Phospholipid profile and production of prostanoids by murine colonic epithelium: Effect of dietary fat

Dietary fat and abnormal production of various prostanoids have been linked to various disease states of the large bowel, including cancer of the colon. Studies were conducted to determine the effect of dietary fat (beef tallow or corn oil) on the lipid composition and prostanoid production of the murine colon. Female C57BL/6J mice were fed high-fat (HF) diets (47% of calories as fat) or low-fat (LF) diets (10% of calories as fat). After four wk of dietary treatment, the mucosa was scraped, and lipids were extracted from the mucosal and muscle layers. The fat content of the diets did not significantly alter the amount of phospholipid (PL) or neutral lipid in the colonic tissue. However, the HF affected the PL profile of the colonic mucosa. For example, the ratio of phosphatidylcholine (PC) to phosphatidylethanolamine (PE) was significantly higher for both the HF groups compared with that of the two LF groups (0.76±0.15 and 0.80±0.13 vs 0.31±0.20 and 0.34±0.18). Production of 13,14-dihydro-15-keto-PGE₂ (measured as bicyclic PGE₂) and TXB₂ (a stable metabolite of TXA₂) and PGF_1α (a stable metabolite of PGI₂) was unaffected by the dietary treatments. The muscle had a different PL profile (PC:PE is 2.6±0.1) than the mucosa and contributed a larger proportion of the prostanoids formed. This study demonstrates that the phospholipid polar head group composition of normal colonic mucosa is altered by dietary fat, but the ability of the mucosa to synthesize metabolites of PGE₂, TXA₂ and PGI₂ is not affected. ARC Contribution No. 1504.     

Influence of dietary fat and protein on serum cholesterol of cholesterol-fed chicks

Summary Cottonseed oil, partially hydrogenated cottonseed oil and corn oil, were fed at 4% and 10% of the diets with two levels of protein, 19% and 25%, and with 0.5% cholesterol to cockerels 21 days of age for a period of 38 days. Blood samples were obtained at 0, 14, 24, and 38 daysvia heart puncture. The data indicate that the serum cholesterol value, irrespective of the level or type of fat, was significantly lower in those groups of birds which were fed the higher level of protein. Excluding the combination of 19% protein and 10% cottonseed oil, the degree of saturation did not have any apparent effect upon serum cholesterol because the mean differences among the oils and between the fat levels were not statistically significant.     

Effect of dietary fat on the fluidity of platelet membranes

Dietary fat type was reflected in the phospholipid fatty acid composition of the plasma membrane of rabbit platelets and apparently controlled the fluidity of these membranes. Rabbits were maintained for 6 months on diets that varied in stearic and polyunsaturated fatty acids and thus had different potentials for thrombosis. Microviscosities at 37 C, calculated from the anisotropy of fluorescence from the probe 1,6-diphenyl-1,3,5-hexatriene, were 3.5, 3.4, 2.8 and 2.2. poise for platelet membranes isolated from rabbits whose only source of dietary fat was cocoa butter, milkfat, coconut oil, or corn oil, respectively. The relative fluidities of the membrane isolates were correlated with the polyunsaturated fatty acid contents of the membrane phospholipids.     

Effect of dietary fat on phospholipid class distribution and fatty acid composition in rat fat cell plasma membrane

The effect of dietary fats on phospholipid class distribution and fatty acid composition was studied in rat fat cell plasma membrane. Three groups of male Wistar weanling rats were fed for 8 wk three diets differing in the amount and nature of the fats: 1.5% sunflower oil (low fat control; LFC), 10% sunflower oil (high fat, unsaturated; HFU), 1.5% sunflower oil+8.5% cocoa butter (high fat, saturated; HFS). Plasma membranes were prepared from epididymal adipocytes. The amount and type of dietary fat significantly altered membrane phospholipid distribution. Phospholipid content was lowered with HFU as compared to LFC or HFS diets, but no changes were observed for cholesterol. Phosphatidylinositol (PI) and phosphatidylserine (PS) were less affected by dietary changes than were other phospholipid classes. Major changes were detected for phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin (SM) contents. No large changes in PC and PE fatty acid compositions were observed between the LFC and HFS groups, but the HFU diet induced several changes. Correlations with plasma membrane 5′-nucleotidase activities are discussed.     

Activation of rat liver cholesterol ester hydrolase by cAMP-dependent protein kinase and protein kinase C

Short term regulation of hepatic cholesterol ester hydrolase by reversible phosphorylation is described. Two different kinase systems seem to be involved in this regulation. The addition of ATP, cyclic AMP and Mg²⁺ to rat liver 104,000× g supernatant (S104) produced a 100–140% increase in cholesterol ester hydrolase activity. This stimulation was abolished when protein kinase inhibitor was added prior to the addition of ATP, cyclic AMP and Mg²⁺. Cholesterol ester hydrolase activity was also stimulated when calcium ions, phosphatidylserine, and diolein were added to S104 along with ATP and Mg²⁺. Diolein in this reaction could be substituted by phorbol 12-myristate 13-acetate. Preincubation of S104 with alkaline phosphatase resulted in a deactivation of cholesterol ester hydrolase. The addition of increasing concentrations of Mg²⁺ to S104 produced increasing inhibition of cholesterol ester hydrolase activity, and this effect was blocked by NaF. It is suggested that rat liver cholesterol ester hydrolase is activated by cyclic AMP dependent protein kinase and protein kinase C. Deactivation is accomplished by dephosphorylation catalyzed by a phosphoprotein phosphatase, dependent on Mg²⁺. This work was presented at the Twenty-Third Southeastern Regional Lipid Conference, held October 26–28, in Cashiers, North Carolina.     

Derivatives of Di-O-octanoylglycerol and mono-O-octylglycerol as modulators of protein kinase C and diacylglycerol kinase activities

Twelve analogs of 1,2-di-O-octanoylglycerol modified at C-3 and three quaternaryN-alkyl-ammonium derivatives of glycerol were synthesized. The compounds were testedin vitro as potential modulators of the calcium activated, phospholipid dependent protein kinase C (PKC) and diacylglycerol (DAG) kinase activities in order to understand the molecular interactions of these enzymes with their natural activators, inhibitors, or substrates. PKC activity was assayed by measuring histone H₁ phosphorylation, and the compounds synthesized were tested either in the presence (inhibitors) or in the absence (activators) of 1,2-di-O-octanoyglycerol analogs with the phosphatidylserine/Ca²⁺ mixture. DAG kinase activity was measured by the incorporation of phosphate into 1,2-di-O-oleoyl-sn-glycerol in the presence of the various analogs synthesized. In regard to PKC activity, the assays revealed that 1,2-di-O-octanoylglycerol analogs are inactive when modified at C-3 with groups which do not permit hydrogen bonding. Under our conditions, di-O-octanoylthioglycerol, which has been reported as inactive, was able to activate PKC in the presence of phosphatidylserine. It has been shown to give a synergistic activation with diacylglycerol and had no affinity for the phorbol ester receptor binding site, suggesting thatO-octanoylthioglycerol interacts with the enzyme at a different site from the phorbol ester receptor binding site. PKC and DAG kinase activities are inhibited byN-alkyl-ammonium compounds (IC₅₀ 24 μM) only when either two 8-carbon alkyl or acyl chains are present at the 1- and 2-positions of the glycerol backbone. The fact that these compounds have a strong effect on the binding of [³H]phorbol 12,13-dibutyrate to protein kinase C, and also inhibit DAG kinase, may suggest binding to the DAG site of the regulatory domain of PKC.     

Effect of dietary components on the pathobiology of colonic epithelium: Possible relationship with colon tumorigenesis

The concept that diet plays an important role in the initiation and/or development of various types of tumors in man and experimental animals is well documented. The etiology of colon cancer is complex and multifactorial in nature, and these is little information on the dietary components that may act as initiators during colon tumorigenesis. We have evaluated various dietary heterocyclic mutagenic amines present in a typical “Western” diet for their nuclear damaging effect (presumably a genotoxic response) on the colonic epithelium of C57BL/6J mice in vivo. Among the mutagenic amines studied, 2-amino-3,4-dimethylimidazo(4,5-f)quinoline and 2-amino-3-methylimidazo(4,5-f)quinoline were very potent inducers of nuclear aberrations. These observations provide us with clues that our daily diet may contain colon-specific genotoxic components. Promotional effects of dietary fat and/or bile acids on colon tumorigenesis have been well studied. Dietary levels of calcium (0.1, 0.5 or 1.0% by weight) appear to modify the toxicity of orally administered fat or cholic acid (assessed by quantifying cell proliferation). The colons of animals consuming 0.1% or 0.5% calcium diet were more susceptible to the toxicity, whereas the colons of those consuming a 1.0% calcium diet appeared more like control colons. These studies demonstrate a profound effect of dietary constituents on the pathobiology of the colonic epithelium which may have a marked influence on the colon tumorigenesis.     

Effect of age and dietary fat (fish,corn and coconut oils) on tocopherol status of C57BL/6Nia mice

The effect of age and dietary fat type on tocopherol status was investigated using young and old C57BL/6Nia mice fed semipurified diets containing 5% (by weight) fish, corn or coconut oils and supplemented with 30, 100 or 500 ppm dl-α-tocopheryl acetate for 6 wk. Tocopherol levels in the diets, plasma, liver, kidney and lung were measured by high performance liquid chromatography following appropriate extractions. The results indicate that mice fed fish oil maintain lower plasma and tissue tocopherol concentrations than those fed corn and cononut oils (fish<corn oil<coconut oil). The difference was not due to a loss of tocopherol prior to consumption, but rather appeared to occur during the absorption process. Old mice had lower plasma and liver tocopherol concentrations than young mice. Old mice fed fish oil, however, maintained plasma tocopherol levels better than young mice fed fish oil, presumably due to their larger tocopherol pool. No age effect was detected on kidney and lung tocopherol levels. It is concluded that tocopherol status is affected by age and dietary fat type, especially fish oil.     

The effect of dietary fat on the glyceride structure of rat carcass fat

Carcass fats were obtained from weanling rats fed a complete diet for 8 weeks, which consisted of 2% cottonseed oil and 10% of the following fats: (1) corn oil; (2) the fatty acids of corn oil; (3) triricinolein; (4) ricinoleic acid; (5) the hydrogenated fatty acids of castor oil ; and (6) commercial hydrogenated shortening. The fats were subjected to both pancreatic lipase and nonspecific hydrolysis ; the resulting acids converted into methyl esters by conventional methods, and subjected to gas Chromatographie analysis. From these data, the positional distri-bution of the component fatty acids, glyceride types, and isomeric forms were calculated. The results indicated a preferential placement of un-saturated acids in the 2- position of the carcass triglycerides and that the carcass fat composition in terms of unsaturated (U) and saturated (S) fatty acid composition is not greatly influenced by the S and U compositions of the dietary fat. It was found that hydroxy acids or their tri-esters are metabolized much the same as are normal triglycerides and exert no particular in-fluence upon the fat structure of the rat. Some type of relationship between the dietary U and the U₃ in the carcass fat appears to be present. The glycerides of the carcass fats examined here are essentially a random mixture of the major glyceride types, but the isomeric forms (SUS, S SU, USU and UUS) are a definite non-random mixture. Carried out at the Food Res. Div., Armour & Co., and at The Burnsides Research Laboratory under research grant No. EF 225 from the National Institutes of Health, U. S. Public Health Service, and Deparmtent of Health, Education, and Welfare.     

Effect of dietary fat on individual long-chain fatty acyl-CoA esters in rat liver and skeletal muscle

The effect of dietary fat on the long-chain acyl-CoA ester profile of liver and skeletal muscle was investigated by feeding weanling rats 12%-fat diets composed of high-linoleic safflower oil (73% 18∶2n−6), high-oleic safflower oil (70% 18∶1n−9) or olive oil (70% 18∶1n−9) for six and ten weeks. Approximately 50% of both hepatic and skeletal muscle acyl-CoA esters comprised linoleoyl-CoA or oleoyl-CoA with high-linoleic or oleic feeding, respectively. Total hepatic acyl-CoA ester concentration was 40% higher (p<0.05) in rats fed 12% fat compared with controls fed a 4%-fat diet. These data demonstrate that the long-chain acyl-CoA ester profile of liver and skeletal muscle reflects the dietary fatty acid profile.     

Effect of dietary fat on rat liver microsomal and mitochondrial/lysosomal dolichol,phospholipid and cholesterol

The influence of different fat diets on liver phospholipid, cholesterol and dolichol was studied. Rats were separated into four groups and fed standard laboratory chow (control), a diet containing linolenic acid, a coconut oil diet, or a corn oil-containing diet. After five weeks, microsomes and mitochondrial/lysosomal fractions were prepared from the liver, and lipid compositions were analyzed. No changes in phospholipid content were observed. In control animals, the fatty acid compositions of phosphatidylcholine and phosphatidylethanolamine in the two subfractions were similar. However, these two phospholipids showed different fatty acid patterns, which were altered independently upon dietary treatment. The dietary treatments resulted, in most cases, in decreased cholesterol and dolichol contents and, especially in microsomes, in a decreased level of esterification of both lipids. The fatty acid compositions of cholesteryl esters in the two subfractions showed significant differences and cholesterol was esterified to a large extent with linolenic acid when this fatty acid was supplied in the diet. The same dietary treatment exerted different effects on the cholesterol localized in the two different intracellular compartments. This difference was most pronounced in rats fed the corn oil-containing diet; microsomal cholesteryl esters exhibited increased saturation, whereas cholesteryl esters in the mitochondrial/lysosomal fraction displayed decreased saturation. Dolichyl esters in the two cellular compartments had different fatty acyl compositions, with a considerably higher degree of saturation in microsomes. The various diets influenced the nature of the fatty acid moieties present in the isolated fractions and the effects on the two subfractions were opposite. The diet containing linolenic acid decreased the degree of saturation in microsomal dolichyl esters and increased the degree of saturation in the mitochondrial/lysosomal fraction. The results demonstrate that the fatty acid compositions of both dolichyl and cholesteryl esters display organelle specificity. Both the content of these lipids and their fatty acid compositions are greatly influenced by dietary conditions, and the esterification processes at different cellular locations exhibit independent regulation, regardless of the fatty acid content of the diet.     

Interaction between dietary protein and fat in triglyceride metabolism in the rat: Effects of soy protein and menhaden oil

The objective of the present study was to determine the mechanisms by which dietary proteins interact with dietary lipids in the regulation of triglyceridemia in rats. Male Sprague-Dawley rats (n=56) were subjected to 28-d experimental diets containing different combinations of proteins (20% w/w) and lipid sources (14% w/w): (i) casein-menhaden oil, (ii) casein-beef tallow, (iii) soy protein-menhaden oil, and (iv) soy protein-beef tallow. Significant protein-lipid interactions were observed on triglyceridemia and hepatic cholesterol in fasted rats. The combination of casein and beef tallow was associated with high plasma TG and hepatic cholesterol concentrations, which were reduced by substitution either of soy for casein or of menhaden oil for beef tallow. Therefore, triglyceridemia and liver cholesterol remained low with soy protein feeding, independently of the lipid source, as well as with menhaden oil feeding, regardless of the protein source. The menhaden oil diets reduced plasma cholesterol, hepatic TG, and TG secretion compared with beef tallow diets independently of the dietary protein source. Modifying the source of dietary proteins and lipids had no effect on post-heparin plasma lipoprotein lipase activity. These results demonstrate that soy protein can lower rat triglyceridemia relative to casein when associated with beef tallow consumption, whereas menhaden oil can attenuate hypertriglyceridemia when rats are fed casein. The data further suggest that part of the hypotriglyceridemic effect of soy protein in the rat may be mediated by reduced hepatic lipid synthesis, as is the case for menhaden oil.     

Effect of dietary fat on the lipid composition and utilization of short-chain fatty acids by rat colonocytes

The objective of the present studies was to examine the effect of dietary fat on the lipid composition of rat colonocytes and their utilization of short-chain fatty acids (SCFA). Rats were fed 14% beef fat, fish oil or safflower oil plus 2% corn oil in a semi-synthetic base diet for 4 wk. Colonocytes were isolated and their lipid composition was examined. Feeding beef fat and fish oil resulted in an increase in monounsaturated fatty acids and a reduction in ω-6 fatty acids. Feeding fish oil resulted in an enrichment with ω-3 fatty acids. These was no dietary influence on the amount of either cholesterol or phospholipids of colonocytes. Fish oil feeding resulted in significant increase in colonocyte free fatty acids (FFA) as compared to other diets. Dietary fat was found to have no effect on SCFA utilization by colonocytes. Colonocytes were found to utilize SCFA in the order of butyrate ≥acetate ≥propionate. The presence of acetate and propionate in the medium had no effect on the rate of butyrate utilization.     

Production and protein kinase C activation of diacylglycerols containing polymethylene-interrupted PUFA

Sciadonic acid (20∶3, Δ-5c,11c,14c) is a polymethylene-interrupted PUFA (PMI-PUFA) that is present in conifer seeds and known to be incorporated into animal cells and to accumulate in membrane PI as a substitute for arachidonate. In this study, we investigated whether PI having sciadonate could serve as source of DAG that could activate protein kinase C (PKC). When Swiss 3T3 cells cultured with sciadonic acid were stimulated with 100 nM of bombesin, 1-stearoyl-2-sciadonoyl-glycerol (G) and 1-stearoyl-2-arachidonoyl-G were produced. The net increments of these two molecular species of DAG reflected the levels of the two molecular species in the PI in the cells. When cells cultured with juniperonic acid (20∶4, Δ-5c,11c,14c,17c) were stimulated 1-stearoyl-2-juniperonoyl-G was produced in proportion to the level of this molecular species in PI in the cells. We also examined PKC activation by synthetic DAG using a partially purified PKC fraction from rat brain and found that both 1-stearoyl-2-sciadonoyl-G and 1-stearoyl-2-juniperonoyl-G could activate PKC comparably to 1-stearoyl-2-arachidonoyl-G. These results indicate that 1-stearoyl-PI having these C₂₀ PMI-PUFA residues can serve as sources of potential signaling molecules.     

Effects of hexadecylphosphocholine on protein kinase C and TPA-induced differentiation of HL60 cells

Several structural analogs of alkylphosphocholine (APC) were studied for their effects on protein kinase C (PKC) and 12-O-tetradecanoylphorbol-13-acetate (TPA) elicited biochemical and cellular events in HL60 cells. Hexadecylphosphocholine (He-PC₂), the APC prototype, inhibited PKC competitively with respect to phosphatidylserine and noncompetitively with respect to CaCl₂, both with an apparent K_i of about 15 μM. Inhibition of PKC by He-PC₂ was selective, since cyclic AMP dependent protein kinase and Ca²⁺/calmodulin dependent protein kinase II were relatively unaffected. He-PC₂ inhibited TPA-induced depletion of PKC and TPA-stimulated phosphorylation of cellular proteins in HL60 cells. TPA-induced differentiation of HL60 cells was also inhibited by He-PC₂, and this inhibition was synergistic or additive to the effects of 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine (H-7), a PKC inhibitor. The present findings are consistent with the hypothesis that inhibition of PKC might be related, in part, to the antineoplastic effect of He-PC₂ and ether lipid analogs such as ET-18-OCH₃ (1-octadecyl-2-methyl-glycero-3-phosphocholine).     

Effect of dietary fat upon aflatoxicosis in rats fed torula yeast containing diet

This study was designed to study the possible interrelationships between Torula yeast, vitamin E, and the dietary fat source on aflatoxin-induced tumors. Rats were fed Torula yeast-containing basal diets which included 1.7 ppm aflatoxin B₁ with either lard, corn oil or no fat, and with or without vitamin E supplements for 3 months. Thereafter, the respective diets without aflatoxin were fed for ca. 9 months. Animals receiving the vitamin E-deficient diets had a high mortality. Although the vitamin E-deficient, aflatoxin-treated rats had lower wt gains than did the vitamin E-deficient controls, they lived twice as long. In addition, regardless of the dietary fat source, the kidneys and adrenals of these vitamin E-deficient, aflatoxin-supplemented rats were found to be significantly heavier than the controls, and plasma cholesterol levels were elevated. Increased amounts of liver lipid were observed in response to aflatoxin in both corn oil-fed and fat-deficient rats. No such differences were observed in the responses of the vitamin E-supplemented groups to aflatoxin. On the corn oil diet, aflatoxin administration resulted in an increased deposition of polyunsaturated fatty acids in cholesteryl ester and phospholipid fractions in livers of vitamin E-deficient rats and the phospholipid fraction of vitamin E-sufficient rats. The vitamin E-deficient rats exhibited necrosis of the liver, which was alleviated when aflatoxin was included in the diet, and calcification of the kidneys, which was potentiated by the dietary aflatoxin. No tumors were observed in these animals. In animals maintained on vitamin E-sufficient diets for 1 year, growth was depressed as a result of aflatoxin administration with the greatest depression occurring in the group fed corn oil. Spleen wt were decreased in all groups given aflatoxin. However, there were no changes in either plasma or liver cholesterol or total liver lipids which could be attributed to aflatoxin administration. When aflatoxin was fed with lard, the cholesteryl ester, triglyceride, and free fatty acid fractions of plasma had decreased amounts of the C20:4 acid. In the cholesteryl ester fraction only, this change was accompanied by increased levels of C16:0, C18:0, and C18:1 acids. In the liver phospholipids, there were increased levels of mono- and polyunsaturated fatty acids and decreases in the saturated fatty acids. All of the animals receiving aflatoxin exhibited severe necrosis and tumor formation in the kidneys; the animals fed lard had the highest level of involvement and those in the fat-free group the least. Liver pathology was the least marked among the rats fed the fat-free diet. Since aflatoxin-induced tumors are rich in lipids, the fat-free diet may be protective to the animal.     

Separate effects of dietary protein and fat on serum cholesterol levels: another view of amino acid content of proteins

Casein or soy protein with vegetable or animal fat were used to determine the dietary protein or fat effects and their possible interaction on serum cholesterol levels. Young, male New Zealand white rabbits with a mean weight of 2.1 kg were divided into groups of six and fed one of four different diets containing 20% of the calories as protein, 30% as fat (according to dietary guidelines for the United States) and 50% as carbohydrate. The diets contained casein or soy (lysine/arginine ratio = 2.2 or 0.9, respectively) as the protein sources with fat from either almond oil or butter. There was no significant difference in weight gain among the diet groups. Total serum cholesterol level was highest among animals fed the diet containing butter with casein (177 +/- 25 mg/dl) or soy protein (189 +/- 50 mg/dl), it was intermediate in animals fed the vegetable oil with casein (121 +/- 14 mg/dl), and lowest in the soy protein with vegetable oil group (58 +/- 12 mg/dl). There was a significant difference in serum cholesterol levels due to the protein effect when vegetable oil was used (p less than 0.05) but not with butter. There was also a significant fat effect on serum cholesterol when the diet contained soy protein (p less than 0.005) but not when the protein was casein. No significant interaction was observed between the dietary fat and protein sources on serum cholesterol levels, which suggests that dietary protein and fat independently affect the levels of serum cholesterol. Thus, dietary protein has a significant effect on serum cholesterol levels and may be a factor in the low levels of serum cholesterol observed among vegetarians and in humans of Third World countries where the diets is primarily of vegetable origin.