Sources of triacylglycerol accumulation in livers of rats fed a cholesterol-supplemented diet

The source of free fatty acids (FFA) and the pathways contributing to the accumulation of neutral fats in livers of rats fed a cholesterol-enriched diet were investigated in this report. Supplementation with 1% cholesterol in the diet for four weeks resulted in hepatomegaly in the rats. The contents of cholesterol and triacylglycerols (TG) per gram liver measured in rats fasted overnight increased by 48 mg (∼tenfold) and 66 mg (∼fourfold), respectively. The activities of glycerophosphate acyltransferase and diacylglycerol acyltransferase, the two key enzymes for TG synthesis in liver microsomes, were found to increase by 23 and 19%, respectively, in the cholesterol-fed rats. The secretion of plasma TG present predominantly in very low density lipoprotein was found to decrease by ∼30%. The incorporation of tritium from tritiated water in liver FFA increased by twofold in rats fed the cholesterol-supplemented diet, whereas the activity of CPT I in liver mitochondria decreased by 23%. The uptake of plasma FFAin vivo in livers of fasted rats maintained on the cholesterol-supplemented diet decreased by 60%. Our data thus indicate that the excess TG accumulated in livers of rats fed the cholesterol-enriched diet resulted from increased synthesis and decreased secretion of TG. To meet the demand of fatty acids for this purpose,de novo lipogenesis increased, whereas β-oxidation decreased. Although difference in the uptake of extrahepatic FFA may be discounted, a difference in the uptake of chylomicron remnants between the control and cholesterol-fed rats may not be ruled out.     

Changes in liver lipid composition of male rats fed rapeseed oil diets

Studies are reported on the effect of feeding diets containing rapessed oils differing in their erucic acid content to male weanling rats for 16 weeks. Rapeseed oil high in erucic acid depressed growth. Total lipids, lipid phosphorous and cholesterol, in the livers were not significantly different between the experimental groups. The fatty acid composition of the total liver lipids, the neutral lipids, phosphatidylethanolamine and phosphatidylcholine are documented. Erucic and eicosenoic acids were found in all lipid classes at the same relative concentration; the amount being incorporated was proportional to that found in the dietary oil. The positional analysis of phosphatidylethanolamine and phosphatidylcholine are presented. Erucic acid was incorporated preferentially at position two of these phospholipids, whereas, twice the level of eicosenoic acid was found at position one, compared to that which occurred at the two position. This article represents part of an extensive experiment carried out by Agriculture Canada to investigate the nutritional value of rapeseed oils (see ref. 15). Contribution No. 497 from the Animal Research Institute.     

The effect of a histidine-excess diet on cholesterol synthesis and degradation in rats

Feeding a diet high in excess histidine (5% L-histidine) resulted in hypercholesterolemia and enlargement of the liver in rats. To clarify the mechanism of the hypercho-lesterolemia cholesterol synthesis and degradation were followed. We found that hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in histidine-excess diet rats was significantly higher than in rats fed a basal diet. Incorporation of [³H]water into cholesterol of liver slices from rats fed the histidine-excess diet was higher than incorporation into liver slices from rats fed the basal diet (expressed per liver per 100 g body weight).In vivo incorporation of [³H]water into hepatic cholesterol was also higher, but the incorporation into cholesterol of the small intestine was lower in histidine-fed rats than in rats fed the basal diet (expressed per liver per 100 g body weight). Hepatic cholesterol 7α-hydroxylase activity was similar in both groups. The data suggest that the hypercholester-olemia caused by histidine-excess diet appears to be due to the stimulation of cholesterol synthesis in the liver.     

Changes in fatty acid composition of phospholipids from liver microsomes and nuclei in rats fed a choline-free diet

Male F-344 rats were fed a choline-free (CF) diet, and changes in phospholipid content, phospholipid fatty acids and phospholipase A₂ activity in liver nuclei and microsomes were examined during the first 72 hr. Both nuclei and microsomes showed a decrease in phosphatidylcholine (PC) content. Microsomes showed an increase in PC arachidonate while nuclei showed a decrease. Also, microsomes showed increased activity of phospholipase A₂ (PLA₂) while nuclei did not. These observations are consistent with the hypothesis that the absence of diene conjugates in liver microsomes in the rats on the CF diet may reflect the increased rate of removal of peroxidized fatty acids by phospholipase A₂.     

Molecular species of phosphoglycerides in liver microsomes of rats fed a fat-free diet

The influence of a fat-free diet on the molecular species composition of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI) of rat liver microsomes was studied by using reversed-phase high-pressure liquid chromatography. In the three phosphoglyceride classes analyzed, the fat-free diet produced a large decrease in the 18:0/20:4n−6 species but less important changes were found in the 16:0/20:4n−6 species. In PC, the most abundant phosphoglyceride class of rat liver microsomes, the fall in the 18:0/20:4n−6 species was counterbalanced mainly by an enhancement in the 16:0/18:1n−9 species although it was not evident in PE. In PI, the decrease in the 18:0/20:4n−6 species was counterbalanced by an increase in the 18:0/20:3n−9 species. Fluorescence polarization measurements of 1,6-diphenyl-1,3,5-hexatriene in liposomes of 16:0/18:1n−9-, 18:0/18:1n−9-, 16:0/20:4n−6-, and 18:0/20:4n−6-PC indicated that the change in the saturated fatty acid in the sn-1 position accompanying the replacement of 20:4n−6 by 18:1n−9 could be very important for a homeoviscous compensation, maintaining the membrane physical properties without large alterations in spite of the essential fatty acid deficiency due to the fat-free diet.     

Liver and serum lipids and lipoproteins of rats fed 5% L-lysine

Soybean protein and casein supplemented with 1% Arg were compared for their ability to prevent fatty livers caused by excess dietary Lys. The concentrations of serum lipids and lipoproteins of rats fed 5% Lys and having vatty livers were also compared with those of rats fed the identical diet but lacking fatty livers when killed. The total liver lipids, triglycerides and cholesterol of rats fed 15% casein +5% Lys were 3.9, 12.4 and 2 times control values, respectively. Rats fed 5% Lys +1% Arg or 5% Lys with 15% soybean protein had liver lipid concentrations similar to controls fed no supplemental Lys. Serum total lipids, triglycerides, cholesterol, phospholipids and free fatty acids also did not change, and serum ketone bodies were slightly elevated with Lys feeding whether the rats had fatty livers or not. The concentrations of circulating HDL were slightly depressed in all rats fed 5% Lys while LDL were significantly elevated, particularly in rats without fatty livers. Serum VLDL did not change with 5% dietary Lys. Overall, excessive dietary Lys caused fatty livers which were prevented by varying the diet or length of feeding. Excess Lys feeding altered lipoprotein metabolism shown by decreased serum HDL and a substantial elevation in LDL. The latter was more apparent when the fat accumulation in liver was less severe or absent. The data suggest that the fatty liver from Lys excess is probably unrelated to increased fat mobilization from storage, decreased fat oxidation or to a major block in the transport of triglycerides from the liver to the circulation.     

The effect of clofibrate on heart and plasma lipids in rats fed a diet containing rapeseed oil

The effect of clofibrate on heart and plasma lipids in rats fed a diet containing 30% of the calories as peanut oil (PO) or rapeseed oil (RSO) (42.7% erucic acid and 0.5% eicosenoic acid) was studied. A decrease of erucic acid content to one-third and concomitant increase in the content of 18∶1, 16∶1 and 16∶0 fatty acids in plasma triacylglycerols were observed after administration of clofibrate to rats fed the RSO-diet. It is suggested that these changes reflect the increased capacity of the liver to chainshorten very long chain length fatty acids. The extent of lipidosis in the heart of rats fed the RSO-diet was decreased by 50% by clofibrate. However, the concentration of erucic acid in heart triacylglycerols decreased much less (30%) than the concentration of all other fatty acids (50–65%). It is concluded that the clofibrate administration increased the oxidative capacity of the heart mitochondria and that the heart cell does not have an efficient system to handle very long chain length monounsaturated fatty acids as does the liver.     

Fatty acid composition of liver lipids in rats fed brominated fatty acids

Feeding rats diets containing brominated corn oil or di- or tetrabromostearate as the monoglyceride produced changes in fatty acid composition of liver lipids. Those changes associated with the feeding of brominated corn oil or tetrabromosterates could be explained by the accumulation of triglyceride, and the changes associated with the feeding of dibromostearate could result from the proliferation of a membrane system. A unique response to the feeding of diets containing brominated corn oil is an increase in the level of γ-linolenic acid.     

Response of plasma lipids to dietary cholesterol and wine polyphenols in rats fed polyunsaturated fat diets

This experiment was designed to evaluate the effects of dietary red wine phenolic compounds (WP) and cholesterol on lipid oxidation and transport in rats. For 5 wk, weanling rats were fed polyunsaturated fat diets (n−6/n−3=6.4) supplemented or not supplemented with either 3 g/kg diet of cholesterol, 5 g/kg diet of WP, or both. The concentrations of triacylglycerols (TAG, P<0.01) and cholesterol (P<0.0002) were reduced in fasting plasma of rats fed cholesterol despite the cholesterol enrichment of very low density lipoprotein + low density lipoprotein (VLDL+LDL). The response was due to the much lower plasma concentration of high density lipoprotein (HDL) (−35%, P<0.0001). In contrast, TAG and cholesteryl ester (CE) accumulated in liver (+120 and +450%, respectively, P<0.0001). However, the cholesterol content of liver microsomes was not affected. Dietary cholesterol altered the distribution of fatty acids mainly by reducing the ratio of arachidonic acid to linoleic acid (P<0.0001) in plasma VLDL+LDL (−35%) and HDL (−42%) and in liver TAG (−42%), CE (−78%), and phospholipids (−28%). Dietary WP had little or no effect on these variables. On the other hand, dietary cholesterol lowered the α-tocopherol concentration in VLDL+LDL (−40%, P<0.003) and in microsomes (−60%, P<0.0001). In contrast, dietary WP increased the concentration in microsomes (+21%, P<0.0001), but had no effect on the concentration in VLDL+LDL. Cholesterol feeding decreased (P<0.006) whereas WP feeding increased (P<0.0001) the resistance of VLDL+LDL to copper-induced oxidation. The production of conjugated dienes after 25 h of oxidation ranged between 650 (WP without cholesterol) and 2,560 (cholesterol without WP) μmol/g VLDL+LDL protein. These findings show that dietary WP were absorbed at sufficient levels to contribute to the protection of polyunsaturated fatty acids in plasma and membranes. They could also reduce the consumption of α-tocopherol and endogenous antioxidants. The responses suggest that, in humans, these substances may be beneficial by reducing the deleterious effects of a dietary overload of cholesterol.     

Acetoacetate metabolism of rats fed high fat or restricted calorie diets

Ethyl-¹⁴C-acetoacetate was used to trace oxidation and metabolism of acetoacetate when rats were fed a high fat diet (80% of total calories from beef tallow or corn oil, carbohydrate free), a high carbohydrate diet (2% corn oil) or a high carbohydrate diet with restriction of calories to one half of ad lib. consumption for two weeks. The rate of expiration of¹⁴CO₂ in all groups of animals did not differ significantly and was not related to plasma concentration of acetoacetate. The high fat diets slightly enhanced the oxidation of acetoacetate to¹⁴CO₂ over a 3 hr period compared to other diets. Incorporation of acetoacetate into fatty acids did not differ significantly among groups. Rats fed the high carbohydrate diet ad lib. incorporated into liver cholesterol more acetoacetate than did any other group, but dietary unsaturated fat resulted in greater incorporation of acetoacetate into cholesterol than saturated fat. High calorie and high beef tallow groups were ketonemic but the low concentration of plasma acetoacetate in rats fed a high corn oil diet indicates that unsaturated fatty acids are not ketogenic. The data show that utilization of acetoacetate is not significantly reduced in a ketonemic condition and support the premise that overproduction of ketone bodies is the cause of ketonemia. Rats appeared to be normal during the two-week period when no carbohydrate was included in the diet. Presented at the AOCS Meeting, Chicago, October, 1967.     

Changes in serum lipids in rats treated with oral cooper

Disturbances in lipid metabolism during copper deficiency in rats are well recognized. Copper deficiency is associated with the spontaneous retention of hepatic iron. Previous studies have reported that hypercholesterolemia and hypertriglyceridemia are associated with elevated hepatic iron concentrations in copper deficient rats. There was a direct relationship between the magnitude of blood lipids and the concentration of hepatic iron. Based on these data, it has been hypothesized that iron was responsible for the development of lipemia of copper deficiency. In this study was determined the effect of increasing doses of Cu(10, 20 and 50 ppm) in the diet, on the serum total lipids, total cholesterol, triglycerides (triacylglicerols), phospholipids, non-esterified fatty acids (NEFA) and liver iron and zinc concentrations in normal rats. The results were compared with normal rats that received a balanced diet containing 0.6 and 6 ppm of Cu, respectively. The results show that Cu-supplement diminished the cholesterol and triglyceride serum levels, increased the level of phospholipids, NEFA and concomitantly decreased the hepatic concentrations of Fe and Zn. There was a statistically significant (p < 0.05) simple correlation between triglycerides and liver Fe (r = 0.917; R2 = 64.03%), cholesterol and liver Zn (r = 0.872; R2 = 76.07%), cholesterol and liver Fe (r = 0.995; R2 = 99.10%), liver Fe and liver Cu (r = -0.612), liver Fe and liver Zn (r = 0.837), liver Cu and liver Zn (r = -0.612), and serum triglycerides and liver Zn (r = 0.967). The mechanism(s) by which Fe and Zn determine these changes is not known; none of the enzymes that act in cholesterol and triglyceride metabolism and biosynthesis require Fe and/or Zn. The increase of NEFA is due to changes in the process of lipolysis and re-esterification of the fatty acids in blood. However, additional studies are needed for the precise mechanisms of this interrelationships to be clarified.     

Myocardial lipids and nucleotides of rats fed olive oil or rapeseed oil

After 1 week, the level of myocardial fatty acids was 4 times greater in young rats fed high erucic rapessed oil than in those fed olive oil. The proportion of erucic acid was 5.6% in the mitochondrial fraction, 15.1% in the microsomal fraction, and 34.8% in the floating fat fraction. This incorporation of erucic acid into triglycerides of the floating fat was evidence of esterification. The changes in the mitochondrial lipids did not alter the content of adenine nucleotides of the myocardium nor its apparent capacity to oxidize substrates.     

Liver phospholipids of rats fed a choline-deficient diet supplemented with choline or methionine

The low amount of arachidonic acid in the total phospholipids in the liver of rats fed a standard type of choline-deficient diet was corrected by either choline or methionine, which also increased food intake. Choline increased the content of this fatty acid in the phosphatidyl ethanolamine but not in the phosphatidyl choline. Methionine increased both the amount of phosphatidyl choline and its content of arachidonic acid.     

Activity of fatty acyl CoA-lysophospholipid acyltransferases in liver microsomes of rats fed a choline-deficient diet

Feeding a choline-deficient diet to rats has no effect on the activity of fatty acyl CoA-lysophospholipid acyltransferases, even though liver microsomes are severely depleted of lecithins. Thus lecithins appear to have no functional role in the activity of these transferases. It can be excluded that the latter enzymes are involved in the production of the changes in the fatty acid composition of liver phospholipids occurring in choline-deficient rats. These changes result most likely from alterations in the biosynthesis of liver lecithins and cephalins.     

Blood pressure,serum lipids,and fatty acids in populations on a lake-fish diet or on a vegetarian diet in Tanzania

Major risk factors for coronary heart disease were assessed in two populations of Tanzania, one on a fish diet (FD) living along the coast of Lake Nyasa, and the other, mainly on a vegetarian diet (VD), living in a farming area. Lower blood pressure values were found in the FD subjects (n=618) vs. VD (n=618) (systolic blood pressure, SBP, 120±15 vs. 135±20,P<0.01; diastolic blood pressure, DBP, 70±9 vs. 78±11,P<0.01, respectively). In an FD subgroup (n=61), total cholesterol (TC) (122 vs. 136 mg/dL,P<0.01); triglycerides (TG) (82 vs. 105 mg/dL,P<0.01); and lipoprotein (a) [Lp(a)] (19.9±18.4 vs. 32.3±22.4,P<0.001) were lower than in a VD subgroup (n=55). Serum fatty acids (FA) in the FD subgroup were as follows: eicosapentaenoic acid (EPA) (20∶5) 2.48 vs. 0.72%, docosahexaenoic acid (DHA) (22∶6) 5.93 vs. 1.49%, vs. the VD, respectively. Arachidonic acid (AA) (20∶4n-6) also was higher in the FD vs. the VD group (9.85 vs. 8.30%,P<0.05), whereas 18∶2n-6 was about double (23.97 and 14.85%) in VD vs. FD. The peculiar serum FA pattern in FD reflected the FA of dietary fish. In fact, in four main species of lake fish, DHA was 8–19%, higher than EPA (1.8–4.2%), in contrast with the situation in cold-water fish, and AA was 5.8–8%, higher than in cold-water fish. The data, obtained in populations strictly on natural, unprocessed, low-fat diets, show that a diet based on freshwater fish results in lower BP, serum TC, TG, and Lp(a), and suggests that serum AA is not reduced when the major dietary n-3 is DHA rather than EPA.     

Effect of cocaine intake on the development of fatty liver in rats fed a low-protein diet

It has been shown that cocaine given in the diet is able to reduce fat accumulation in the liver of protein-malnourished rats (Arch. Latinoamer. Nutr. 19: 69-79, 1969). This study was, therefore, designed to approach the probable action of the drug upon the process (increased triglycerides synthesis and normal/decreased capacity for exporting triglycerides from the liver into the blood) which leads to an increased fat accumulation in the liver under this physiological condition. To accomplish this purpose, the total and fractioned lipids in the liver and total lipids as well as lipoproteins in serum were determined in female Wistar rats (120-130 g) fed either a 5% corn protein diet or a 20% casein diet, with and without cocaine (15 mg HCl cocaine/10 g of diet) for 18 days. The results, aside from confirming the reduction (p less than 0.001) of fat accumulation in the liver of rats fed on the 5% corn protein diet plus cocaine, revealed that this drug also reduced triglycerides concentration (significantly, (p less than 0.001, when results were calculated by difference, and slightly reduced them when results were determined) in this tissue. Nevertheless, it increases both total lipids (p less than 0.05) and triglycerides-rich pre-beta lipoprotein (p less than 0.10) levels in the serum of these animals. Otherwise, these lipidic parameters were not modified by cocaine in rats on the 20% casein diet, except for the total cholesterol level in liver and the cholesterol-rich beta lipoprotein level in serum. Respectively, these were slightly and significantly (p less than 0.001) reduced by the drug. These evidences and their statistical significance suggest that cocaine given chronically with the 5% corn-protein diet for 18 days, reduces at least partially (other biochemical event in the liver could have also accounted for its effect at this level) the liver fat accumulation, by increasing the triglycerides output from the liver into the blood. Elsewhere, cocaine appears to be able to induce some metabolic alterations in the hepatic cholesterol of well-nourished rats.     

Lung surfactant and fatty acid composition of lung tissue and lavage of rats fed diets containing different lipids

Two nutritional models, essential fatty acid (EFA) deficiency and the feeding of saturated vs unsaturated fats, were used to determine the effects of dietary lipids on the fatty acid composition of rat lung and lavage. Semipurified diets containing 7% corn oil, 7% hydrogenated coconut oil (EFA-deficient), 10% butter or 10% safflower oil were fed to dams during lactation and thereafter to their offspring for a total of 24 weeks. Lipids were extracted from the lung lavage and lung tissue and their fatty acid composition was determined. The content of dipalmitoylphosphatidylcholine (DPPC), the main surfactant in the lungs, was also determined. The results show that the levels of DPPC in the lungs of rats fed 10% butter decreased although the decrease in the EFA-deficient rats was greater. Comparing rats fed butter with those fed corn oil, there were also modifications in the fatty acid composition of the total lipids and phospholipids of lung tissue and lavage as well as in phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol +phosphatidylserine fractions isolated from the lung tissue. The changes in fatty acid composition were somewhat fewer in rats fed butter then in those fed an EFA-deficient diet. The results suggest that a marginal EFA deficiency produced in rats by long-term feeding of 10% butter may account for the reduction in DPPC levels and in the changes in fatty acid composition in the lung tissue and lavage.     

Increased plasma triglyceride secretion in EFA-deficient rats fed diets with or without saturated fat

Metabolic responses to essential fatty acid-deficiency in rats include an increased rate of triglyceride secretion into the plasma, a large reduction in the HDL₁ plasma lipoprotein concentration, and increased concentrations of liver triacylglycerols and cholesteryl esters. Because of differences in the types of EFA-deficient diets used, it is not clear whether these responses were solely due to the absence of EFA from the diet or whether saturated fat, or differences in acyl group chain length in this fat, might be responsible. Therefore, we fed rats diets differing only in amounts and kinds of fat, and measured triacylglycerol secretion rates and liver concentrations of triacylglycerols and cholesteryl esters, for comparison with our earlier measurements of plasma high density lipoprotein subpopulations in rats fed exactly the same diets. The purified diets contained either no fat, 5% by weight hydrogenated coconut oil, 5% hydrogenated cottonseed oil, or each of these three diets supplemented with 1% safflower oil, or 5% corn oil. We also fed some rats a nonpurified stock diet for comparison with literature reports. The present results indicate that the metabolic responses to essential fatty acid deficiency described above are definitely due to essential fatty acid-deficiency and not to the presence or chain length of acyl groups in saturated fat in the diet. Presented in part at the May 1984 meeting of the American Oil Chemists' Society in Dallas, Texas.     

Short-Term changes in hepatic HMG-CoA reductase in rats fed diets containing cholesterol or oat bran

In vivo regulation of hepatic HMG-CoA reductase (HMGR) (mevalonate: NADP⁺ oxidoreductase [acylating CoA]; EC 1.1.1.34] by phosphorylation/dephosphorylation has not been demonstrated. Rats were meal-fed semipurified diets; effects of inclusion of cholesterol (2%) or oat bran (15%) in a single meal on expressed (phosphorylated) and total (dephosphorylated) activities of HMGR were measured from 15 min to 4 hr after presentation of the meal. Expressed activity was not significantly altered in response to the control diet during the time periods examined, while total HMGR activity declined by 15 min and increased through 4 hr to an activity about 1.5 times control levels. Addition of cholesterol resulted in little change in expressed activity but a greater and more sustained reduction in total activity. Oat bran caused reductions in both total and expressed activities, which were maintained through 4 hr. Total HMGR activity was best correlated with apparent demand for cholesterol synthesis.     

Hypercholesterolemia in rats fed cholesterol in agar gel diets

Female (Exp. I) or male (Exp. II) weanling rats were fed diets containing either 2% Solka-Floc or 2% agar for 28-day periods. Some groups received 1% cholesterol, either added in crystalline form or first dispersed in the oil portion of the diet, and some agar groups received their diet in a gelled form. Feces were collected for a 3-day period after 2 weeks (Exp. II) or during the fourth week (Exp. I) of experimentation. Serum and liver cholesterol, total liver lipids, fecal lipids, and fecal sterols were determined. The results indicated that cholesterol feeding increased serum cholesterol, total liver, and fecal lipids, liver cholesterol, and fecal sterols. Substitution of agar for Solka-Floc in dry (nongelled) diets further increased total liver lipids (Exp. I), but had no significant effect upon any other measured parameter. Gelling of 1% cholesterol agar diets, in contrast to the 1% cholesterol dry agar diet, resulted in reduced liver cholesterol in both experiments. Gelling significantly increased fecal sterols after 2 weeks feeding (Exp. II), but no significant differences were observed after 4 weeks feeding (Exp. I) when compared to 1% cholesterol-fed groups. Small, nonsignificant increases of liver cholesterol and total liver lipids with similar reduction of fecal sterols resulted from dispersing the cholesterol in the oil portion of the diet prior to mixing. The results indicate that (a) inclusion of 2% agar in rat diets and (b) dispersing cholesterol in oil had little effect upon serum, liver, or fecal lipids in cholesterol-fed rats. However, gelling the agar diets reduced liver cholesterol, possibly by initial reduction of dietary cholesterol absorption.