Effects of oxidative stress on glycerolipid acyl turnover in rat hepatocytes

Lipid peroxidation was induced in freshly isolated rat hepatocytes by incubation in the presence of Fe³⁺, resulting in accumulation of thiobarbituric acid reactive substances. Analysis of lipid classes revealed that the levels and fatty acid compositions of the two major phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), remained unchanged but the levels of triacylglycerols (TAG) were significantly reduced, and some of their polyunsaturated fatty acids were selectively lost as the result of oxidant treatment. Acyl turnover in PC and PE as determined by ¹⁸O incorporation from H₂ ¹⁸O-containing media remained largely unchanged during oxidant treatment, while some increased turnover of the saturated fatty acids in TAG was observed. We hypothesize that constitutive recycling of membrane phospholipids rather than selective in situ repair eliminates peroxidized species of PC and PE, TAG could serve as an expendable fatty acid reserve, providing a limited but very dynamic pool of polyunsaturated fatty acids for the resynthesis of phospholipids.     

Plasma membrane phospholipid,cholesterol and fatty acyl composition of differentiated and undifferentiated L6 myoblasts

The lipid composition of plasma membranes isolated from differentialted and undifferentiated L₆ myoblasts has been compared. In general, the plasma membranes of differentiated L₆ myoblasts have a higher cholesterol to phospholipid molar ratio than plasma membranes of undifferentiated cells. Differentiated L₆ myoblasts have increased relative amounts of phosphatidylethanolamine and phosphatidylcholine in their plasma membrane and a decreased relative amount of sphingomyelin when compared with the plasma membranes of undifferentiated myoblasts. In addition, preliminary results show that differentiated L₆ myoblasts plasma membrane phospholipid shows differences in the fatty acyl composition, specifically there appears to be relatively more 17∶0 and 24∶1 and less 16∶1 and 18∶1 than in plasma membrane phospholipids of undifferentiated L₆ myoblasts. These observations indicate that significant changes in plasma membrane lipid composition occur during myoblast differentiation. The role that changes in lipid composition play in control of cellular differentiation, however, remains to be elucidated.     

Growth hormone and liver mitochondria: Effects on phospholipid composition and fatty acyl distribution

Effects of growth hormone on phospholipid composition and fatty acyl distribution were studied in liver mitochondria of hypophysectomized rats. After hypophysectomy, only cardiolipin showed a 25% decrease. Its fatty acyl distribution, which consisted mainly of linoleic acid (55–60%) and oleic acid (20%), was unchanged. In phosphatidylcholine and phosphatidylethanolamine fractions the contents of docosahexaenoic and arachidonic acids were decreased with a concomitant increase in linoleic acid content. These changes could be accounted for by small but significant decreases in the activities of Δ⁹-desaturase (sucrose-induced), Δ⁵-desaturase and mitochondrial elongation enzymes. The activities of Δ⁶-desaturase NADH cytochrome b₅ ferri-reductase, cytochrome b₅, NADH cytochrome c reductase and microsomal elongation enzymes remained virtually unchanged. Injection of bovine growth hormone daily for seven days restored cardiolipin and fatty acyl distribution and the enzyme activities. From these and other results, we conclude that growth hormone-dependent increase of respiratory activity of liver mitochondria may be partly mediated by the hormonal effects on membrane lipid distribution.     

Effects of diets high in saturated fat and cholesterol on the lipid composition of canine platelets

The phospholipid composition of platelets from dogs on various experimental diets was determined. Thyroidectomized foxhounds were fed a control diet or the control diet supplemented with (1) beef tallow, (2) beef tallow and cholesterol, or (3) beef tallow, cholesterol, and safflower oil for 23 weeks prior to isolation of platelets. Platelets from animals fed the control diet contained 36.7% phosphatidylcholine (PC), 22.8% phosphatidylethanolamine (PE), 18.4% sphingomyelin (Sph), 11.8% phosphatidylserine (PS), 6.3% phosphatidylinositol (PI), and 2.2% lysophosphatidylcholine. The PE was 77.6% in the plasmalogen form. No highly significant changes in the phospholipid class composition resulted from the experimental diets. Cholesterol supplementation of the diets, however, caused consistent alterations in the fatty acid compositions of the platelet phospholipids including increases in the percentages of 18∶1ω9 (oleic acid), 18∶2ω6 (linoleic acid), and 20∶3ω6 (homo-gamma linolenic acid) and a decrease in the percentage of 20∶4ω6 (arachidonic acid). Addition of safflower oil to the tallow-cholesterol diet partially reversed these effects. These cholesterol-induced alterations in fatty acid composition could be due to exchange with plasma lipids, de novo synthesis, or altered platelet metabolism. The mechanism remains to be determined. Der. Nelson’s current affiliation is the Lipid Metabolism Branch, Division of Heart and Vascular Diseases, National Heart, Lung, and Blood Institute.     

Differences in the phospholipid,cholesterol, and fatty acyl composition of 3T3 and SV3T3 plasma membranes

An analysis of the phospholipid, cholesterol, and phospholipid fatty acyl composition of isolated plasma membranes of 3T3 and SV3T3 mouse embryo cells has been performed. The results show that the plasma membrane of SV3T3 cells contain relatively less phosphatidylethanolamine and sphingomyelin and more cholesterol than 3T3 plasma membranes. The fatty acyl composition of individual phospholipid classes as determined by gas liquid chromatography also showed differences between 3T3 and SV3T3 plasma membranes. The plasma membranes of SV40 transformed 3T3 cells contain: (a) a higher percentage of 18∶1 and less 20∶3 and 20∶4 in phosphatidylethanolamine; (b) a higher percentage of 18∶1 in phosphatidylserine; and (c) a higher percentage of 18∶2 and 20∶4 in phosphatidylinositol.     

Effects of fructose and troglitazone on phospholipid fatty acid composition in rat skeletal muscle

Skeletal muscle phospholipid fatty acid (PLFA) composition is associated with insulin sensitivity in animal models and in man. However, it is not clear whether changes in insulin sensitivity cause a change in PLFA composition or vice versa. The present studies have examined the effects of agents known to increase or decrease insulin sensitivity on PLFA composition of the major phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), in soleus and extensor digitorum longus muscle. Four groups of Sprague-Dawley rats— control, 0.2% troglitazone (Tgz), 60% fructose fed, and fructose + Tgz—were treated for 3 wk. Fructose feeding was associated with a decrease in muscle membrane polyunsaturated fatty acids (PUFA) and n-3 fatty acids in both PC and PE. Administration of Igz alone resulted in an increase in liver (3.75±0.93 to 6.93±1.00 μmol/min/mg tissue, P<0.05) and soleus muscle (0.34±0.03 to 0.67±0.09 μmol/min/mg, P<0.01) elongase activity, which would be expected to increase membrane PUFA. However, Tgz decreased PLFA associated with greater insulin sensitivity (e.g., PUFA and n-3 fatty acids) and increased PLFA associated with decreased insulin sensitivity (16∶0 and n-6 fatty acids) in both PC and PE. Co-administration of fructose and Tgz did not reverse the decrease in PUFA observed with fructose alone. We conclude that the improvement in insulin sensitivity reported with Tgz is associated with an apparently paradoxical effect to decrease PUFA and n-3 PLFA composition in rat skeletal muscle. These studies suggest that Tgz-mediated increases in insulin sensitivity do not result in improved PLFA composition.     

Reduced plasma lecithin cholesterol acyl transferase activity in rats fed iron-deficient diets

An iron-deficient diet containing no fat (FF?Fe) or containing either 14% hydrogenated coconut oil (HCNO?Fe) or 14% corn oil (CO?Fe) was fed to separate groups of rats for 10 weeks. In the control group, the corresponding iron-supplemented diets were fed FF+Fe, HCNO+Fe, CO+Fe. When rats were fed iron-deficient diets, their plasma lecithin cholesterol acyl transferase (LCAT) activity was significantly reduced as compared to controls. Their plasma also contained relatively more cholesteryl esters (CE) than free cholesterol (CH). In rats fed FF+Fe and CO+Fe diets, plasma contained similar levels of CE and CH. In those fed HCNO+Fe diet, plasma had 40% less CE than CH. Red cell CH content was significantly greater in the CO?Fe group. Iron deficiency, as indicated by low blood hemoglobin (Hb) and hematocrit (Hct) values, was also observed only in this group. The triglyceride and phospholipid contents of plasma in rats fed iron-deficient diets were significantly lower than of those in the control groups. Thus, changes in LCAT activity and CE/CH ratio in plasma showed the effect of iron-deficient diet consumption even before the blood Hb and Hct levels were reduced.     

Altered microsomal phospholipid composition in the streptozotocin diabetic rat

Streptozotocin diabetes in the rat alters liver microsomal membrane fatty acid composition. The present study was undertaken to determine if such changes in fatty acid composition were due to changes in the amount of individual phosphoglycerides or to disproportionate changes in fatty acid composition in any of the individual phosphoglycerides. The diabetic animals showed a small increase in total microsomal phospholipid, which is due to a selective increase in the phosphatidylethanolamine fraction. The changes in fatty acid composition in the total lipid extract (decreased palmitoleic, oleic and arachidonic acids and increased linoleic and docosahexaenoic acids) from the diabetic animals were present in both the major phosphoglycerides, phosphatidylcholine and phosphatidylethanolamine, with very little change in fatty acid composition in the phosphatidylserine and inositol fraction. Further studies are necessary to delineate the cause of the abnormal membrane phospholipid composition in the diabetic animal. Abbreviations: The abbreviated fatty acid nomenclature refers to the number of carbon atoms in the chain, the number of unsaturated bonds, and the position of the first unsaturated bond counting from the terminal methyl group; thus arachidonic acid, 5,8,11,14-eicosatetraenoic acid, is 20∶4ω6.     

Tissue phospholipid fatty acid composition in the diabetic rat

Tissue phospholipid fatty acid compositions in streptozotocin-induced diabetic rats were studied. The major changes in liver, plasma, erythrocyte and heart were increased proportions of linoleic and dihomo-γ-linolenic acids and a decreased proportion of aracchidonic acid. The latter was not significantly changed in phospholipids of kidney, adrenal gland and testis. Skin fatty acids in diabetic rats showed an increase in the proportion of arachidonic acid and a reduction in the proportion of linoleic acid. The fatty acid desaturating activity in diabetes may be regulated differently in different tissues.     

l-Carnitine effects on chemical composition of plasma lipoproteins of rabbits fed with normal and high cholesterol diets

l-Carnitine plays an important role in the mitochondrial uptake of long-chain fatty acids in mammals. It has recently been shown that this compound has a marked hypo-cholesterolemic effect when used in conjunction with lipid-rich diets. The aim of this study was to investigate the effects of l-carnitine on the fatty acid composition of plasma lipoproteins in rabbits fed with different diets. Four different groups were investigated: group I (standard diet), group II (standard diet supplemented with l-carnitine at 80 mg/kg), group III (standard diet supplemented with 0.5% cholesterol), and group IV (standard diet supplemented with 0.5% cholesterol plus l-carnitine at 80 mg/kg). The feeding period was 126 d. Total plasma cholesterol was indistinguishable in groups I and II, but increased nearly 40-fold in group III. This increment was reduced by 50% in group IV. Correspondingly, total cholesterol content in lipoprotein fractions [very low density lipoprotein (VLDL), low density lipoprotein (LDL), high density lipoprotein (HDL) separated by agarose gel chromatography was the same for groups I and II, while for animals fed a cholesterol-rich diet (III) total cholesterol in VLDL+LDL increased nearly 100-fold when compared with groups I and II but, again, the increment was reduced by 50% in group IV. In contrast, total cholesterol in HDL increased only fivefold for both groups III and IV when compared with groups I and II, indicating no effects of l-carnitine on this parameter. The reduction of total cholesterol in VLDL+LDL particles in animals fed a cholesterol-rich diet plus l-carnitine was associated with a marked decrease in the ratio of cholesteryl ester to free cholesterol and a dramatic increase in their phospholipid content; opposite effects were observed for HDL. l-Carnitine induced a marked decrease in the saturated to unsaturated C₁₆+C₁₈ fatty acid ratio in cholesteryl esters associated with VLDL and LDL from animals fed with both normal and cholesterol-rich diets. The opposite effect (a large increase in the saturated to unsaturated fatty acid ratio) was observed for both cholesteryl esters and phospholipids associated with HDL in animals fed with both diets. The results suggested that the hypocholesterolemic effects of l-carnitine could be associated with increased systemic breakdown of cholesteryl esters, a probable increase in reverse cholesterol transport, and the stabilization of a phospholipid-based structure of VLDL+LDL particles.     

Changes in the cholesterol and phospholipid content of mouse spleen after Rauscher Leukemia Virus infection

The effect of Rauscher Leukemia Virus (MuLV-R) infection on the lipid composition of mouse spleen from BALB/c mice was investigated. Drastic changes in the lipid composition of the spleen as a result of tumor growth induced by the virus could be demonstrated at 21 days after infection. The molar ratio of cholesterol to phospholipids was found to be low, while a shift within the choline containing phospholipid classes resulted into a lower sphingomyelin and a higher phosphatidyl choline content of the MuLV-R infected spleen. The cholesterol ester content increased more than two-fold during tumor growth, and shifts in the fatty acid patterns of the lipids were demonstrated.     

Effects of various dietary fats on cardiolipin acyl composition during ontogeny of mice

Cardiolipin (CL) is a unique mitochondrial phospholipid, containing up to 85 wt% 18∶2n−6 in mammals. The influence of maternal dietary fatty acids on the acyl composition of offspring CL has not been examined previously. Adult female mice were thus fed diets rich in 18∶1n−9 (olive oil), 18∶2n−6 (safflower oil), 18∶3n−3 (linseed oil) or 20∶5n−3 and 22∶6n−3 (fish oil/safflower, 9∶1, w/w), for a five month period, encompassing two breeding cycles. Offspring from the second breeding cycle were then fed these diets. The acyl composition of CL, phosphatidylcholine and phosphatidylethanolamine from liver and heart was evaluated from mice killed 3, 18 and 42 days after parturition. The primary nutrient sources at these three time points were transplacental nutrients, breast milk and the diet, respectively. Maternal diet was found to influence the acyl composition of CLvia both placental transfer of fatty acids and breast milk. Fish oil feeding resulted in replacement of a substantial portion of 18∶2n−6 with 22∶6n−3; after 42 days, the area% of 18∶2n−6 in heart CL was reduced from 62% in safflower oil fed mice to 12%. In comparison to fish oil feeding, linseed oil feeding resulted in a much lower accumulation of 22∶6n−3. Olive oil feeding resulted in substantial replacement of 18∶2n−6 with 18∶1n−9 (18∶2n−6 was reduced from 62% to 31%). Physiologically, these findings are relevant because changes in CL acyl composition may influence the activity of associated inner mitochondrial membrane enzymes. This work was presented in part as an Honored Student Award paper at the 82nd Annual AOCS Meeting, Chicago, IL, May 1991.     

Liver and intestinal fatty acid-binding protein expression increases phospholipid content and alters phospholipid fatty acid composition in L-cell fibroblasts

Although fatty acid-binding proteins (FABP) differentially affect fatty acid uptake, nothing is known regarding their role(s) in determining cellular phospholipid levels and phospholipid fatty acid composition. The effects of liver (L)- and intestinal (I)-FABP expression on these parameters were determined using stably transfected L-cells. Expression of L- and I-FABP increased cellular total phospholipid mass (nmol/mg protein) 1.7- and 1.3-fold relative to controls, respectively. L-FABP expression increased the masses of choline glycerophospholipids (ChoGpl) 1.5-fold, phosphatidylserine (PtdSer) 5.6-fold, ethanolamine glycerophospholipids 1.4-fold, sphingomyelin 1.7-fold, and phosphatidylinositol 2.6-fold. In contrast, I-FABP expression only increased the masses of ChoGpl and PtdSer, 1.2- and 3.1-fold, respectively. Surprisingly, both L- and I-FABP expression increased ethanolamine plasmalogen mass 1.6- and 1.1-fold, respectively, while choline plasmalogen mass was increased 2.3- and 1.7-fold, respectively. The increase in phospholipid levels resulted in dramatic 48 and 33% decreases in the cholesterol-to-phospholipid ratio in L- and I-FABP expressing cells, respectively. L-FABP expression generally increased polyunsaturated fatty acids, primarily by increasing 20∶4n−6 and 22∶6n−3, while decreasing 18∶1n−9 and 16∶1n−7. I-FABP expression generally increased only 20∶4n−6 proportions. Hence, expression of both I- and L-FABP differentially affected phospholipid mass, class composition, and acyl chain composition. Although both proteins enhanced phospholipid synthesis, the effect of L-FABP was much greater, consistent with previous work suggesting that these two FABP differentially affect lipid metabolism.     

Changes in rat heart phospholipid composition after rapeseed oil feeding

The influence of long duration rapeseed oil feeding with high or low levels of erucic acid has been investigated on rat heart phospholipids. The rats treated for 20 wk with rapeseed oil containing 46.2% erucic acid showed a twofold increase in the sphingomyelin content of the heart. Treatment with primor rapeseed oil (3.7% erucic acid) for 20 wk did not modify phospholipid composition of rat heart. The fatty acid patterns of phosphatidylethanolamine and phosphatidylcholine were slightly influenced by the high erucic rapeseed oil; eicosenoic acid was incorporated preferentially into position one, but erucic acid showed a random distribution in both. After high erucic rapeseed oil feeding, 22∶1 was incorporated into cardiolipin (5.6%) and sphingomyelin (10.5%). The incorporation of 22∶1 into sphingomyelin was associated with an increase of the percentage of 24∶1 (14.6%) and a decrease of saturated long chain fatty acid (22∶0, 24∶0) percentages. Primor rapeseed oil caused a slight increase of 24∶1 and a decrease of 22∶0 and 24∶0 in rat heart sphingomyelin. As cardiolipin is localized in the inner membrane of mitochondria and sphingomyelin in plasma and microsomal membranes, the acyl-moiety alterations of both phospholipids might be correlated to the pathological lesions of rat heart after a long duration of rapeseed oil feeding.     

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.     

Fatty acid and cholesterol synthesis from specifically labeled leucine by isolated rat hepatocytes

Hepatocytes isolated from female rats meal-fed a high-glucose diet were incubated in Krebs-Henseleit bicarbonate medium containing 16.5 mM glucose,³H₂O, and¹⁴C-labeled amino acids (−)-Hydroxycitrate depressed the incorporation of³H₂O and [¹⁴C] alanine into fatty acids and cholesterol. Incorporation of [U-¹⁴C] leucine into lipids was not affected but incorporation of³H₂O into lipids was decreased significantly by (−)-hydroxycitrate. (−)-Hydroxycitrate depressed the incorporation of radioactivity from [2-¹⁴C]leucine into fatty acids and cholesterol by 61 and 38%, respectively, and stimulated the incorporation of radioactivity from [4,5-³H]leucine 35 and 28%. As [2-¹⁴C]leucine labels the acetyl-CoA pool and [4,5-³H]leucine labels the acetoacetate pool, it was concluded that mitochondrial 3-hydroxy-3-methylglutaryl-CoA is not incorporated intact into cholesterol, and that acetoacetate can be activated effectively in the liver cytosol for support of cholesterol and fatty acid synthesis.     

Effects of bile salts on rat hepatic acyl CoA:Cholesterol acyltransferase

Acyl CoA:cholesterol acyltransferase (EC2.3.1.26, ACAT), responsible for intracellular esterification of cholesterol, may play an important role in cholesterol trafficking within the cell, and thus, in maintenance of cellular cholesterol homeostasis. Bile acids are potential regulators of cholesterol trafficking in the liver. Therefore, the effect of bile salts on hepatic ACAT activity was studied in the perfused rat liver. ACAT activity was increased after liver perfusion with either taurocholate or taurochenodeoxycholate. However, addition of these bile salts at physiological concentrationsin vitro had little effect on microsomal ACAT activity. The increase in hepatic ACAT activity due to perfusion with bile salts was accompanied by reduced accumulation of very low density lipoprotein cholesterol in the perfusate, but there was no effect on 3-hydroxy-3-methylglutaryl-CoA reductase activity. Hepatic ACAT activity was decreased after bile diversion for four hours in the intact animal. This treatment had no statistically significant effect on 3-hydroxy-3-methylglutaryl-CoA reductase activity. These data suggest that bile salts induce changes in hepatic compartmentation and traffic of cholesterol within the hepatocyte accompanied by response of ACAT activity to maintain cellular cholesterol homeostasis.     

Effect of nutritional status on the fatty acid composition of rat liver and cultured hepatocytes

The lipid concentration and fatty acid composition of the whole liver and of cultured hepatocytes isolated from the livers of rats fed ad libitum (fed), fasted for 24 hr (fasted), or fasted for 48 hr and then refed a fat-free, high carbohydrate diet for 48 hr (refed) was studied. Hepatocytes were maintained as monolayer cultures in serum-free, lipid-free media and their fatty acid composition was analyzed at 3, 24, 48, 72 and 96 hr. The livers of fed animals, as well as their hepatocytes, contained less total lipid than those from animals on either of the other dietary regimes. Livers of fasted animals had three times the amount of lipid found in the livers of fed animals, and the livers of refed animals contained five times the amount of lipid as the livers of fed animals (all based on mg lipid/g wet weight of liver). The fatty acid composition of hepatocytes after 3 hr of culturing was very similar to that of fresh liver when compared in each of the dietary regimes. However, while the fatty acid compositions of livers and hepatocytes from fed and fasted animals were similar, the pattern in liver of refed animals was quite distinct from that of the fed animals. In the fed and fasted animals palmitic acid (16∶0), stearic acid (18∶0), oleic acid (18∶1[n-9]), linoleic acid (18∶2[n-6]) and arachidonic acid (20∶4[n-6]) were the major fatty acids of the liver; in refed animals 16∶0, palmitoleic acid (16∶1[n-7]), 18∶0, 18∶1(n-9) andcis-vaccenic acid (the n-7 isomer of oleic acid) were the major fatty acids. During maintenance in culture the 18∶1(n-9) content of the hepatocytes increased in cells from livers of animals on all three dietary regimes. The polyunsaturated fatty acid content was similar in fresh livers and isolated hepatocytes in all samples when compared on the basis of μg fatty acid/mg of hepatocyte or liver protein. It was also found that the polyunsaturated fatty acid content of hepatocytes was remarkedly stable with time of culture when the cells were incubated in serum-free, lipid-free medium. Thus, isolated hepatocytes maintained in serum-free medium appear to be a possible system for the evaluation of the effects of prior nutritional status on fatty acid metabolism in the whole animal, not subject to hormonal and other somatic influences which often complicate the interpretation of such nutritional studies.     

Dietary ω3 fatty acids and cholesterol modify enterocyte microsomal membrane phospholipids,cholesterol content and phospholipid enzyme activities in diabetic rats

Diabetes-associated changes in intestinal uptake of nutrients are modified by isocaloric variations in the type of dietary lipids, and are associated with alterations in the phospholipid and fatty acyl content of the intestinal brush border membrane. The present study was designed to test the hypothesis that diet- and diabetes-associated changes in enterocyte microsomal membrane phospholipids are due to variations in the activity of two phospholipid metabolizing enzymes, 1,2-diacylglycerol: CDP choline cholinephosphotransferase (CPT) and phosphatidylethanolamine methyltransferase (PEMT). Adult female Wistar rats were fed one of four semisynthetic diets—beef tallow low in cholesterol (BT), beef tallow high in cholesterol (BTC), fish oil low in cholesterol (FO) or fish oil high in cholesterol. In half of the animals, diabetes mellitus was produced by injection of streptozotocin. Jejunal and ileal enterocyte microsomes (EMM) were isolated and analyzed for cholesterol and phospholipids, as well as for CPT and PEMT activities. In control animals, feeding FO reduced EMM total phospholipids including phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol. Feeding FO resulted in a greater than 95% reduction in the activity of CPT. Diabetes was associated with increased jejunal EMM total phospholipids including sphingomyelin (SM) and PE, without associated changes in CPT or PEMT. Dietary cholesterol supplementation did not effect EMM total cholesterol or phospholipid composition in control rats fed BT or FO, but was associated with an increase in EMM cholesterol in diabetic rats fed BT or FO. A decrease in total phospho-lipids due to a decline in SM, PC and PE in diabetic rats fed FO was not associated with changes in the activities of CPT or PEMT in EMM. Thus (i) enterocyte microsomal membrane cholesterol and phospholipid contents are influenced by diabetes, dietary cholesterol and the type of fatty acid in the diet, and (ii) changes in phospholipid composition are not fully explained by alterations in the activities of CPT and PEMT.