Effects of essential fatty acid deficiency on prostaglandin synthesis and fatty acid composition in rat renal medulla

Studies are reported on the capacity of isolated rat renal papilla (inner medulla) to synthesize and release prostaglandin (PG) E from endogenous and exogenous precursor(s) during development of an essential fatty acid (EFA) deficiency in the rat. Weanling (21-day-old) male Sprague-Dawley rats were fed a fat-free diet supplemented with either 5% hydrogenated coconut oil (HCO) or 5% safflower oil (SO). At approximately 3, 6 and 7 weeks (6, 9 and 10 weeks of age), groups of animals fed each diet were killed for studies of PGE synthesis in the renal papillae. Differences in the fatty acid composition of the papillae lipids of the animals of each group were also determined. The in vitro production of PGE from endogenous precursor(s) was significantly reduced in the papillae from the 6-week-old rats fed the HCO diet compared to the control (SO) rats, and appeared to be near maximally depressed in the 10-week-old animals compared to that of animals fed an EFA deficient diet for over a year in an accessory experiment. Analyses of the fatty acids of the papillae lipids of the HCO groups showed that the levels of 18∶2 and 20∶4 were markedly reduced, and those of 16∶1, 18∶1 and 20∶3 were elevated compared to the controls even in the 6-week-old animals, typical of an EFA deficiency. The papillae lipids of the animals fed the HCO diet were also depleted of their stores of 22∶4ω6. A fatty acid believed to be derived by chain elongation of 20∶3ω9, 22∶3, was found in large concentrations in the papillae triglycerides of the EFA deficient rats. Incubations of exogenous arachidonic acid (20∶4) in homogenates and tissue slices of the papillae of the HCO dietary groups showed that the PG synthetase was not impaired by an EFA deficiency. The rate of PGE synthesis in the papillae of the EFA deficient animals was generally enhanced when exogenous 20∶4 was added, indicating that the concentration of available precursor(s) is a primary factor in the control of PGE synthesis in the papilla of the rat.     

Lysosomes and essential fatty acid deficiency

The hydrolytic activity usually associated with lysosomes increased in the homogenates and subcellular fractions of rat liver as a result of essential fatty acid (EFA) deficiency. The proportion of the total (tissue homogenate) activity found in each subcellular fraction, however, was unchanged by EFA deficiency. Lysosomes isolated from normal and EFA-deficient rat livers differed significantly in their stability to thermal and osmotic variations. This suggested that lysosomal membranes, like other membranes, were altered by EFA deficiency. In spite of increased tissue-bound hydrolytic activity and altered lysosomal membranes, hydrolytic activity of the serum was not markedly changed in EFA deficiency. These minor changes in hydrolytic activity and in lysosomal membrane stability seemed insufficient to explain the general lesions of EFA deficiency. Published with the approval of the Director of The Wisconsin Agricultural Experiment Station.     

Effect of essential fatty acid deficiency on the size and distribution of rat plasma HDL

Rat plasma high density lipoproteins (HDL) are comprised of two major particle size subpopulations, HDL₁ (255 Å?140 Å) and HDL₂ (140 Å?84 Å), in which the proportion of arachidonate in fatty acids of cholesteryl esters is greater than 50%. To determine whether decreased availability of arachidonate for cholesterol esterification would alter the distribution and/or amounts of the HDL subpopulations, we compared HDL subpopulations in EFA-deficient and control rats. To separate the effects of EFA deficiency and fat deficiency and to evaluate effects of different saturated fats, we used EFA-deficient diets that were fat-free or that contained 5% saturated fat. The control diets were the EFA-deficient diets plus 1% safflower oil. The saturated fats were hydrogenated coconut oil, hydrogenated cottonseed oil and saturated medium-chain triglycerides. All EFA-deficient diets decreased the proportion of the HDL₁ subpopulation and the peak diameter of the HDL₂ subpopulation. These changes appeared after quite brief EFA depletion in young rats and may be related to the increased liver cholesteryl ester concentrations typical of EFA-deficient rats.     

Effect of essential fatty acid deficiency on myelin proteins

The effect of essential fatty acid (EFA) deficiency on rat-brain myelin proteins was studied. Rats were maintained on a lipid-free diet and compared with control rats fed the same diet supplemented with 3% corn oil. At 17 days of age, each pup was injected with [³H]leucine and rats from each group were killed over a period of 163 days. Although a large decrease occurred in the total amount of myelin protein per brain, the proportions of constituent myelin proteins remained relatively unchanged. Metabolic studies showed a decrease in the net turnover of myelin proteins analogous to that previously demonstrated for myelin phospholipid (PL).     

Effects of essential fatty acid administration on cardiovascular responses to stress in the rat

This study examined the effects of 18∶2(n−6), 18∶3(n−6), 20∶4(n−6) and 18∶3(n−3) on cardiovascular responses to isolation stress in male rats. Group-acclimated rats were fasted for 2 days, then placed on a fat-free diet. Two wk later animals were divided into six groups (six animals per group) and given eight-wk intraperitoneal osmotic pumps releasing 1.47×10⁻⁷ mol/hr of either olive oil (OL), or of 18∶2(n−6), 18∶3(n−6), 20∶4(n−6) or 18∶3(n−3) in OL. Another group received dummy pumps. Two wk after pump implantation, animals were isolated for four wk. Blood pressure (BP), heart rate and body weight were followed before and during stress. Following the stress period, animals were assessed for cardiovascular reactivity to norepinephrine (NOR) and angiotensin (ANG). Prior to isolation, 18∶3(n−6) lowered BP vs OL (p<0.01). Stress increased BP within 24 hr in all groups except 18∶3(n−6) and 20∶4(n−6). Treatment with 20∶4(n−6) vs OL prevented the BP rise (p<0.001) only for the first two wk of stress. Administration of 18∶3(n−6) vs OL prevented any BP increase over the four-wk stress period (p<0.001). Stress increased heart rate in all groups except 20∶4(n−6). Heart rate was lowered by 18∶3(n−6) vs OL (p<0.01) before and during stress. Vascular reactivity to NOR was unaffected by treatment, but OL and 18∶3(n−6) decreased responses to ANG infusion. These data suggest that 18∶3(n−6) supplementation attenuates cardiovascular responses to chronic stress, and that Δ6- and Δ5-desaturase activity are inhibited during chronic psychological stress.     

Biochemical,functional, and histochemical effects of essential fatty acid deficiency in rat kidney

The present study was designed to examine the effects of EFA deficiency (EFAD) on biochemical, functional, and structural aspects of the kidney in growing and adult rats fed a normal or EFAD diet for 9 wk after weaning. Food and fluid intake (FI), urine volume, and Na⁺ and K⁺ excretions were measured weekly from weeks 4 to 8 by placing the rats in individual metabolic cages for 24 h. At week 9, Li⁺ and a 5% water load, respectively, were administered at 14 and 1.5 h prior to glomerular and proximal tubular function studies, as assessed by 3-h creatinine (C_Cr) and Li⁺ (C_Li+) clearances. Hematocrit and urine volume; serum and urine [Cr], [Li⁺], [Na⁺], and [K⁺]; and renal FA distribution were also measured. Data [corrected to 100 g/body weight (bw) and presented as means ±SEM] were significant, at P<-0.05. Despite a similar ingestion of solids from weeks 4 to 7 (weeks 7 to 10 of life), the rats on the EFAD diet showed a decreased body weight from week 5. From weeks 4 to 8, Fl and urine volume were similar for both groups, but the Fl increased at week 6 in the EFAD group; 24-h Na⁺ and K⁺ excretions were similar at all weeks, except for an increase in the EFAD group for both ions at week 7. In the EFAD group, C_Cr and C_Li+ decreased by 27 and 56.3%, respectively (385.7±33.4 vs. 280±21.1, and 21.0±2.1 vs. 9.2±1.1 μL/min/100 g; n=9 vs. 10), the latter result suggesting increased proximal reabsorption. The 3-h Na⁺ and K⁺ excretions were similar, but the Li⁺ decreased (0.78±0.06×10⁻² vs. 0.32±0.03×10⁻² μeq/min/100 g) in the EFAD group, giving additional support to the suggestion. Renal structure was normal and similar for both groups, but the EFAD group showed a more prominent proximal tubule brush border, together with heavier periodic acid-Schiff staining in all specimens from weeks 5 to 9. In the EFAD group, FA of the n−9 and n−7 series were higher, but most of the n−6 series were lower as a percentage of total lipids in the medulla and cortex. Medullary levels of 20∶4n−6 were maintained, 22∶4n−6 declined twice, arachidonic acid was maintained, and 20∶5n−3 was lower. The EFAD diet affected glomerular function, proximal tubular structure and function, and FA distribution in the rat kidney.     

Rat testicular lipids and dietary isomeric fatty acids in essential fatty acid deficiency

Weanling rats were fed essential fatty acid-deficient diets, either completely fat-free, or with partially hydrogenated fish oil (PHFO, 28 wt %), or with fractions derived from PHFO containing primarily positional isomers oftrans-eicosenoate (20∶1, 3 wt %) ortrans-docosenoate (22∶1, 3 wt %). Control animals were fed a peanut oil-containing diet (28 wt %). After 5 or 15 weeks on the diet, the content of neutral and phosphorus-containing lipids in the testes was determined. The fatty acid distribution in major lipid classes was analyzed for animals fed the diets for 15 weeks. The testicular stage of maturation or degeneration was assessed by histology. The group fed PHFO exhibited signs of complete testicular degeneration, or lack of maturation, already after 5 weeks, whereas the animals on the diets with the very long chain monoenoic acids suffered severe degenerations only after 15 weeks. In the PHFO-fed rats, a sharp decline in the concentration of testicular triacylglycerols was observed. In all of the essential fatty acid-deficient groups, an increase in testicular sphingomyelin was observed. Cholesterol levels were fairly similar among all dietary groups. The total testicular fatty acids of the PHFO-fed animals contained somewhat more eicosadienoic acid than found in the other groups, and somewhat less (n−9)-acids. In all EFA-deficient groups, (n−6)-acids were lowered, in particular in triacylglycerols and phosphatidyl cholines. The PHFO group did not show a lower (n−6)-concentration than the other deficient groups, in spite of the more severe symptoms of deficiency. There was no evidence of a major accumulation of long chain isomeric fatty acids in the degenerated testes of the PHFO-, 20∶1, and 22∶1-fed groups.     

Studies on the metabolism of linoelaidic acid in the essential fatty acid-deficient rat

Male weanling rats of the Sprague-Dawley strain were made essential fatty acid (EFA)-deficient by feeding them a fat-free diet for five months. The animals were then fed a supple-ment of methyltrans-9,trans-12-octadecadienoate (methyl linoelaidate), as 5% of the dietary calo-ries (approximately 400 mg/animal/day) for 19 days, and killed by exsanguination. The com-position of the liver, kidney, epididymal and plasma lipids was determined and compared with that obtained from EPA-deficient rats given oral supplements of methylcis-9,cis-12-octadeca-dienoate (linoleate) and methylcis-9,trans-12- octadecadienoate. Linoelaidic acid was depos-ited in the phospholipids, sterol esters and tri-glycerides in all of the tissues examined. Iso-lation analysis of the fatty acids showed no evidence that linoelaidic acid was converted to higher polyunsaturated fatty acids in the EFA-deficient rat. Supported in part by U. S. Public Health Service Grant No. AM 04942.     

Increased liver lipase activity in rats with essential fatty acid deficiency

Liver lipase activity was measured in EFA-deficient rats (long-term) and in control rats and rats fed an EFA-deficient diet for two weeks (short-term). Liver lipase activity was significantly enhanced by EFA deficiency, both in long-term and short-term experiments. The enhanced liver lipase activity could be normalized by feeding these rats normal laboratory chow for 14 days. Since during EFA deficiency prostaglandin synthesis is impaired, the possible involvement of prostaglandins in the observed changes in liver lipase actvity during EFA deficiency was studied. Administration of the prostaglandin synthesis inhibitor indomethacin (5 mg/kg body weight, i.p.) to normally fed rats for two days led to an increase of liver lipase activity. Prostaglandin E₂ was found to inhibit the secretion of liver lipase activity by freshly isolated parenchymal liver cellsin vitro. These results indicate that the increase in liver lipase activity during EFA deficiency may be due to an impairment of the prostaglandin synthesis.     

A relationship between essential fatty acid and vitamin E deficiency

To test whether vitamin E deficiency might influence the course of essential fatty acid (EFA) deficiency, Long Evans rats were fed diets containing a marginal amount (1.5% of calories) of 18∶2ω6 or 18∶3ω3 fatty acid with complete absence of the other and with or without vitamin E. Vitamin E contents decreased continuously in serum and liver in all rats fed the E-free diets but in the brains of only the rats fed the marginal 18∶3ω3, E-free diet. It is considered that the vitamin E is cooxidized in the liver with 22∶6ω3, since this fatty acid is very low in livers of the rats fed the marginal 18∶2ω6 diet but much higher in livers of the rats fed the marginal 18∶3ω3 diet. Brain 22∶6ω3 values are comparable for both groups. The source of 22∶6ω3 is evidently in the mother's milk, since following weaning there is a precipitous drop in 22∶6ω3 in serum, liver and carcass of rats on the 18∶2ω6-containing diet. No significant signs of EFA deficiency were seen in the E-deficient rats. Operated for the U.S. Department of Energy by the University of California under contract no. DE-AC03-76-SF00012.     

Rat neutrophil function,and leukotriene generation in essential fatty acid deficiency

Since the essential fatty acid linoleic acid is the precursor of arachidonic acid and thus of leukotriene B₄ (LTB₄), essential fatty acid deficiency (EFAD) may result in decreased synthesis of this stimulator of neutrophil granulocyte functions. Peritoneal and blood neutrophils from rats fed a diet with only 0.3% of energy requirements as linoleic acid and exhibiting biochemical evidence of EFAD showed substantial functional impairments compared to neutrophils from rats maintained on a diet with 3% of the energy requirement as linoleic acid. Oxidative burst activation (assessed by chemiluminescence), chemotaxis and aggregation were impaired upon stimulation with formylpeptides or the ionophore A23187. In contrast, these functions were intact on stimulation with exogenous LTB₄. Chemiluminescence was slightly but not significantly enhanced in EFAD rat neutrophils compared to controls when stimulated with phorbol myristate acetate (PMA). There were no differences between EFAD and control peritoneal neutrophils in the number of f-metleu-phe (fMLP) receptors, or in their affinity for the ligand, assessed with fML(³H)P. The fraction of responding cells also were similar, assessed with dichlorofluorescein diacetate fluorescence. Moreover, the endogenous LTB₄ production in response to A23187 or fMLP was decreased by 57.7% and 63.5%, respectively, in EFAD peritoneal neutrophils. Thus, EFAD was associated with reductions of LTB₄ production and neutrophil responsiveness to A23187 and formylpeptides but not to LTB₄ or PMA, which supports the hypothesis that endogenous LTB₄ may contribute to the activation of neutrophil functions involved in inflammation and host defense.     

Composition and concentration of lipoproteins in the serum of normal rats and rats deficient in essential fatty acids

A comparison is made of the concentration and chemical composition of serum lipoproteins of normal rats and rats deficient in essential fatty acids. The concentration of very low density lipoproteins (VLDL) and of low density lipoproteins (LDL) in serum of deficient rats is about half that found in normal rats, but the concentration of high density lipoproteins (HDL) is higher than normal and they contain an increased amount of cholesterol esters. The proportion of cholesterol that is esterified is much greater than normal in the serum of deficient rats. The deficiency of essential fatty acids also appears to result in compensating changes occurring in the composition of serum lipoproteins. In both VLDL and LDL of deficient rats the proportion of protein is raised and that of phospholipid lowered compared to normal, while the proportions of trigly ceride and cholesterol esters are unchanged.     

Functional and ultrastructural effects of essential fatty acid deficiency in kidney epithelial cells

Madin-Darby canine kidney (MDCK) epithelial cells were grown in culture medium supplemented with 1% fetal bovine serum (FBS) to provide a cell culture model of essential fatty acid deficiency (EFAD). 5,8,11-Eicosatrienoic acid (20∶3n−9) accumulated in cellular phospholipids, and arachidonic acid (20∶4) decreased. A large increase in cellular cholesterol/phospholipid ratio was observed. Hemicyst formation was greatly reduced from normal levels in the EFAD-MDCK cells. Scanning and transmission electron microscopy revealed that EFAD-MDCK were much flatter than their normal counterparts. They had much less dense surface microvilli, mitochondria and other organelles were very sparse, except in the perinuclear area, and much of the peripheral cytoplasm was amorphous. The EFAD was rapidly reversed by the addition of as little as 10 μM linoleic or arachidonic acid to the medium. Cells supplemented with 10% FBS, the usual culture condition, displayed borderline EFAD, with intermediate levels of 20∶3n−9 and 20∶4 and hemicyst formation. These studies suggest that EFAD reduces water and electrolyte transport in renal tubular epithelium.     

Short term essential fatty acid deficiency in rats. Influence of dietary carbohydrates

The effects of long term (8–14 wk) essential fatty acid (EFA)-deprived diets in rats are well documented. In the present study, we compared, in weanling rats, the effect of a short term (two wk) hydrogenated coconut oil, EFA-deprived, diet (D) with that of a corn oil, EFA-adequate, diet (A), using either sucrose (SU) or starch (ST) as carbohydrate. After two wk, rats fed the sucrose/hydrogenated coconut oil diet developed some characteristic features of EFA deprivation: slower growth rate, decreases in linoleic and arachidonic acid of plasma phospholipids and an increase in n−9 eicosatrienoic acid of plasma phospholipids. When rats ate the starch/hydrogenated coconut oil diet, there was a similar decrease in linoleic acid of plasma phospholipids, but only a small effect on growth rate and no change in the arachidonic acid content of plasma phospholipids. EFA deprivation and sucrose had opposite effects on plasma triglyceride (TG) levels: deprivation induced a decrease, whereas the sucrose induced an increase in very low density lipoprotein (VLDL) triglycerides. The observed decrease in plasma triglyceride during EFA deporivation might result from an activation of lipoprotein lipase during the early stages of deprivation.     

Effect of chronic ingestion of DDT on physiological and biochemical aspects of essential fatty acid deficiency

Male weanling rats were fed semipurified diets with and without essential fatty acid (EFA) and DDT (150 ppm) for 14 weeks to determine the effects of the pesticide on physiological and biochemical aspects of EFA deficiency (EFAD). DDT did not affect EFAD-induced reduction in growth rate or final body weight, nor did the pesticide affect EFAD-induced changes in feed efficiency or skin dermatitis. The pesticide did increase liver/body mass ratios, but did not interact with EFAD, which also increased this ratio. The pesticide produced complex changes in total fatty acid composition of liver and tail skin: liver levels of 18∶0, 18∶2 and 20∶3ω9 were increased, whereas levels of 12∶0, 14∶0 and 16∶0 were decreased. In both tissues, DDT interacted with EFA to increase 18∶2 levels. DDT did not change the total fatty acid 20∶3ω9/20∶4ω6 ratio in either tissue. In this study, although DDT did not exacerbate the physiological aspects of EFAD, DDT-induced changes in fatty acid composition of liver and tail skin indicated that 150 ppm DDT in the diets did alter lipid metabolism of the rats in an unexplained manner. Scientific contribution No. 811, Storrs Agricultural Experimental Station, University of Connecticut, Storrs, CT 06268.     

Reduction of essential fatty acid deficiency in rats fed a low iron fat free diet

Young male rats were fed ad libitum for 8 weeks a low iron fat-free (FF-Fe) diet or a fat-free diet supplemented with iron (FF+Fe). The relative levels of 16∶1 to 16∶0 and 18∶1 to 18∶0 in the total fatty acids of liver and other tissues (plasma, erythrocytes and intestinal mucosa) were considerably decreased because of a lack of dietary iron. In rats fed the FF-Fe diet, the levels of essential fatty acids (18∶2ω6+20∶4ω6) in tissues were 2-to 3-fold greater than in the corresponding tissues of rats fed the FF+Fe diet. Eicosatrienoic acid (20∶3ω9) levels in tissue lipids from rats fed the FF+Fe diet were high (8–16%), whereas they were low (2–5%) in the case of animals fed the FF-Fe diet. The proportion of 20∶4 in total fatty acids of tissues was 2-to 3-fold greater in rats fed the FF-Fe diet than when they were fed the FF+Fe diet. Therefore, the relative levels of 20∶3ω9/20∶4ω6 varied from 1-2.9 in tissue lipids of rats fed the FF+Fe diet, while it varied only from 0.2–0.3 in animals fed the FF-Fe diet. These results suggest that a lack of dietary iron may reduce the synthesis of 16∶1, 18∶1, 20∶3 and 20∶4 and the metabolism of 20∶4.     

Effects of zinc deficiency and castration on fatty acid composition and desaturation in rats

The effects of zinc deficiency and testosterone on fatty acid composition of plasma lipids and microsomes of liver, intestine and testes were studied. The activities of fatty acid desaturase (Δ6 and Δ5) in rat liver and testes were also measured. A significant decrease in the level of arachidonic acid was observed in plasma of normal rats fed the zinc-deficient diet. Castration significantly decreased arachidonic acid but increased 20∶3 fatty acid, which is negligible in normal rats. Testosterone and zinc administration restored arachidonic acid to normal values. Zinc deficiency does not significantly change the fatty acid profile in liver, but castration decreased both arachidonic and 22∶6 fatty acid. Intestinal mucosal microsomes showed that the predominant fatty acid in this tissue, palmitic acid, is independent of zinc status, whereas polyunsaturated fatty acids 18∶2 and 20∶4 were decreased by zinc-deficient diet or castration. Zinc deficiency sharply decreased 22∶5 fatty acid and to some extent, other polyunsaturated fatty acids in testis microsomes. These changes in fatty acids are in agreement with increased Δ9 desaturation and decreased Δ5 desaturase activity. In testes, both Δ6 and Δ5 desaturase activities are decreased in zinc deficiency. It appears that zinc influences the conversion of linoleic to arachidonic acid, whereas testosterone influences Δ6 desaturase activity. The data suggest that zinc deficiency may be one of the important factors in the causation of polyunsaturated fatty acid deficiency, which in turn, may induce serum hypertriglyceridemia.     

Effects of perfluorodecanoic acid onde novo fatty acid and cholesterol synthesis in the rat

Perfluorodecanoic acid (PFDA) is a peroxisome proliferator that causes a dose-dependent (20–80 mg/kg) increase in hepatic triacylglycerol and cholesteryl ester levels in the rat. We hypothesized that PFDA may cause an increase in thede novo synthesis of fatty acids and cholesterol in this species, which would explain observed effects. The incorporation of³H₂O into tissue lipids was examined 7 days after rats received vehicle or 20 or 80 mg/kg of PFDA. PFDA treatment decreased the rate of synthesis of cholesterol and fatty acids in the liver and in epididymal fat pad. At a PFDA dose (20 mg/kg) that decreasedde novo synthesis of fatty acids and cholesterol, there was no effect on the concentration of fatty acids and cholesterol in the liver, epididymal fat pads, and plasma. We conclude that PFDA induced fatty liver is due to either a decrease in the oxidation of fatty acids in the liver, or an impairment of triacylglycerol catabolism and/or export from the liver, and is not the result of an increase inde novo synthesis of fatty acids and cholesterol.     

Effect of essential fatty acid deficiency on lipid metabolism in isolated fat cells of epididymal fat pads of rats

Lipogenesis, lipolysis, and stimulation of glucose conversion into lipid by insulin or prostaglandin E₁ were studied in isolated fat cells of the epididymal fat pads of rats fed a fat-free diet or this diet supplemented with 10% hydrogenated coconut oil or 10% safflower seed oil. Changes in fatty acid composition, characteristic of an essential fatty acid deficiency, were well advanced in the neutral lipid but had only started in the polar lipid of the fat cells of the epididymal fat pads of animals 3 months after weaning. Cellularity of the epididymal fat pads, as indicated by protein to lipid ratio of the fat cells, was influenced greatly by hydrogenated coconut oil in the diet irrespective of an essential fatty acid deficiency. Lipogenesis was increased in the fat cells of the animals fed the hydrogenated coconut oil diet 5 weeks after weaning but was not significantly different from that of the safflower fed animals 3 months after weaning. Incorporation of glucose into lipid, oxidation to CO₂, and basal lipolysis were not significantly different in the fat cells of the essential fatty acid deficient animals from those fed safflower oil 3 months after weaning, except in animals of the fat-free group based upon cell lipid. However, conversion of glucose to free fatty acid was significantly greater in the isolated fat cells of animals fed either the hydrogenated coconut oil or the fat-free diet than in those of animals fed the safflower oil supplement. The incorporation of glucose into lipid by isolated fat cells was stimulated significantly by insulin in young animals fed a fat-free diet, but the effect on lipogenesis appeared to be reversed in the fat cells of animals receiving safflower seed oil 3 months after weaning. Prostaglandin E₁ also appeared to stimulate the incorporation of glucose into lipid in the fat cells of the older animals receiving safflower seed oil. Differences in osmolarity produced large differences in utilization of glucose and release of lipid from isolated fat cells, but no significant differences were observed between the cells from animals fed the fat-free diet and those from the controls fed safflower oil. The results demonstrated the effects of diets containing fat or no fat on enzyme activities and membrane properties of fat cells of the epididymal fat pads of essential fatty acid deficient rats.