of nitric oxide in endotoxin-induced hepatic microvascular dysfunction in rats chronically fed ethanol

BACKGROUND: Nitric oxide (NO) appears to be involved in the pathogenesis of endotoxin-induced liver injury. However, little is known about how NO acts on the hepatic microcirculation, especially in alcohol-fed animals. We examined the roles of NO in endotoxin-induced hepatic microvascular dysfunction in control and ethanol-fed rats. METHODS: One lobe of the liver was observed with an intravital microscope. Flow velocity of fluorescein isothiocyanate-labeled erythrocytes in sinusoids was measured with an off-line velocimeter. Portal pressure and mean arterial pressure also were measured. RESULTS: After administration of endotoxin to control, the flow velocity decreased after 30 min. Portal pressure increased after 45 min. However, in ethanol-fed rats, both the flow velocity and portal pressure temporarily increased in the early phase. Thereafter, the flow velocity decreased and portal pressure increased. At 30 min after administration of the endotoxin, pretreatment with 10 mg/kg of an NO synthase inhibitor, NG-monomethyl-L-arginine (L-NMMA), enhanced the endotoxin-induced decrease in the velocity of erythrocytes in the midzonal region of both control and ethanol-fed rats. Although 0.5 mg/kg of L-NMMA enhanced the endotoxin-induced reduction of erythrocyte velocity in the midzonal region of ethanol-fed rats, L-NMMA enhanced the endotoxin-induced reduction of erythrocyte velocity in the pericentral region of control rats. At 60 min after the endotoxin administration, L-NMMA did not affect the endotoxin-induced decrease of erythrocyte velocity in either control or ethanol-fed rats. Although 10 mg/kg of L-NMMA increased mean arterial pressure both in control and ethanol-fed rats, 0.5 mg/kg of L-NMMA did not change mean arterial pressure in either control or ethanol-fed rats. CONCLUSIONS: These results suggest that NO is involved in endotoxin-induced hepatic microvascular dysfunction, which may contribute to the sequential liver injury, especially in alcohol-fed animals.     

terbal cardiotonic pills prevent gut ischemia／reperfusion-induced hepatic microvascular dysfunction in rats fed ethanol chronically

AIM: Cardiotonic Pill (CP), an oral herbal medicine that includes Danshen (Salviae Miltiorrhizae), Panax notoginseny and Dyroblanops aromatica gaettn, has been clinically used for vascular diseases such as occlusive vasculitis, coronary diseases, atherosclerosis, and cerebral infarction. The main component, Salviae Miltiorrhizae, has been reported to prevent cerebral and intestinal reperfusion injury. However, little is known about the effect of CP on hepatic microcirculation. Thus, this study aimed to determine whether CP could affect hepatic microvascular dysfunction elicited by gut ischemia/ reperfusion (I/R) in rats fed ethanol chronically. METHODS: Male Wistar rats were pair-fed with a liquid diet containing ethanol or isocaloric control diet for 6 wk. After laparotomy, one lobe of the liver was examined through an inverted intravital microscope. The rats were exposed to 30 min of gut ischemia followed by 60 min of reperfusion. Rhodamine-6G-labeled leukocytes in the sinusoids were observed 90 min after the onset of superior mesenteric artery occlusion. Plasma tumor necrosis factor (TNF)-α and endotoxin levels were measured 1 h after the onset of reperfusion. Plasma alanine aminotransferase (ALT) activities were measured 6 h after the onset of reperfusion. In another set of experiments, CP (0.8 g/kg, intragastrically) was administered 1 and 24 h before the onset of ischemia. RESULTS: In control rats, gut I/R elicited increases in the number of stationary leukocytes, and plasma TNF-α and endotoxin levels and plasma ALT activities. These changes were mitigated by pretreatment with CP. In ethanol-fed rats, the gut I/R-induced increases in the number of stationary leukocytes, plasma endotoxin levels and ALT activities were enhanced. Pretreatment with CP attenuated the enhancement of gut I/R-induced responses by chronic ethanol consumption. CONCLUSION: These results suggest that CP prevents the gut I/R-induced hepatic microvascular dysfunction and hepatocellular injury. A reduction of inflammatory responses such as TNF-α production via reduction of blood endotoxin levels appears to be involved in the mechanisms. Chronic ethanol consumption enhances gut I/R-induced hepatic microvascular and hepatocellular injury. CP also attenuates an enhancement of gut I/R-induced responses by chronic ethanol consumption via the reduction of blood endotoxin levels.     

Lipid peroxidation and antioxidant defense systems in rat liver after chronic ethanol feeding

The effects of chronic ethanol feeding on hepatic lipid peroxidation, ascorbic acid, glutathione and vitamin E levels were investigated in rats fed low or adequate amounts of dietary vitamin E. Hepatic lipid peroxidation was significantly increased after chronic ethanol feeding in rats receiving a low-vitamin E diet, indicating that dietary vitamin E is an important determinant of hepatic lipid peroxidation induced by chronic ethanol feeding. No significant change was observed in hepatic non-heme iron content, but hepatic content of ascorbic acid and glutathione was increased by ethanol feeding. Both low dietary vitamin E and ethanol feeding significantly reduced hepatic alpha-tocopherol content, and the lowest hepatic alpha-tocopherol was found in rats receiving a combination of low vitamin E and ethanol. Plasma alpha-tocopherol was elevated after ethanol feeding, probably because of the associated hyperlipemia. Both the ratio of plasma alpha-tocopherol/plasma lipid and the red blood cell alpha-tocopherol were reduced by ethanol feeding. Furthermore, ethanol feeding caused a marked increase of hepatic alpha-tocopheryl quinone, a metabolite of alpha-tocopherol by free radical reactions. Ethanol feeding caused little changes of alpha-tocopherol and alpha-tocopheryl quinone content in mitochondria, whereas a striking increase in alpha-tocopheryl quinone was observed in microsomes. These data suggest that ethanol feeding causes a marked alteration of vitamin E metabolism in the liver and that the combination of ethanol with a low-vitamin E intake results in a decrease of hepatic alpha-tocopherol content which renders the liver more susceptible to free radical attack.     

Lipid membrane peroxidation in beta-thalassemia major.

The composition of membrane lipids was studied in 17 splenectomized and eight unsplenectomized patients with beta-thalassemia major and compared to normal controls. The results showed a nearly twofold increase in total cell lipids; a reduction in the percentage, but not the absolute amount of phosphatidylethanolamine, and a corresponding increase in phosphatidylcholine in the lipids; a considerable increase in the percentage of the saturated fatty acid, palmitic acid, and a reciprocal decrease in the polyunsaturated fatty acid, arachidonic acid; a twofold increase in the amount of malonyldialdehyde (MDA) generated after peroxide threat to the RBC when calculated either per gram hemoglobin or per cell; no change in the amount of MDA generated when calculated per microgram of membrane phosphorus at risk per cell; and a considerable decrease in serum alpha-tocopherol (vitamin E) levels. Thalassemic erythrocytes contain more lipid per cell which is susceptible to peroxidation. In addition, the distribution of fatty acids in these cells suggests that autooxidation of that lipid may have occurred. Autooxidation may be initiated by free radicals, which are constantly formed in the normal red cell, and may be especially prevalent when unstable hemoglobins are present. The low MCHC or some other intracellular defect of thalassemic cells may allow such potent oxidants to find their way to the cell membrane. Vitamin E, a biologic antioxidant is decreased in these patients, and clinical supplementation may be indicated to prevent some of the membrane damage in thalassemia.     

Lipid peroxidation in nicotinamide-deficient and nicotinamide-supplemented rats with streptozotocin-induced diabetes

Reactive oxygen species have been related to the pathogenesis of various diseases, including diabetes mellitus. Nicotinamide has been used for the prevention of the diabetogenic effects of streptozotocin (STZ) in animals. In the present study we assessed the effect of diets with deficient, normal or 17-fold supplemented nicotinamide concentrations on the rate of lipopoeroxidation in animals with STZ-induced diabetes. Male Wistar rats were divided into three groups kept on one of the diets for six weeks: DD, diabetic rats on a nicotinamide-deficient diet; DN, diabetic rats on a normal nicotinamide diet; and DS, diabetic rats on a nicotinamide-supplemented diet. During the fourth week of the experiment all animals were fasted for 24 hours and injected into the tail vein with a single STZ dose (40 mg/kg weight). Eight animals from each of the six groups were then sacrificed 24 hours, 1 week and 2 weeks after STZ injection. Mean pancreatic thiobarbituric acid reactive substances (TBARS) (nmol/mg tissue) were significantly lower in the DS group (p < 0.05) compared to the DN and DD groups at 24 hours and during the first week. Hepatic TBARS concentrations (nmol/mg protein) did not differ between groups. Mean hepatic reduced glutathione (GSH) levels were significantly higher (46.76 ± 12.33 nmol/mg protein) in the DS group compared to the DD (32.90 ± 6.70) and DN (24.55 ± 6.41) groups, but only after the 24-hour period. Hepatic vitamin E consumption (Μg/g tissue) was considerable in the groups not supplemented with nicotinamide, whereas vitamin E levels were unchanged in the supplemented group. In contrast, plasma vitamin E levels were decreased in the normal and supplemented groups after 1 and 2 weeks. A higher N-methylnicotinamide excretion (μg/ 24 hours) occurred in the supplemented group. We conclude that, after induction of diabetes with STZ, nicotinamide supplementation protected from the damage caused by the toxic action of STZ, promoting lower lipid peroxidation. Received: 27 September 1999 / Accepted in revised form: 3 March 2000     

Lipid peroxidation: a mechanism for ethanol-associated testicular injury in rats

Chronic alcohol administration leads to hepatic membrane alterations which, at least in part, are due to lipid peroxidation and may contribute to the toxicity of ethanol at the level of the hepatocyte. Because changes in testicular function also occur after chronic administration of ethanol to rats, we evaluated testicular mitochondria for evidence of alcohol-associated peroxidation injury which might contribute to the gonadal injury that occurs with prolonged use of the drug. Lipid peroxidation was assessed through measurement of diene conjugates, polyenoic fatty acid composition, malonaldehyde formation, and testicular reduced glutathione levels. Compared to isocalorically matched dextrimaltose-fed controls (ISO), rats fed alcohol (ETOH) for 50 days had a decreased content of polyenoic acids and a compensatory increase in saturated fatty acids [ETOH, 50.69 +/- 0.65% (by wt); ISO, 52.93 +/- 0.72 (mean +/- SE); P less than 0.01]. This decrease in polyunsaturated fatty acid content was accompanied by an increase in diene conjugates in testicular mitochondria (ETOH, 0.455 +/- 0.053 OD units at 233 nm/mg lipid; ISO, 0.382 +/- 0.045; P less than 0.05). An increase in malonaldehyde formation also was observed in the alcohol-fed rats compared to the control level (ETOH, 21.39 +/- 1.67 nmol/mg protein; ISO, 17.50 +/- 1.39; P less than 0.05) as well as a decrease in glutathione content (ETOH, 1218 +/- 89 micrograms GSH/testes; ISO, 1638 +/- 89; P less than 0.05). Taken together, these findings support the concept that lipid peroxidation may be an important mechanism responsible, at least in part, for the toxic effect of ethanol on the testes.     

Lipid peroxidation in early experimental diabetes in rats: effects of diabetes and insulin.

The degree of lipid peroxidation was measured in organs from diabetic rats receiving no treatment, and in those from insulin-treated diabetic rats and controls. Lipid peroxidation was measured as organ content of malondialdehyde, a degradation product of polyunsaturated fatty acids. In the kidney, lipid peroxidation was increased after one week of diabetes; insulin treatment reduced the level of lipid peroxidation to levels lower than seen in controls. In the liver, diabetes caused an increased lipid peroxidation, which could be reversed by insulin; no additional effect of insulin was found. In heart and pancreas no effects of diabetes or insulin were demonstrated. The present paper provides evidence that lipid peroxidation is increased in the early stages of experimental diabetes and is reversible by insulin treatment. Hyperinsulinaemia may, in itself, counteract lipid peroxidation in kidney.     

Lipid peroxidation in normoxic and ischaemic-reperfused hearts of fish oil and lard fat fed pigs

The in situ and in vitro rate of lipid peroxidation of hearts were determined in two groups of pigs which had been fed diets which differed only in fatty acid composition for 8 weeks. During the dietary period venous plasma levels of malondialdehyde and lipofuscin were not higher in pigs receiving the highly unsaturated fatty acid-containing mackerel oil than those receiving lard fat. Malondialdehyde was produced in the coronary system of the mackerel oil fed animals. After the heart was subjected to a sequence of short periods of ischaemia (5 min) and reperfusion (10 min), myocardial malondialdehyde production in the mackerel oil fed pigs did not increase. Contribution of prostaglandin synthesis products to myocardial malondialdehyde formation is probably of minor importance. Recovery of regional heart function after the ischaemic periods was similar for both dietary groups. In the phospholipids of sarcolemmal preparations isolated from the left ventricle of mackerel oil fed animals 18:2 n-6 and 20:4 n-6 were partially replaced by 20:5 n-3 and 22:6 n-3. Ischaemia-reperfusion did not alter sarcolemmal fatty acid composition and Ca2+ pumping ATPase activity. Sarcolemmal membrane from mackerel oil fed pigs exposed in vitro to a free radical generating system showed a higher malondialdehyde production than that from lard fat fed pigs. Thus, in spite of the increased susceptibility of heart membranes to free radical generated peroxidation in mackerel oil fed animals, recovery of left ventricular function was similar following multiple short-term periods of ischaemia.     

Lipid peroxidation in mouse models of atherosclerosis.

Over the past decade, oxidative modification of low-density lipoprotein (LDL) has been implicated in atherogenesis. This hypothesis has been supported indirectly by a number of in vitro and ex-vivo observations demonstrating the pro-atherogenic properties of oxidized LDL, their occurrence in atherosclerotic lesions and some positive results in animal studies with antioxidants. Only recently, however, have small mammalian models (mouse) of atherosclerosis been created by gene manipulation and widely used by investigators to better elucidate this pathogenetic aspect of such a complex disease. Transgenic animal models combined with the availability of more reliable, specific and sensitive markers of in vivo lipid peroxidation have now provided consistent evidence for a direct and functional role of oxidant stress in atherogenesis. The identification of yet unknown initiating event(s) of in vivo oxidant stress will be the challenge for the future. This should finally provide the basis for the development of most effective antioxidant agents that could modulate atherogenesis.     

Long-term hypertriglyceridemia and glucose intolerance in rats fed chronically an isocaloric sucrose-rich diet

We have previously shown that short-term feeding [20 to 25 day induction period (IP)] normal rats a sucrose-rich diet (SRD) results in an increase of plasma (P), liver (L), and heart (H) triacylglycerol (TG) levels, accompanied by a drop in plasma postheparin total (T-TGL) and hepatic (H-TGL) triglyceride lipases activities, IV glucose intolerance (low Kg) and hyperinsulin responses both in vivo and in vitro, suggesting that a state of insulin resistance had developed. Since normalization of P-TG ensued in the medium term [40 to 55 day adaptation period (AP)] we decided to carry out a longitudinal, long-term (90 to 120 day) follow-up study to observe the dynamic behavior of the above metabolic and hormonal parameters as compared to the appropriate time course control rats were fed the standard chow (STD).(ABSTRACT TRUNCATED AT 250 WORDS)     

Alcoholic pancreatitis in rats fed ethanol in a nutritionally adequate liquid diet

In an effort to develop a model of chronic alcoholic pancreatitis in Sprague-Dawley rats fed a nutritionally adequate diet, 3 groups of 15 animals each were fed Wayne Rodent-Blox ad libitum, Lieber-DeCarli diet with 40% of carbohydrate calories replaced by ethanol ad libitum and isocaloric amounts of Lieber-DeCarli diet respectively for a period of 18 months. Rats were anesthetized and basal and secretin-stimulated pancreatic juice was obtained. Pancreatic glands were isolated and divided into portions for histology, biochemical analyses, and cell fractionation. The homogenate, zymogen granule fraction, mitochondrial-lysosomal fraction, microsomal fraction and postmicrosomal supernatant as well as aliquots of pancreatic juice were analyzed for cathepsin B, acid phosphatase, beta-D-glucoronidase, arylsulphatase and leucine naphthylamidase. All of the ethanol-fed animals developed morphological changes akin to human chronic pancreatitis. There were focal areas of parenchymal degeneration with fibrosis, protein plug formation and tubular complexes. In the pancreatic tissue of animals fed ethanol, total protein, trypsinogen (and free trypsin) were increased and amylase was decreased. While acid phosphatase was increased in all of the particulate fractions, cathepsin B was increased in the zymogen granule and mitochondrial-lysosomal fractions. Basal and post-secretin pancreatic juice did not show a significant increase in digestive or lysosomal enzymes. It is suggested that focal degenerative changes may be due to trypsin generated by intracellular activation of digestive enzymes by lysosomal enzyme cathepsin B.     

Alcoholic pancreatitis in rats fed ethanol in a nutritionally adequate liquid diet

Summary In an effort to develop a model of chronic alcoholic pancreatitis in Sprague-Dawley rats fed a nutritionally adequate diet, 3 groups of 15 animals each were fed Wayne Rodent-Blox ad libitum, Lieber-DeCarli diet with 40% of carbohydrate calories replaced by ethanol ad libitum and isocaloric amounts of Lieber-DeCarli diet respectively for a period of 18 months. Rats were anesthetized and basal and secretin-stimulated pancreatic juice was obtained. Pancreatic glands were isolated and divided into portions for histology, biochemical analyses, and cell fractionation. The homogenate, aymogen granule fraction, mitochondrial-lysosomal fraction, microsomal fraction and postmicrosomal supernatant as well as aliquots of pancreatic juice were analyzed for cathepsin B, acid phosphatase, β-d-glucoronidase, arylsulphatase and leucine naphthylamidase. All of the ethanol-fed animals developed morphological changes akin to human chronic pancreatitis. There were focal areas of parenchymal degeneration with fibrosis, protein plug formation and tubular complexes. In the pancreatic tissue of animals fed ethanol, total protein, trypsinogen (and free trypsin) were increased and amylase was decreased. While acid phosphatase was increased in all of the particulate fractions, cathepsin B was increased in the zymogen granule and mitochondrial-lysosomal fractions. Basal and post-secretin pancreatic juice did not show a significant increase in digestive or lysosomal enzymes. It is suggested that focal degenerative changes may be due to trypsin generated by intracellular activation of digestive enzymes by lysosomal enzyme cathepsin B.     

Food restriction in female Wistar rats: V. Lipid peroxidation and antioxidant enzymes in the liver

The activities of antioxidant enzymes as well as the levels of basal and enzyme induced peroxidation have been investigated in liver of female Wistar undernourished rats. Food restriction was applied starting from the age of 3.5 months by feeding the animals on every-other-day schedule (EOD). Diet restriction prevented the age-dependent increase of basal and enzyme induced lipid peroxidation in both mitochondrial and microsomal liver membrane preparations. The activities of antioxidant enzyme, i.e. superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) of liver decreased during aging in ad libitum fed rats. In the diet conditioned animals, a small increase of SOD and a complete recovery of CAT activities were observed. Present data support that food restriction improved the protection against peroxidation, and this may be in close relationship with the life prolonging effect of such a treatment.     

Lipid peroxidation in the heart of adult and old rats during immobilization stress

This study was designed to assay the lipid peroxidation (LPO) in the heart from adult (10--12 months) and old (22--25 months) Wistar male rats during immobilization stress. A value of LPO was estimated according to the concentration of conjugate dienes, TBA-positive substances and fluorescent products such as Schiff base. Investigations have shown that immobilization of rats was accompanied by increase in the concentration of all studied LPO intermediates in myocardium from both the age groups. Accumulation of these metabolites in old rat hearts was considerably higher than in adult ones. Dimethyl sulfoxide injection has limited stress-induced stimulation of LPO. Changes revealed in LPO-stimulation have different consequences in pathogenesis of stress-dependent injury in myocardium from adult and old rats. Utilization of LPO metabolites in old rats takes place mainly with formation of Schiff base. Accumulation of these products in myocardium is accompanied by modification of intracellular macromolecules and thus promotes the enhancement of stress-induced injury of myocardial cells. In adult rats during stress, the utilization of malonic dialdehyde and substances homologous to it is not accompanied by Schiff base accumulation in myocardium. It may be important for the restriction of stress-induced injury of myocardial cells.     

Lipid peroxidation in human red cells

In this review we have discussed the chemistry and biochemistry of lipid peroxidation as well as lipid repair mechanisms in human RBCs. We have presented findings relating to the effect of lipid peroxidation on the RBC membrane and on several properties that are determinants of RBC survival in vivo. Since we have not discussed how oxidative damage to membrane proteins or hemoglobin may affect RBC survival, the role of lipid oxidation must be considered in a broader perspective. Considerable evidence has recently been reported to indicate that oxidative hemoglobin denaturation plays an extremely important role in RBC survival. Since all cellular components are susceptible to peroxidative damage, it is likely that multiple reactions will be important with regard to RBC oxidant injury, just as they have been implicated in many degenerative processes, and that certain "compartments" of the membrane may be more susceptible than others due to congenital or acquired defects in membrane structure.