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Rationale: Although dietary fatty acids are a major fuel for the heart, little is known about the direct effects of dietary fatty acids on gene regulation in the intact heart., Objective: To study the effect of dietary fatty acids on cardiac gene expression and explore the functional consequences., Methods and Results: Oral administration of synthetic triglycerides composed of one single fatty acid altered cardiac expression of numerous genes, many of which are involved in the oxidative stress response. The gene most significantly and consistently upregulated by dietary fatty acids encoded Angiopoietin-like protein (Angptl)4, a circulating inhibitor of lipoprotein lipase expressed by cardiomyocytes. Induction of Angptl4 by the fatty acid linolenic acid was specifically abolished in peroxisome proliferator-activated receptor (PPAR)beta/delta(-/-) and not PPARalpha(-/-) mice and was blunted on siRNA-mediated PPARbeta/delta knockdown in cultured cardiomyocytes. Consistent with these data, linolenic acid stimulated binding of PPARbeta/delta but not PPARalpha to the Angptl4 gene. Upregulation of Angptl4 resulted in decreased cardiac uptake of plasma triglyceride-derived fatty acids and decreased fatty acid-induced oxidative stress and lipid peroxidation. In contrast, Angptl4 deletion led to enhanced oxidative stress in the heart, both after an acute oral fat load and after prolonged high fat feeding., Conclusions: Stimulation of cardiac Angptl4 gene expression by dietary fatty acids and via PPARbeta/delta is part of a feedback mechanism aimed at protecting the heart against lipid overload and consequently fatty acid-induced oxidative stress.

We investigated the effects of dietary trans fatty acids, PUFA, and SFA on body and liver fat content, liver histology, and mRNA of enzymes involved in fatty acid metabolism. LDL receptor knockout weaning male mice were fed for 16 wk with diets containing 40% energy as either trans fatty acids (TRANS), PUFA, or SFA. Afterwards, subcutaneous and epididymal fat were weighed and histological markers of nonalcoholic fatty liver disease (NAFLD) were assessed according to the Histological Scoring System for NAFLD. PPARalpha, PPARgamma, microsomal triglyceride transfer protein (MTP), carnitine palmitoyl transferase 1 (CPT-1), and sterol regulatory element binding protein-1c (SREBP-1c) mRNA were measured by quantitative RT-PCR. Food intake was similar in the 3 groups, although mice fed the TRANS diet gained less weight than those receiving the PUFA diet. Compared with the PUFA- and SFA-fed mice, TRANS-fed mice had greater plasma total cholesterol (TC) and triglyceride (TG) concentrations, less epididymal and subcutaneous fat, larger livers with nonalcoholic steatohepatitis (NASH)-like lesions, and greater liver TC and TG concentrations. Macrosteatosis in TRANS-fed mice was associated with a higher homeostasis model assessment of insulin resistance (HOMA(IR)) index and upregulated mRNA related to hepatic fatty acid synthesis (SREBP-1c and PPARgamma) and to downregulated MTP mRNA. Diet consumption did not alter hepatic mRNA related to fatty acid oxidation (PPARalpha and CPT-1). In conclusion, compared with PUFA- and SFA-fed mice, TRANS-fed mice had less adiposity, impaired glucose tolerance characterized by greater HOMA(IR) index, and NASH-like lesions due to greater hepatic lipogenesis. These results demonstrate the role of trans fatty acid intake on the development of key features of metabolic syndrome.

Luminal perfusion with the long-chain fatty acid (LCFA) oleate in concentrations similar to that found in premature infant formula produces a dose- and age-dependent mucosal injury in developing intestine. To investigate whether this lipid-induced phenomenon is a function of the degree of saturation and/or chain length of the fatty acid, 51Cr-EDTA plasma-to-lumen clearance was measured in jejunum and ileum of 1-d-, 3-d-, 2-wk-, and 1-mo-old piglets after perfusion with 5-mM solutions of different medium-chain saturated fatty acids and saturated and unsaturated LCFA. Mono- and polyunsaturated LCFA produced significant increases in jejunal permeability. In general, this effect was greater in piglets < or = 2 wk old compared with 1-mo-old animals, but no differences were observed among the unsaturated LCFA within an age group. In contrast, the alterations in mucosal permeability induced by medium-chain fatty acids were overall more attenuated than those induced by LCFA. Our results suggest that developing intestine is vulnerable to the injurious effect of dietary fatty acids and that the lipid-induced changes in mucosal permeability appear to be a function of the fatty acid chain length. The degree of saturation of the fatty acid does not alter its cytotoxic effects.

Both dietary unsaturated fat and raw soybean products are known to enhance pancreatic carcinogenesis when fed during the postinitiation phase. A comparison of these two dietary components was made to evaluate the relative potency of each ingredient for enhancing pancreatic carcinogenesis and to determine if this enhancement was correlated with an increase in plasma cholecystokinin (CCK) levels. Male Wistar rats were initiated with a single dose of azaserine (30 mg/kg body weight) at 14 days of age. The rats were weaned to test diets formulated from purified ingredients. Dietary protein at 20% by weight was either casein or soy protein isolate (heat treated or raw). Corn oil was the unsaturated fat of major interest and it was fed at either 5 or 20% by weight. Pancreases were quantitatively evaluated for carcinogen-induced lesions at 2- and 4-month postinitiation. In a second experiment designed to closely mimic the above experiment, rats were implanted with cannulae which allowed plasma to be repetitively sampled over a 2.5-week period during which the test diets were fed. Plasma was collected both prior to introduction of the test diets and afterwards. Plasma CCK was measured by a specific radioimmunoassay. Both the 20% corn oil diet and the raw soy protein isolate diet enhanced pancreatic carcinogenesis. The effects of the raw soy protein isolate on the growth of the carcinogen-induced lesions were significantly greater than the effects of the 20% corn oil diet. Plasma CCK values were not elevated in the rats fed the 20% corn oil diet, but they were significantly elevated in the rats fed the raw soy protein isolate. Heat-treated soy protein isolate neither enhanced carcinogenesis nor elevated the plasma CCK level. This study demonstrates that certain plant proteins enhance the growth of carcinogen-induced pancreatic foci and that this effect is considerably greater than the enhancement by high levels of dietary unsaturated fat. Furthermore, the enhancement by the raw soy protein isolate may be mediated by CCK; but this does not appear to be the mechanism by which the unsaturated fat, corn oil, enhances pancreatic carcinogenesis.

A diet of low polyunsaturated-to-saturated fatty acid (P/S) ratio known to alter fatty acid composition of lymphocyte membranes and retard the progressive changes which result in loss of suppressor capacity in SJL/J female mice, was fed to these mice from conception to study its effect on the development of lymphoma. Between 12 and 14 months, at a time when 100% of mice on a diet of high polyunsaturated-to-saturated fatty acid ratio showed lymphoma, only 70% of the mice fed the low P/S diet had developed lymphoma. Dietary fatty acid levels would appear to have a significant effect on the rate of progression of lymphoma in SJL/J female mice.

Light microscopy of paraffin embedded and frozen sections, supplemented with electron microscopy, was performed on the heart muscle of young rats fed rapeseed oil in short-term experiments. It was confirmed that high levels of rapeseed oil, which contains erucic acid, produce severe lipoidosis of the heart muscle fibres within 10 days. An attempt was made to find out the lowest level of erucic acid in the rat diet to give rise to pathological fatty accumulation. Several frozen sections from each heart or serial sections in combination with electron microscopy were used for this evaluation. The level found to give rise to pathological fatty accumulation was about 2% by weight (w/w), while rats fed 1% erucic acid showed normal myocardium. No direct proof that erucic acid is of importance in human pathophysiology has hitherto been presented. It is concluded, however, that the similarity in reaction among the many species of experimental animals tested by different workers, as well as the basic metabolic disturbances demonstrated, are in strong favour of a similar effect in man.

The influence of dietary rapeseed oil on the lipid classes and fatty acid pattern of rat heart homogenate and mitochondria has been investigated after feeding a diet with 9.8 weight- % erucic acid for 10 days and 1.4 and 2.6% erucic acid for 28 days. The rats treated with 9.8% erucic acid showed a significant increase in the triglycerides of the heart mitochondria. This tendency was much less pronounced in rats treated with 1.4 resp. 2.6% erucic acid. These results confirm those of other investigators. A slight increase in the cholesterol esters of the mitochondria could be seen in all the treated rats. The total phospholipids were decreased in the experiment with 9.8% erucic acid and slightly increased in experiments with 1.4 and 2.6% erucic acid. The concentration of phosphatidylcholine showed a tendency to increase and the concentration of phosphatidylethanolamine to decrease in the experiment with 9.8% erucic acid in the diet. The concentration of cardiolipin was mainly unchanged. In all experiments the triglycerides of the heart mitochondria showed a high content of erucic acid. The fatty acids of the cholesterol esters of the heart mitochondria were also influenced of dietary rapeseed oil but to a less extent than the triglycerides. The fatty acids of phosphatidylcholine, phosphatidylethanolamine and cardiolipin were all influenced by the dietary rapeseed oil, but the erucic acid seemed to have a specific affinity to cardiolipin. Cardiolipin of rat heart mitochondria was isolated and identified with gas chromatography and mass spectrometry. The isolated cardiolipin was found to contain 12 per cent erucic acid after feeding 9.8% erucic acid as rapeseed oil for 10 days. Similar results were obtained after feeding glyceryl trierucate for 5 days to rats. The incorporation of erucic acid into cardiolipin was followed by a corresponding decrease of linoleic acid. This observation is of great interest because the molecular structure of fatty acids in lipid molecules has a profound influence of the packing of these molecules in a bilayer. Since cardiolipin is a component of the inner membrane of mitochondria its high affinity for erucic acid might influence the normal function of the inner membrane of heart mitochondria.

Rats fed rapeseed oil, pure erucic acid, or a control diet of sunflowerseed oil during 24-26 weeks were studied for effects upon mechanical behavior of the isolated perfused heart and upon myocardial performance and hemodynamics in intact animals both under basal and stimulated conditions. In spite of focal myocardial fibrotic lesions due to rapeseed oil, no changes were found with respect to the intrinsic myocardial contractility in vitro and and in vivo. After inotropic intervention, only the rapeseed oil fed animals showed less contractile reserve capacity. The absence of this effect in the erucic acid-treated animals is in agreement with the histological studies showing no epicardiac fibrotic lesions in these animals. It appears that erucic acid is able to interfere with the contractile system of the peripheral vascular system. Both in the rapeseed oil-treated group and the erucic acid-treated group, the vasoconstrictor response toward norepinephrine was profoundly reduced. In all three oil fed groups, isoproterenol reduced myocardial contractility which has been attributed to a lowered perfusion pressure in the coronary blood supply of the myocardium with simultaneous increased energy demand. Neither rapeseed oil nor erucic acid feeding led to electrocardiographic changes in comparison with the control sunflowerseed oil group. It is concluded that rapeseed oil and not erucic acid is responsible for loss of contractile reserve capacity without changes in the myocardial conductance system and further, that erucic acid might interfere with the peripheral vascular system. Finally, it appears that a fat rich diet might result in reduced myocardial function during a state of energy demand coupled with a blood pressure decrease.

In long-term studies covering up to 160 days young Sprague-Dawley rats were fed diets containing 40 cal% of fat. The fat component consisted of either conventional rapeseed oil, or Canadian rapeseed oil low in erucic acid, or arachis oil. Myocardial fatty accumulation was demonstrated in light microscopic studies throughout the experiments in rats fed conventional rapeseed oil, but the number of fat droplets decreased with time. The controls fed arachis oil showed no fatty accumulation. In the rats fed conventional rapeseed oil focal myocardial lesions appeared after 40 days on the diet. These consisted of histiocytic infiltration, occurrence of macrophages, myolysis, proliferation of fibroblasts and finally scarring. Such foci were found widely spread in the myocardium of these rats. In the experimental groups given Canadian rapeseed oil from the cultivar Oro no histiocytic foci or scarring were observed. Small myocardial lesions were occasionally found in the control rats. These latter findings were observed on serial sections. It was concluded that this type of lesion is a "normal" finding. The number and size of the foci observed in animals fed conventional rapeseed oil (10% and 2% (w/w) erucic acid in the diet) indicate, however, that they have to be considered pathological under such circumstances. The pathogenesis of the myocardial alteration is discussed and it is concluded that the long-chain fatty acids are responsible. No direct proof has been presented that the described events are of importance in human pathophysiology, However, several circumstances pointing in this direction are discussed. It is concluded that on the basis of our present knowledge a pathological effect of erucic acid and its homologues in man cannot be excluded.

The effects of erucic acid on the oxidative metabolism of rat-heart mitochondria have been investigated using intact animals, perfused beating heart, isolated mitochondria and mitochondrial extracts. Feeding rats with a diet containing erucic acid was found to lead to a diminished ability of the isolated heart mitochondria to oxidize various substrates, in accordance with previous reports (Houtsmuller et al., Biochim. Biophys. Acta 218 (1970) 564). This effect was almost pronounced with palmitylcarnitine as substrate, in which case the rate of oxidation was decreased by more than 50% at such a low erucic acid content in the diet as 1.4% given over 2-4 weeks. Oxidation of palmitylcarnitine was also found to be inhibited when erucylcarnitine was added to isolated heart mitochondria from control animals, in agreement with earlier observations (Christophersen and Bremer, FEBS Lett. 23 (1972) 230; Biochim. Biophys. Acta 280 (1972) 506). The inhibition was accompanied by a decrease in the rate and extent of reduction of mitochondrial flavoprotein. Experiments with perfused beating rat-heart likewise revealed an inhibition of flavoprotein reduction, as well as nicotinamide nucleotide reduction, when erucate was added to the perfusing medium of the beating heart respiring with oleate--but not with octanoate--as substrate. These data together with those earlier published in the literature indicate that erucic acid may interfere with the enzyme system involved in the mitochondrial oxidation of long-chain fatty acids, probably at the level of acyl-CoA dehydrogenase. Kinetic data supporting this conclusion, obtained with extracts of rat-heart mitochondria containing the acyl-CoA dehydrogenase and electron-transferring flavoprotein system, are presented. The possible implications of these results for the known effect of dietary erucic acid in causing an accumulation of fat in the heart are discussed.

Rapeseed oil has a growth retarding effect in animals. Some investigators claim that the high content of erucic acid in rapeseed oil alone causes this effect, while others consider the low ratio saturated/monounsaturated fatty acids in rapeseed oil to be a contributory factor. Normally erucic acid is not found or occurs in traces in body fat, but when the diet contains rapeseed oil erucic acid is found in depot fat, organ fat and milk fat. Erucic acid is metabolized in vivo to oleic acid. The effects of rapeseed oil on reproduction and adrenals, testes, ovaries, liver, spleen, kidneys, blood, heart and skeletal muscles have been investigated. Fatty infiltration in the heart muscle cells has been observed in the species investigated. In long-term experiments in rats erucic acid produces fibrosis of the myocardium. Erucic acid lowers the respiratory capacity of the heart mitochondria. The reduction of respiratory capacity is roughly proportional to the content of erucic acid in the diet, and diminishes on continued administration of erucic acid. The lifespan of rats is the same on corn oil, soybean oil, coconut oil, whale oil and rapeseed oil diet. Rats fed a diet with erucic acid or other docosenoic acids showed a lowered tolerance to cold stress (+4 degrees C). In Sweden erucic acid constituted 3-4% of the average intake of calories up to 1970 compared with about 0.4% at present.

The morphological effects on the myocardium of feeding rapeseed oil were compared in conventional and germ-free rats in short-term experiments (10 days). It was concluded that the fatty accumulation in the heart muscle cells occurring in rats fed rapeseed oil was not influenced by the presence or absence of a normal intestinal flora. In long-term experiments (80 days) under similar conditions, the myocardial effects of feeding germ-free rats with conventional rapeseed oil, rapeseed oil from the Canadian cultivar Oro very low in erucic acid, or arachis oil were studied in serial sections. Severe myocardial lesions developed in the group of rats fed conventional rapeseed oil, while in the other two groups the myocardium was completely normal. These results give no support to the theory that other factors than C22:1 acids in rapeseed oil are responsible for the myocardial lesions.

The functional effects of a diet containing rapeseed oil (40% of total energy intake) were studied in rats, starting at age 28 days. There were no effects on the electrocardiogram in spite of morphological changes in the myocardium. In 10 female rats the urine osmolality following 16 hours of dehydration was approximately 20% lower than in 10 control rats during the 9th, 10th and 20th week of the experiment. It is suggested that erucic acid in the rapeseed oil inhibits beta-oxidation of fatty acids in the kidney, thereby depriving the kidney of energy involved in sodium transport.

The effects of dietary polyunsaturated fat in BALB/c mice were determined for several stages of mammary tumorigenesis: a) growth of intact and transplanted normal mammary tissue, b) growth of hyperplastic alveolar nodule (HAN) lines D1/ UCD and Z5C1 , c) incidence of tumors occurring in D1/ UCD , and d) growth rate of the tumors. Growth of transplanted normal mammary epithelium as well as intact glands was faster in mice fed a 10% corn oil (CO) diet, which contains linoleate, than in those fed 10% hydrogenated cottonseed oil ( HCTO ), a diet free of the polyunsaturated fatty acid. We concluded that transplantation itself does not alter the response of mammary tissue to dietary fat. The growth of both HAN lines was unchanged by the presence of CO in the diet. For ascertainment as to whether the difference in response was specific to the tissue, ducts and HAN line D1/ UCD were transplanted into left and right gland-free fat pads of the same mice. The duct tissue grew faster when the hosts were fed the 10% CO diet than in the hosts fed the 10% HCTO diet; however, no effect of dietary fat was noted with the HAN. Although indomethacin retarded the growth of normal tissue in the mice fed CO diet, this prostaglandin inhibitor had no effect on the growth of the HAN transplants. Both incidence and growth rate of tumors arising from transplants of HAN line D1/ UCD were greater in mice fed diets containing either 0.3, 1, or 10% CO than in those fed 10% HCTO . The smaller number of tumors arising in mice fed with 10% HCTO diet may be the result of the slower growth rate of the neoplasms in mice fed diets devoid of linoleate.

Cynomolgi (Macaca fascicularis) were fed diets containing 25% rapeseed oil (RSO), partially hydrogenated herring oil (PHHO), or a 3:1 mixture of lard and corn oil as control for 4 months. The RSO contained approximately 25% of the fatty acids as erucic acid; the PHHO contained a similar concentration of mainly cetoleic acid. The control diet did not include such fatty acids. At the time of necropsy, the RSO- and PHHO-fed monkeys showed myocardial and skeletal muscle lipidosis. Foci of mononuclear cell infiltration, although infrequent, occurred in all three groups and were thought to be nonspecific. The only significant intergroup difference in serum biochemical or hematologic parameters was an increase in serum glutamic-oxaloacetic transaminase activity in both RSO and PHHO groups. Ultrastructural studies confirmed the presence of lipidosis in cardiac and skeletal muscle and revealed mild mitochondrial degeneration, causing a depression of the P/O ratio of the RSO group and a State III respiratory rate depression of the PHHO group. The difference in the exposure/life span ratio represented by this experiment may account for the absence of clear intergroup differences such as are reported in rats used in similar studies, but a true species difference in regard to dietary oils containing docosenoic acids has to be considered as well.