Differential modulation of rat heart mitochondrial membrane-associated enzymes by dietary lipid

Diets supplemented with high levels of saturated fatty acids derived from sheep kidney (perirenal) fat or unsaturated fatty acids derived from sunflower seed oil were fed to rats and the effect on heart mitochondrial lipid composition and membrane-associated enzyme behaviour was determined. The dietary lipid treatments did not change the overall level of membrane lipid unsaturation but did alter the proportion of various unsaturated fatty acids. This led to a change in the omega 6/omega 3 unsaturated fatty acid ratio, which was highest in the sunflower seed oil fed rats. Arrhenius plots of the mitochondrial membrane associated enzymes succinate-cytochrome c reductase and oligomycin-sensitive adenosinetriphosphatase (ATPase) after dietary lipid treatment revealed different responses in their critical temperature. For succinate-cytochrome c reductase, the critical temperature was 29 degrees C for rats fed the sheep kidney fat diet and 20 degrees C for rats fed the sunflower seed oil diet. In contrast, no shift in the critical temperature for the mitochondrial ATPase was apparent as a result of the differing dietary lipid treatments. The results suggest that the discontinuity in the Arrhenius plot of succinate-cytochrome c reductase is induced by some change in the physical properties of the membrane lipids. In contrast, mitochondrial ATPase appears insensitive, in terms of its thermal behaviour, to changes occurring in the composition of the membrane lipids. However, the specific activity of the mitochondrial ATPase was affected by the dietary lipid treatment being highest for the rats fed the sheep kidney fat diet. No dietary lipid effect was observed for the specific activity of succinate-cytochrome c reductase. This differential response of the two mitochondrial membrane enzymes to dietary-induced changes in membrane lipid composition may affect mitochondrial oxidative phosphorylation.     

Membrane lipid fluidity and its effect on the activation energy of membrane-associated enzymes.

1. The fatty acid composition of mitochondrial membranes from sheep and rats was altered by feeding these animals diets which were rich in unsaturated fatty acids. Changes in membrane lipid fluidity resulting from the altered membrane lipid composition were assessed by determining the upper temperature limit of the disorder-order transition (Tf) and the Arrhenius activation energy (Ea) of succinate oxidase. 2. After feeding the unsaturated fatty acid-rich diet to sheep the Ea, in the temperature range above Tf, increased from 8 to 63 kJ . mol-1 while Tf decreased from 32 to 15 degrees C. Rats fed an unsaturated fatty acid-rich diet exhibited an increase in Ea from 17 to 63 kJ . mol-1 and a decrease in Tf from 23 to 4 degrees C. 3. This decrease in Tf was related to an increase in the ratio of linoleic acid to stearic acid in the membrane lipid. Tf was not related to the proportion of unsaturated fatty acids in the membrane lipids, although an increase in unsaturation usually led to a decrease in Tf. 4. The results show that membrane lipid fluidity has a direct influence on the conformation of the active site of some membrane-associated enzymes, with the result that such enzymes display a higher Ea when the membrane lipids are comparatively more fluid. The increase in Ea of membrane-associated enzymes which accompanies changes in the physical state of membrane suggests that some proteins may phase separate with the more fluid lipids at low temperatures.     

The influence of storage temperature on the transition, activation enthalpy, and activity of enzymes associated with inner mitochondrial membranes

The effects of storage at low temperature on the transition in enzyme function, Tf*, and the Arrhenius activation energy, Ea, were determined for several enzymes associated with the inner membrane of rat liver mitochondria. The enzymes studied were succinate:cytochrome c reductase, cytochrome c oxidase, beta-hydroxybutyrate dehydrogenase, and oligomycin-sensitive, Mg2+-activated ATPase. For freshly isolated mitochondria the Tf*, for succinate:cytochrome c reductase and cytochrome c oxidase, occurred at approximately 23 degrees C and was coincident with a transition in structure, Ts*, determined as the change in temperature coefficient of motion for a spin label intercalated with the membrane lipids. This suggest that the change in thermal response of the membrane-associated enzymes is related to a change in molecular ordering of the membrane lipids. When mitochondria were stored at -12 degrees C, the specific activities of succinate:cytochrome c reductase and cytochrome c oxidase decreased. Concomitant with these changes the Ea, above Tf*, increased. After 100 days storage at -12 degrees C, Ea above Tf* approached the value for Ea below Tf* such that the transition in thermal response could no longer be detected. In contrast, for mitochondria stored at -196 degrees C, although the specific activity declined over the 100 days storage, no changes in either Ea or Tf* were evident. The results indicate a need for caution in evaluating comparative studies of Tf and Ea, for membrane-associated enzymes, using mitochondria which have been frozen and stored.     

The effect of dietary lipids on the thermotropic behaviour of rat liver and heart mitochondrial membrane lipids

Diets supplemented with relatively high levels of either saturated fatty acids derived from sheep kidney fat (sheep kidney fat diet) or unsaturated fatty acids derived from sunflower seed oil (sunflower seed oil diet) were fed to rats for a period of 16 weeks and changes in the thermotropic behaviour of liver and heart mitochondrial lipids were determined by differential scanning calorimetry (DSC). The diets induced similar changes in the fatty acid composition in both liver and heart mitochondrial lipids, the major change being the omega 6 to omega 3 unsaturated fatty acid ratio, which was elevated in mitochondria from animals on the sunflower seed oil diet and lowered with the mitochondria from the sheep kidney fat dietary animals. When examined by DSC, aqueous buffer dispersions of liver and heart mitochondrial lipids exhibited two independent, reversible phase transitions and in some instances a third highly unstable transition. The dietary lipid treatments had their major effect of the temperature at which the lower phase transition occurred, there being an inverse relationship between the transition temperature and the omega 6 to omega 3 unsaturated fatty acid ratio. No significant effect was observed for the temperature of the higher phase transition. These results indicate that certain domains of mitochondrial lipids, probably containing some relatively higher melting-point lipids, independently undergo formation of the solidus or gel phase and this phenomenon is not greatly influenced by the lipid composition of the mitochondrial membranes. Conversely, other domains, representing the bulk of the membrane lipids and which probably contain the relatively lower melting point lipids, undergo solidus phase formation at temperatures which reflect changes in the membrane lipid composition which are in turn, a reflection of the nature of the dietary lipid intake. These lipid phase transitions do not appear to correlate directly with those events considered responsible for the altered Arrhenius kinetics of various mitochondrial membrane-associated enzymes.     

NADH-cytochrome c reductase, succinate cytochrome c reductase and phospholipids.

Phospholipid peroxidation of isolated rat liver inner mitochondrial membranes induced by either ascorbate or cysteine was accompanied by a release of flavins and coenzyme Q. A straight correlation between this release and the alteration of molecular species of phosphatidylcholine and phosphatidylethanolamine containing one saturated and one unsaturated fatty acid has been found. Peroxidation induced on molecular species of phosphatidylcholine and phosphatidylethanolamine containing only unsaturated fatty acids were accompanied by losses in enzyme activities of NADH-cytochrome c reductase and succinate cytochrome c reductase.     

Response of rat heart membranes and associated ion-transporting ATPases to dietary lipid

The effects of different dietary fat intake on the lipid composition and enzyme behaviour of sarcolemmal (Na+ + K+)ATPase and sarcoplasmic reticulum Ca2+-ATPase from rat heart were investigated. Rat diets were supplemented with either sunflower seed oil (unsatd./satd. 5.6) or sheep kidney fat (unsatd./satd. 0.8). Significant changes in the phospholipid fatty acid composition were observed in both membranes after 9 weeks dietary lipid treatment. For both membranes, the total saturated/unsaturated fatty acid levels were unaffected by the dietary lipid treatment, however the proportions of the major unsaturated fatty acids were altered. Animals fed the sunflower seed oil diet exhibited an increase in n-6 fatty acids, including linoleic (18:2(n-6] and arachidonic (20:4(n-6] while the sheep kidney fat dietary rats were higher in n-3 fatty acids, principally docosahexaenoic (22:6), with the net result being a higher n-6/n-3 ratio in the sunflower seed oil group compared to sheep kidney fat dietary animals. Fluorescence polarization indicated that the fluidity of sarcoplasmic reticular membrane was greater than that of sarcolemmal membrane, with a dietary lipid-induced decrease in fluidity being observed in the sarcoplasmic reticular membrane from sheep kidney fat dietary animals. Despite these significant changes in membrane composition and physical properties, neither the specific activity nor the temperature-activity relationship (Arrhenius profile) of the associated ATPases were altered. These results suggest that with regard to the parameters measured in this study, the two ion-transporting ATPases are not modulated by changes which occur in the membrane lipid composition as a result of the diet.     

The interaction of dietary fatty acid and cholesterol on catecholamine-stimulated adenylate cyclase activity in the rat heart

Diets supplemented with high levels of saturated or unsaturated fatty acids supplied by addition of sheep kidney fat or sunflower seed oil, respectively, were fed to rats with or without dietary cholesterol. The effects of these diets on cardiac membrane lipid composition, catecholamine-stimulated adenylate cyclase and beta-adrenergic receptor activity associated with cardiac membranes, were determined. The fatty acid-supplemented diets, either with or without cholesterol, resulted in alterations in the proportion of the (n-6) to (n-3) series of unsaturated fatty acids, with the sunflower seed oil increasing and the sheep kidney fat decreasing this ratio, but did not by themselves significantly alter the ratio of saturated to unsaturated fatty acids. However, cholesterol supplementation resulted in a decrease in the proportion of saturated and polyunsaturated fatty acids and a dramatic increase in oleic acid in cardiac membrane phospholipids irrespective of the nature of the dietary fatty acid supplement. The cholesterol/phospholipid ratio of cardiac membrane lipids was also markedly increased with dietary cholesterol supplementation. Although relatively unaffected by the nature of the dietary fatty acid supplement, catecholamine-stimulated adenylate cyclase activity was significantly increased with dietary cholesterol supplementation and was positively correlated with the value of the membrane cholesterol/phospholipid ratio. Although the dissociation constant for the beta-adrenergic receptor, determined by [125I](-)-iodocyanopindolol binding, was unaffected by the nature of the dietary lipid supplement, the number of beta-adrenergic receptors was dramatically reduced by dietary cholesterol and negatively correlated with the value of the membrane cholesterol/phospholipid ratio. These results indicate that the activity of the membrane-associated beta-adrenergic/adenylate cyclase system of the heart can be influenced by dietary lipids particularly those altering the membrane cholesterol/phospholipid ratio and presumably membrane physico-chemical properties. In the face of these dietary-induced changes, a degree of homeostasis was apparent both with regard to membrane fatty acid composition in response to an altered membrane cholesterol/phospholipid ratio, and to down regulation of the beta-adrenergic receptor in response to enhanced catecholamine-stimulated adenylate cyclase activity.     

Effect of dietary fat saturation on acylcoenzyme A:cholesterol acyltransferase activity of rat liver microsomes

The saturation of the fat contained in the diet has been observed to affect the acylcoenzyme A:cholesterol acyltransferase (ACAT) activity of rat liver microsomes. ACAT activity in microsomes (Mp) prepared from livers of rats fed a polyunsaturated fat-enriched diet containing 14% sunflower seed oil was 70-90% higher than in microsomes (Ms) prepared from livers of rats fed a saturated fat-enriched diet containing 14% coconut oil. This difference was observed within 20 days after the diets were begun, the earliest time tested, and persisted throughout the 70-day experimental period. The difference was noted at all [1-14C]palmitoyl CoA concentrations tested, 2.5-33 micronM, and at temperatures between 18 and 40 degrees C. Arrhenius plots revealed a single transition in enzyme activity, occurring at 29 degrees C in both microsomal preparations. Likewise, the activation energy above this transition was the same in Mp and Ms, 12.5 KCal/mol. Addition of albumin to the incubation medium increased the ACAT activity of both microsome preparations, but the difference between Mp and Ms persisted. Mp was enriched in polyenoic fatty acids, primarily 18:2 and 20:4, while Ms was enriched in monoenoic acids. Although the 20:4 increase in Mp occurred in all phosphoglycerides, it was especially pronounced in the serine and inositol phosphoglyceride fraction. There were no differences in the phospholipid or cholesterol content, phospholipid head group composition, or protein composition of the two microsomal preparations. The possibility is discussed that the changes in ACAT activity result from the differences in fatty acid composition of the microsomes. Other microsomal enzymes exhibited varying responses to these dietary fatty acid modifications. Palmitoyl CoA hydrolase and NADPH cytochrome c reductase activities were unchanged. UDP glucuronyl transferase activity was 50% higher in Mp, but glucose-6-phosphatase and NADH cytochrome b5 reductase activities were 25% higher in Ms. Therefore, dietary fat modifications do not produce a uniform effect on the activity of microsomal enzymes.     

Effects of dietary omega-6 and omega-3 fatty acids on levels of long chain polyunsaturated fatty acids in erythrocytes]

C18:2 omega 6/C18:3 omega 3 ratio was lowered in the diet of Elderly subjects. This was done by the replacement of usual sunflower oil by rapeseed oil or by supplementing soybean oil. This diet modification induced an increase of EPA (C20:5 omega 3) and DHA (C22:6 omega 3) in red cell phospholipids. The omega 6 fatty acids (C18:2 and C20:4) were slightly modified. Therefore, dietary C18:2 omega 6/C18:3 omega 3 ratio, seems to play an important role in the determination of membrane highly unsaturated fatty acid levels.     

Lizards, lipids, and dietary links to animal function

Our experiments were designed to test the hypotheses that dietary lipids can affect whole-animal physiological processes in a manner concordant with changes in the fluidity of cell membranes. We measured (1) the lipid composition of five tissues, (2) body temperatures selected in a thermal gradient (T(sel)), (3) the body temperature at which the righting reflex was lost (critical thermal minimal [CTMin]), and (4) resting metabolic rate (RMR) at three body temperatures in desert iguanas (Dipsosaurus dorsalis) fed diets enriched with either saturated or unsaturated fatty acids. The composition of lipids in tissues of the lizards generally reflected the lipids in their diets, but the particular classes and ratios of fatty acids varied among sampled organs, indicating the conservative nature of some tissues (e.g., brain) relative to others (e.g., depot fat). Lizards fed the diet enriched with saturated fatty acids selected warmer nighttime body temperatures than did lizards fed a diet enriched with unsaturated fatty acids. This difference is concordant with the hypothesis that the composition of dietary fats influences membrane fluidity and that ectotherms may compensate for such changes in fluidity by selecting different body temperatures. The CTMin of the two treatment groups was indistinguishable. This may reflect the conservatism of some tissues (e.g., brain) irrespective of diet treatment. The RMR of the saturated treatment group nearly doubled between 30 degrees and 40 degrees C. Here, some discrete membrane domains in the lizards fed the saturated diet may have been in a more-ordered phase at 30 degrees C and then transformed to a less-ordered phase at 40 degrees C. In contrast, the RMR of the unsaturated treatment group exhibited temperature independence in metabolic rate from 30 degrees to 40 degrees C. Perhaps the unsaturated diet resulted in membranes that developed a higher degree of disorder (i.e., a certain phase) at a lower temperature than were membranes of lizards fed the saturated diet. Our study demonstrates links between dietary fats and whole-animal physiology; however, the mechanistic basis of these links, and the general knowledge of lipid metabolism in squamate reptiles, remain poorly understood and warrant further study.     

Correlation between changes in membrane lipid composition induced by dietary lipid and membrane-bound enzyme activity in chick liver

The activity of acyl-CoA: cholesterol acyltransferase in the liver-microsomal fraction was considerably reduced in chicks fed on diet containing unsaturated fat, whereas the activity of HMG-CoA reductase and NADPH cytochrome c reductase was not affected. The fatty acid composition of the microsomes was modified appreciably by this dietary condition and there was no change in the phospholipid or cholesterol levels. The addition of cholesterol to the fat supplemented diet resulted in a considerable increase in the microsomal cholesterol content. A decrease in HMG-CoA reductase and an increase ACAT activity was observed compared with the corresponding values from both the groups fed on a standard diet and a fat supplemented diet with no cholesterol. These results suggest that acyl-CoA: cholesterol acyltransferase is modulated by alteration in the fatty acid composition of the microsomal membrane, while the cholesterol content of the microsomes shows a close relationship with the HMG-CoA reductase activity.     

The influence of dietary lipid supplementation on cardiac beta-adrenergic receptor adenylate cyclase activity in the marmoset monkey

Dietary lipid supplements high in either saturated fat derived from sheep kidney fat or unsaturated fat derived from sunflower seed oil, and a low mixed fat reference diet were fed to marmoset monkeys for 20 months and the effects on cardiac membrane lipid composition, and myocardial catecholamine-stimulated adenylate cyclase and beta-adrenergic receptor binding activity were investigated. For cardiac membranes enriched for beta-adrenergic binding activity, the dietary lipid treatment resulted in small changes in the proportion of saturated to unsaturated fatty acids and substantial changes in the (n - 6) to (n - 3) series of unsaturated fatty acids in the membrane phospholipids. The sheep kidney fat diet increased the cholesterol-to-phospholipid ratio in cardiac membranes in comparison to the other diets. This diet also significantly elevated basal and isoproterenol-, epinephrine- and norepinephrine-stimulated adenylate cyclase activity. The value of the dissociation constant (Kd) and the receptor number (Bmax) for the binding of [125I]ICYP to the beta-adrenergic receptor was significantly reduced in marmosets fed the sheep kidney fat diet. These results suggest that dietary lipids can influence the activity of the beta-adrenergic/adenylate cyclase system of the heart. Modulation of this transmembrane signalling system may be induced by changes in the properties of the associated membrane lipids, particularly by alteration in the membrane cholesterol-to-phospholipid ratio. This effect may be limited to those animal species in which the nature of the dietary fatty acid intake may be influencing cardiac membrane cholesterol homeostasis, which is in agreement with previous results in rats following dietary cholesterol supplementation (McMurchie et al. (1987) Biochim. Biophys. Acta 898, 137-153). ICYP, (-)-iodocyanopindolol.     

The regulation of glycerol 3-phosphate oxidase of rat brownadipose tissue mitochondria by long-chain free fatty acids

Added free fatty acids inhibit oxidation of glycerol 3-phosphate, succinate and NADH in brown-adipose tissue mitochondria from 10-day-old rats. The most pronounced is the inhibitory effect of glycerol 3-phosphate cytochrome c reductase (GP-cyto. c reductase). Contrary to other reductases, GP-cyto. c reductase activity of freshly isolated mitochondria is already inhibited by the fraction of endogenous free fatty acids. Both added and endogenous free fatty acids inhibition of GP-cyto. c reductase is fully reversible by the removal of free fatty acids by bovine serum albumine treatment. The inhibition of GP-cyto. c reductase is of strictly non-competitive type. The most inhibitory are unsaturated long-chain free fatty acids-oleic and linoleic acid. Results are discussed with regards to the regulatory importance of free fatty acids in brown-adiposetissue during intensive non-shivering thermogenesis.     

Fatty acids specifically related to the anisotropic properties of plasma membrane from rat urothelium

Four different luminal surfaces of rat urothelium differing in their fatty acid composition were prepared by dietary induction. In order to induce lipid changes, each of four groups of rat received a basal diet rich in one of the unsaturated n-3, n-6 or n-9 fatty acid families and a commercial (control) diet. The effects of the dietary regime on the fatty acid composition of luminal urothelial membranes and their relation to the mobility of fluorescent probes were studied. In comparison with the control diet membrane, all three fatty acid-rich diets induced a decrease of the percentage amount of saturated fatty acid while that of the unsaturated fatty acids was increased. Accordingly, all three diets increased the unsaturation index in comparison with the control diet. The anisotropy across each membrane fraction was assessed using the n-(9-anthroyloxy) fatty acid fluorescent probes 3-AS, 7-AS and 12-AS, which locate at different depths in the membrane. Two different anisotropy profiles were observed. One profile showed the highest anisotropy at the C7 depth, whereas the other exhibited a continuous decrease of the anisotropy from the surface to the center of the bilayer. The molecular properties (isomerization) of 18:2n-9 fatty acid may account, at least in part, for the observed V-shaped profile (the ascending trend) of the membrane anisotropy values as a function of the respective 18:2n-9 fatty acid contents. Nevertheless, the minimum value of the profile did not correspond to the minimum 18:2n-9 fatty acid content, but rather to the higher amount of docosahexaenoic (22:6n-3) fatty acid. Thus, a modulating role of the 22:6n-3 fatty acid on the rigidifying effect of 18:2n-9 fatty acid is suggested, possibly mediated by relationships between fatty acid composition, saturated and unsaturated chain lengths, and freedom of motion of the phospholipid acyl chains.     

High-cholesterol diets induce changes in lipid composition of rat erythrocyte membrane including decrease in cholesterol,increase in alpha-tocopherol and changes in fatty acids of phospholipids

Effects of high dietary cholesterol on erythrocyte membrane lipids were studied. Feeding rats with a diet containing 0.5% cholesterol and 0.15% sodium cholate for two weeks induced changes in erythrocyte membrane lipids including a decrease in cholesterol, an increase in alpha-tocopherol (alpha-Toc) and changes in the fatty acid composition of phospholipids. Oleic acid and linoleic acid increased, while arachidonic acid decreased in phosphatidylcholine. Saturated fatty acids decreased and unsaturated fatty acids increased in phosphatidylethanolamine. Almost the same changes in membrane lipids were also noted after six weeks of feeding rats with the diet. A diet containing 0.5% cholesterol but without sodium cholate caused a decrease in erythrocyte cholesterol and an increase in erythrocyte alpha-Toc after two weeks of feeding, as compared to the basal diet, indicating that high dietary cholesterol, but not sodium cholate, was responsible for these changes in the erythrocyte membrane.     

High-cholesterol Diets Induce Changes in Lipid Composition of Rat Erythrocyte Membrane Including Decrease in Cholesterol,Increase in α-Tocopherol and Changes in Fatty Acids of Phospholipids

Effects of high dietary cholesterol on erythrocyte membrane lipids were studied. Feeding rats with a diet containing 0.5% cholesterol and 0.15% sodium cholate for two weeks induced changes in erythrocyte membrane lipids including a decrease in cholesterol, an increase in α-tocopherol (α-Toc) and changes in the fatty acid composition of phospholipids. Oleic acid and linoleic acid increased, while arachidonic acid decreased in phosphatidylcholine. Saturated fatty acids decreased and unsaturated fatty acids increased in phosphatidylethanolamine. Almost the same changes in membrane lipids were also noted after six weeks of feeding rats with the diet. A diet containing 0.5% cholesterol but without sodium cholate caused a decrease in erythrocyte cholesterol and an increase in erythrocyte α-Toc after two weeks of feeding, as compared to the basal diet, indicating that high dietary cholesterol, but not sodium cholate, was responsible for these changes in the erythrocyte membrane.     

Djungarian hamsters exhibit temperature-dependent dietary fat choice in long days

Previous studies have shown that whole-animal thermal responses of ectotherms and heterotherms (e.g., hibernators), both of which experience a wide range of body temperatures, are related to the saturation level of somatic lipids, which in turn can be influenced by the ratio of saturated and unsaturated fatty acids in the diet. This study demonstrates that Djungarian hamsters held in long days display ambient temperature-dependent choice of dietary fats, increasing their preference for saturated fats when ambient temperature increases (to 27 degrees C) and later reversing this preference when ambient temperature is returned to its original value (8 degrees C). Changes in percent contribution of the unsaturated and saturated diets in response to temperature were accomplished almost solely by changes in the amount of unsaturated diet consumed. Temperature-dependent fatty acid choice occurs at a stage in the annual cycle when Djungarian hamsters do not enter spontaneous daily torpor and therefore experience only small changes in core body temperature. These results suggest that temperature-dependent fatty acid choice may occur in a wide range of animals, including nonheterothermic endotherms.     

Lipid diet and enterocyte microsomal membrane fluidity in rats

Rats were fed on diets more or less enriched with n-3 and n-6 unsaturated fatty acids, before removal of the small intestine. The global protein, cholesterol and phospholipid contents of enterocyte microsomes were measured. Fatty acids of the total lipid extracts were determined. Acyl coenzyme A: cholesterol acyl transferase (ACAT) was chosen as the enzyme whose activity reflects metabolic changes induced by lipid diets. Fluorescence measurements using diphenylhexatriene as the membrane probe were performed. As dietary fat may change the fatty acid composition of membranes, the order parameter S calculated from fluorescence measurements was studied with regard to dietary fatty acid composition. The S values, distributed over a large range, were not different between rat groups. They were positively correlated with the ratios of cholesterol and proteins to phospholipids and the molar percentage of saturated fatty acids. ACAT activity was negatively correlated with S. Variations in S values among rats, whatever the diet, could in part be attributed to individual factors.     

Role of phospholipid fatty acids on the kinetics of high and low affinity sites of cytochrome c oxidase

The nature of the interactions between cytochrome c oxidase and the phospholipids in mitochondrial membranes has been investigated by varying the nature of the fatty acyl components of Saccharomyces cerevisiae. A double fatty acid yeast mutant, FAI-4C, grown in combinations of unsaturated (oleic, linoleic, linolenic, and eicosenoic) and saturated (lauric and palmitic) fatty acids, was employed to modify mitochondrial membranes. The supplemented fatty acids constituted a unique combination of different acyl chain lengths with varying degrees of unsaturation which were subsequently incorporated into mitochondrial phospholipids. Phosphatidylethanolamine and cardiolipin, the predominant phospholipids of the inner mitochondrial membrane, were characterized by their high levels of supplemented unsaturated fatty acids. Increasing the chain length or the degree of unsaturation of mitochondrial membrane phospholipids had no effect on altering the nature of the phospholipid polar head group but did result in a profound change on the specific activity of cytochrome c oxidase. When studied under conditions of different ionic strengths and pHs the enzyme's activity, as documented by Eadie-Hofstee plots, showed biphasic kinetics. The kinetic parameters for the low affinity reaction were greatly influenced by the changes in the membrane fatty acids and only marginal effects were noted at the high affinity reaction site. The discontinuities in the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene, monitored at increasing temperatures, suggested that changes in membrane fluidity were conditioned by alterations in mitochondrial membrane fatty acid constituents. These results indicate that the lipid changes affecting the low affinity binding site of cytochrome c oxidase may be the result of lipid-protein interactions which lead to enzyme conformational changes or may be due to gross changes in membrane fluidity. It may, therefore, follow that this enzyme site may be embedded in or be juxtaposed to the outer surface of the inner mitochondrial membrane bilayer in contrast to the high affinity site which has been shown to be significantly above the membrane plane.     

Effects of saturated and unsaturated fats given with and without dietary cholesterol on hepatic cholesterol synthesis and hepatic lipid metabolism

Hepatic cholesterol synthesis was studied in rats after consuming diets of varying neutral lipid and cholesterol content. Cholesterol synthesis was evaluated by measuring 3-hydroxy-3-methylglutaryl-CoA reductase and by determining the rate of 3H-labeled sterol production from [3H]mevalonate. Results were correlated with sterol balance data and hepatic lipid content. Hepatic cholesterol synthesis was relatively great when cholesterol was excluded from the diet. The source of neutral dietary lipids, saturated vs. unsaturated, produced no change in hepatic sterol synthesis. Values for fecal sterol outputs and hepatic cholesterol levels were also similar in rats consuming either saturated or unsaturated fats. When 1% cholesterol was added to the diet, hepatic cholesterol synthesis was suppressed but the degree of suppression was greater in rats consuming unsaturated vs. saturated fats. This was associated with greater accumulation of cholesterol in livers from rats consuming unsaturates and a reduction in fecal neutral sterol output in this group as opposed to results from rats on saturated fats. Cholesterol consumption also altered the fatty acid composition of hepatic phospholipids producing decreases in the percentages of essential polyunsaturated fatty acids. It is concluded that dietary cholesterol alters cholesterol and fatty acid metabolism in the liver and that this effect is enhanced by dietary unsaturated fats.