- Email: [email protected]
Increased 5′ nucleotidase activity in plasma membranes from rat liver following ingestion of fish oil
NUTRITION RESEARCH, Vol. 5, pp. 277-283, 1985 0271-5317/85 $3.00 + .00 Printed in the USA. Copyright (c) 1985 Pergamon Press Ltd. All rights reserved.
INCREASED 5' NUCLEOTIDASE ACTIVITY IN PLASMA MEMBRANES FROM RAT LIVER FOLLOWING INGESTION OF FISH OIL Janet Flier, Belur R. Lokesh and John E. Kinsella Institute of Food Science Cornell University Ithaca, NY 14853
ABSTRACT
The effect of n-3 fatty acid enriched diets on the membrane compositions of rat liver was studied. Sprague Dawley rats maintained on 10% menhaden oil enriched diet showed two to four fold increase in n-3 fatty acid levels and a 35% decrease in arachidonic acid in liver plasma membrane phospholipids. The specific activity of 5'-nucleotidase enzyme in n-3 fatty acid enriched membranes was two times higher than in liver membranes from control rats. These studies indicate that dietary n-3 fatty acids are incorporated into membrane lipids and influences the membrane associated enzyme activity. Key Words: Menhaden oil, n-3 fatty acids, phospholipids, 5'nucleotidase
liver plasma
membrane,
INTRODUCTION A number of workers have reported that dietary n-3 polyunsaturated fatty acids can alter the fatty acid composition of animal tissues (1-4). Recently we demonstrated that fish oil enriched diets decreased serum thromboxane and prostacyclin levels in rats (5). This dietary treatment also decreased the capacity of lung microsomes to synthesize thromboxane from exogenous arachidonic acid (6). These observations may be caused by the competition of n-3 fatty acids of fish oil with arachidonic acid for cyclooxygenase system (7) or dietary n-3 fatty acids could also alter membrane fatty acid composition (8) which in turn could affect the membrane fluidity (9). It has been reported that changes in membrane fluidity affects prostaglandin synthesis (10) and the binding affinities and capacities of cell membranes for prostaglandins (ii). Conceivably it could also affect the activity of membrane associated phospholipases (12,13) which may regulate the release of arachidonic acid needed for prostaglandin synthesis. In order to assess this we studied the incorporation of dietary n-3 polyunsaturated fatty acids into liver plasma membrane phospholipids and the concomitant effects on arachidonic acid. Because of the difficulties involved in accurately assaying membrane bound phospholipases (1416), we initially monitored the activity of 5'-nucleotidase for assessing the effects of n-3 fatty acids on plasma membrane enzymes. MATERIALS AND METHODS All chemicals used were obtained from Sigma Chemical Company (USA). vents used were analytical reagent grade.
Sol-
Animals and Diets Sprague Dawley rats (Charles River, CDSDBR, Wilmington, MA) weighing 400450 gms were used in three studies. They were maintained at 20-22~ in individual cages in an approved animal facility. Animals had continuous access to
277
278
J. FLIER et al.
food and water.
A 12 hr dark-light cycle was maintained in the room.
The composition of diets was similar to that reported previously, (5). Thus, it contained 20, 57.6, 4.0, 6, 0.2, 0.2, 0.02 and 12% by weight of casein, cornstarch, alphacel AIN 76 mineral mix, choline, methionine, BHT and fat (containing I0 IU vitamin E acetate/g oil), respectively. The control diet contained 2 and 10% safflower oil and hydrogenated coconut oil while the experimental diet contained 2 and 10% safflower oil and menhaden oil aS sources of dietary fatty acids. Safflower oil was used to provide essential fatty acids. The fatty acid composition of dietary fats are summarized in Table i. Fresh diets were fed to the animals everyday, and uneaten food was discarded. TABLE l Fatty Acid Composition of Dietary Fat Fatty Acid
10% Menhaden Oil Enriched Diet
Control wt%
12:0 38.9 14:0 16.0 16:0 i0.6 16:1 O.1 18:0 9.8 18:1 7.8 18:2 15.4 18:3 18:4 20:4 20:5 22:5 22:6 ~The fatty acids are abbreviated as number of carbon atoms : bonds.
0.6 6.2 14.0 9.6 4.2 16.7 14.5 3.2 4.3 l.O 14.3 1.8 6.1 number of double
Lipid peroxidation was routinely monitored by malondialdehyde formation using thiobarbituric acid reagent (17). None of the diets used showed significant increase in malondialdehyde formation during the course of the experiment. Animals were maintained on control and menhaden oil supplemented diet for 3 weeks before they were sacrificed. Food consumption and body weight gain of animals during the course of experiments were similar. Isolation of Plasma Membranes and Lipid Analysis Liver plasma membranes were prepared on a discontinuous sucrose gradient according to the method of Emmelot et al. (18). The yield of plasma membranes was approximately 0.32 mg protein/g wet weight of the liver. Glucose 6-phosphatase and 5'-nucleotidase were assayed according to the method of Aronson and Touster (19) by measuring the release of inorganic phosphate from glucose 6 phosphate and 5'-adenosine monophosphate, respectively. Protein was determined according to Lowry et al. (20) using bovine serum albumin as reference standard. Lipids were extracted by the method of Bligh and Dyer (21). Total phospholipids were determined as described by Stewart (22). Cholesterol was measured by the o-phthalaldehyde method (23). Phospholipids were separated from neutral lipids on silica gel G (Merck) plates in the solvent system chloroform : methanol (8:iv/v). Lipids were eluted from the silica gel with choloroform : methanol (2:Iv/v). Lipids were saponified with 0.5N NaOH in methanol and fatty acids were methylated as described by Bowyer et al. (24). The fatty acid methyl esters were quantified by gas liquid chromatography (Hewlett Packard 5880) using pentadecanoic acid as the internal standard (25).
5' NUCLEOTIDASE
279
RESULTS AND DISCUSSION Purity of Plasma Membrane Initial studies with the liver plasma membranes isolated from rats maintained on regular chow diet indicated that the plasma membranes were enriched in 5'-nucleotidase (12 fold) and Na+/K + ATPase (23 fold), activities compared to the values for liver homogeaates. There was no enrichment of either glucose 6-phosphatase or succinate dehydrogenase activities in plasma membranes indicating a minimal contamination (<5%) from microsomes and mitochondrial fractions. Electron micrographs of plasma membranes indicated that the membranes were isolated mostly in the form of sheets and some in the form of vesicles (Fig. i). No mitochondrial contamination was observed in electron micrograph pictures. Lipid Analysis The fatty acid composition of total phospholipids in plasma membranes were significantly altered in the rats maintained for three weeks on menhaden oil enriched diets (Table 2). Eicosapentaenoic acid and docosahexaenoic acid TABLE 2 Concentration of Phospholipids, Cholesterol and Fatty Acid Composition of Total Phospholipids in Liver Plasma Membranes Menhaden Oil Control Diet Enriched Diet (n=4) (n=4) [nmoles/mg protein (Mean• 14:0 iO • 3 8 16:0 113 • 27 139 16:1 12 • 2 15 18:0 142 • 38 129 18:1 45 • 8 60 18:2 (n-6) 60 • 25 82 20:4 (n-6) 125 • 46 81 20:5 (n-3) 6 • 2 24 22:5 (n-3) Nd 9 22:6 (n-3) 28 • 7 53 Phospholipid (~g/mg protein) 257 • 83 243 Cholesterol (~g/mg protein) 17.3 • 1.5 20.4 Plasma membranes from 3 animals are pooled in each set. Nd: Not detected : *p
• • • • • • • • • • + +
7 24 4 22 27 16 16 8* 3 13" 27 2.8
increased four-fold and two-fold, respectively, whereas there was a concomitant 35% decrease in the arachidonic acid content of membrane phospholipids. No significant differences in the oleic and linoleic acid contents were observed in membrane lipids suggesting that n-3 fatty acid enrichment did not affect the ~-9 and A-6 desaturase enzyme activities. There was a small but significant increase in docosapentaenoic acid levels after feeding the rats with the menhaden oil enriched diet. Saturated fatty acid levels were not affected by the experimental diet. Total phospholipid or cholesterol contents of the plasma membrane were not affected by the dietary fatty acids. These data clearly show that plasma membrane fatty acids can be easily altered by dietary n-3 polyunsaturated fatty acids and thus are consistent with changes in total fatty acids reported earlier (5). Earlier studies have shown that these fatty acid changes in membrane phospholipids are easily detectable after three days feeding of fatty acid enriched diets (4,26,27). Our ongoing studies are in agreement with these observations.
280
J. FLIER et al.
~
i 84)~
~ ~
U~84
/
FIG. I Electron micrograph of liver plasma membrane isolated from on control diets as described in methods [~ 28,000 x , Imm = 357 A]
rats
5' NUCLEOTIDASE
28l
Enzyme Analysis Glucose 6-phosphatase was not enriched in the plasma membrane following purification indicating that the membrane isolated had minimal contamination from microsomes (Table 3). The isolated membranes were significantly enriched in 5'-nucleotidase, an important enzyme (28) frequently used as a marker for the enrichment of plasma membranes. The specific activity of 5'-nucleotidase in n-3 fatty acid enriched plasma membranes was twice that observed in control plasma membranes. The reason for this increased 5'-nucleotidase activity could be the consequence of the changes in the membrane fluidity induced by dietary n-3 fatty acid or due to the specific effect of n-3 fatty acids on the enzyme itself (29-31).
Enzyme
TABLE 3 Enzyme Activity in Liver Plasma Membranes Isolated from Rats Receiving Menhaden Oil Control Menhaden Oil EnrichedDiet Plasma Plasma Homo~enate Membrane Homogenate Membrane ~(nmoles Pi liberated/mg protein/min)
5'-nucleotidase glucose 6-phosphatase ~Mean•
35.7 • 3.3 16.3 • 4.7
313.3 • 70 15.7 • 4.3
34.6 • 5.4 17.2 • 6.8
650.0 • 106.7 16.1 • 5.6
n = 7 for 5'-nucleotidase and n = 4 for glucose 6-phosphatase
This observation is significant since it may indicate that n-3 polyunsaturated fatty acids may affect other membrane bound enzymes such as phospholipase (14) by altering membrane fluidity thereby affecting the release of arachidonic acid and eicosanoid production. The fluidity may also affect adenylcyclase which is important in mediating hormonal responses (32) and Na+/K + ATPase which is critical in maintaining salt balance (33). Preliminary data indicate a decrease in membrane bound ATPase in rats on fish oil diets (unpublished observations, 1984). Further studies to monitor the effects of n-3 fatty acids on arachidonic acid release from plasma membrane are warranted. Acknowledgement: Support Sea Grant Program.
in part by USDA Grant #82-CRCR-I-1036 and New
York
REFERENCES i. TAHIN, Qo S., BLUM, M. and CARAFOLI, E. The fatty acid composition of subcellular membranes of rat liver, heart and brain: Diet induced modifications. Eur. J. Biochem 1981; 121: 5-13. 2. HARRIS, W. S., CONNOR, W. E. AND Mc MURRY, M. P. The comparative reduction of the plasma lipids and lipoproteins by dietary polyunsaturated fats: Salmon oil versus vegetable oil. Metabolism 1983; 32: 179-184. 3. MARSHALL, L. A. and JOHNSTON, P . V . The effect of dietary -linolenic acid in the rat on fatty acid profiles of immunocompetent cell populations. Lipids 1983; 18: 737-742. 4. STUBBS, C. D. and SMITH, A. D. The modification of mammalian membrane polyunsaturated fatty acid composition in relation to membrane fluidity and function. Biochim. Bio~hys. Acta 1984; 779: 89-137. 5. BRUCKNER, G. G., LOKESH, B., GERMAN, B. AND KINSELLA, J. E. Biosynthesis of prostanoids, tissue fatty acid composition and thrombotic parameters in
282
J. FLIER et al. rats fed diets enriched with docosahexaenoic or Thrombosis Res. 1984; 34: 479-497.
eicosapentaenoic
acids.
6. LOKESH, B. R., BRUCKNER, G. and KINSELLA, J. E. Reduction in thromboxane formation by n-3 fatty acids enriched lung microsomes from rat and guinea pig following the ingestion of dietary menhaden oil. Prostaglandins Med. 1984; 15:337-348. 7. COREY, E. J., SHIN, C. and CASHMAN, J. R. Docosahexaenoic acid is a strong inhibitor of prostaglandin but not leukotriene biosynthesis. Proc. Natl. Acad. Sci. 1983; 80: 3581-3585. 8.
MAGRUM, L. J. and JOHNSTON, P. V. Modulation of prostaglandin synthesis in rat peritoneal macrophages with w-3 fatty acids. Lipids 1983; 18: 514-521.
9. STORCH, J. and SCHACHTER, D. Dietary induction of acyl chain desaturases alters the lipid composition and fluidity of rat hepatocyte plasma membranes. Biochemistry ; 23: 1165-1170. i0. DAVE, J. R., BROWN, N. V. and KNAZEK, R. A. Prolactin modifies the prostaglandin synthesis, prolactin binding and fluidity of mouse liver membranes. Biochim. Biophys. Res. Commun. ; 108: 193-199. ii. OPMEER, F. A., ADOLFS, M. J. P. and BONTA, I. L. prostaglandin E 2 receptors in vivo by dietary fatty acids macrophages from rats. J_~.Lipid Res. 1984; 25: 262-268. 12. LIU, M. S., GHOSH, S. and YANG, Y. induced by phospholipase A activation: Life Sci. 1983; 33: 1995-2002.
Regulation of in peritoneal
Change in membrane lipid fluidity A mechanism of endotoxic shock.
13, BORMANN, B. J., HUANG, C. K., MACKIN, W. M. and BECKER, E. L. Receptor mediated activation of phospholipase A 2 in rabbit neutrophil plasma membrane. Proc. Natl. Acad. Sci. 1984; 81: 767-770. 14. IRVINE, R. F. mammalian cells.
How is the level of free arachidonic acid controlled Biochem. J. 1982; 204: 3-16.
15. VAN DEN BOSCH, H. Intracellular phospholipase A. 1980; 604: 191-246.
in
Biochim. Biophys. Acta
16. CHERN, J., BRUCKNER, G. and KINSELLA, J. E. Effects of linoelaidate on fatty acid composition and phospholipase activity liver microsomes. Nutr. Res. 1983; 3: 571-581.
dietary in rat
17. HAMMER, C. T. and WILLS, E. D. The role of lipid components of the diet in the regulation of the fatty acid composition of the rat liver endoplasmic reticulum and lipid peroxidation. Biochem. J. 1978; 174: 585-598. 18. EMMELOT, P., BOS, C. J., VAN HOEVEN, R. P. and VAN BLITTERSWIJK, W. J. Isolation of plasma membranes from rat and mouse livers and hepatomas. Methods Enzymol. 1974; XXXI: 75-90. 19. ARONSON, N. N. and TOUSTER, fragments in isotonic sucrose.
O. Isolation of rat liver plasma membrane Methods Enzymol. 1974; XXXI: 90-102.
20. LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L. and RANDALL, R. J. Protein Measurement with folin-phenol reagent. J. Biol. Chem 1951; 193: 265-275.
5' NUCLEOTIDASE
283
21. BLIGH, E. G. and DYER, W. J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 1959; 37: 911-917. 22. STEWART, J. C. M. Colorimetric determinationof phospholipids with ammonium ferrothiocynate. Anal. Biochem. 1980; 104: 10-14. 23. RUDEL, L. L. phthalaldehyde.
and MORRIS, M. D. Determination of cholesterol using 0J__~.Lipid Res. 1973; 14: 364-366.
24. BOWYER, D. E., LEAT, W. M. F., HOWARD, A. N. and GRESHAM, G. A. The determination of the fatty acid composition of serum lipids separated by thin layer chromatography; and a comparison with column chromatography. Biochim. Biophys. Acta 1963; 70: 423-431. 25. YU, P., MAI, J. and KINSELLA, J. E. The effects of dietary trans, trans methyl octadecadienoate acid on composition and fatty acids of rat heart. Am. J. Clin. Nutrition 1980; 33: 598-605. 26. LOKESH, B. R., MATHUR, S. N. and SPECTOR, A. A. Effect of fatty acid saturation on NADPH-dependent lipid peroxidation in rat liver microsomes. J__LLipid Res. 1981; 22: 905-915. 27. VAN GOLDE, L. M. B., PIETERSON, W. A. and VAN DEENEN, L. L. M. Alterations in the molecular species of rat liver lecithin by corn oil feeding to essential fatty acid deficient rat as a function of time. Biochim. Biophys. Acta 1968; 152: 84-95. 28. SUN, A. S., HOLLAND, J. F., OHRUMA, T. and CHAHINION, M. S. 5'-nucleotidase activity in permanent human lymphoid cell lines. Implication for cell proliferation and aging in vitro. Biochim. Biophys. Acta 1982; 714: 530-535. 29. DIPPLE, I. and HOUSLAY, M. D. The activity of glucagon stimulated adenylate cyclase from rat liver plasma membranes is modulated by the fluidity of its lipid environment. Biochem. J. 1978; 174: 179-190. 30. AWAD, A. B. and CHATTOPADHYAY, J. P. Effect of dietary fats on the lipid composition and enzyme activities of rat cardiac sarcolemma. J. Nutr. : 1983; 113: 1878-1884. 31. LOUIS, S. L., BRIVIO-HAUGLAND, R. P. and WILLIAMS, M. A. Effect on essential fatty acid deficiency on activity of liver plasma membrane enzymes in the rat. J__L Suyf. Molecular. Str. 1976; 4: 487-496. 32. COLARD, O., KERVABON, A. and ROY, C. Effects on adenylate cyclase activities of unsaturated fatty acid incorporation into rat liver plasma membrane phospholipids. Specific modulation by linoleate. Biochim. Biophys. Res. Commun. 1980; 95: 97-102. 33. KEEFE, E. B., SCHARSCHMIDT, B. F., BLANKENSHIP, N. M. and OCKNER, R. K. Studies of relationships smong bile flow, liver plasma membrane Na+/K +ATPase and membrane microviscosity in the rat. J__~.Clin. Invest 1979; 64: 1590-1598.
Accepted for publication January 16, 1985.
INCREASED 5' NUCLEOTIDASE ACTIVITY IN PLASMA MEMBRANES FROM RAT LIVER FOLLOWING INGESTION OF FISH OIL Janet Flier, Belur R. Lokesh and John E. Kinsella Institute of Food Science Cornell University Ithaca, NY 14853
ABSTRACT
The effect of n-3 fatty acid enriched diets on the membrane compositions of rat liver was studied. Sprague Dawley rats maintained on 10% menhaden oil enriched diet showed two to four fold increase in n-3 fatty acid levels and a 35% decrease in arachidonic acid in liver plasma membrane phospholipids. The specific activity of 5'-nucleotidase enzyme in n-3 fatty acid enriched membranes was two times higher than in liver membranes from control rats. These studies indicate that dietary n-3 fatty acids are incorporated into membrane lipids and influences the membrane associated enzyme activity. Key Words: Menhaden oil, n-3 fatty acids, phospholipids, 5'nucleotidase
liver plasma
membrane,
INTRODUCTION A number of workers have reported that dietary n-3 polyunsaturated fatty acids can alter the fatty acid composition of animal tissues (1-4). Recently we demonstrated that fish oil enriched diets decreased serum thromboxane and prostacyclin levels in rats (5). This dietary treatment also decreased the capacity of lung microsomes to synthesize thromboxane from exogenous arachidonic acid (6). These observations may be caused by the competition of n-3 fatty acids of fish oil with arachidonic acid for cyclooxygenase system (7) or dietary n-3 fatty acids could also alter membrane fatty acid composition (8) which in turn could affect the membrane fluidity (9). It has been reported that changes in membrane fluidity affects prostaglandin synthesis (10) and the binding affinities and capacities of cell membranes for prostaglandins (ii). Conceivably it could also affect the activity of membrane associated phospholipases (12,13) which may regulate the release of arachidonic acid needed for prostaglandin synthesis. In order to assess this we studied the incorporation of dietary n-3 polyunsaturated fatty acids into liver plasma membrane phospholipids and the concomitant effects on arachidonic acid. Because of the difficulties involved in accurately assaying membrane bound phospholipases (1416), we initially monitored the activity of 5'-nucleotidase for assessing the effects of n-3 fatty acids on plasma membrane enzymes. MATERIALS AND METHODS All chemicals used were obtained from Sigma Chemical Company (USA). vents used were analytical reagent grade.
Sol-
Animals and Diets Sprague Dawley rats (Charles River, CDSDBR, Wilmington, MA) weighing 400450 gms were used in three studies. They were maintained at 20-22~ in individual cages in an approved animal facility. Animals had continuous access to
277
278
J. FLIER et al.
food and water.
A 12 hr dark-light cycle was maintained in the room.
The composition of diets was similar to that reported previously, (5). Thus, it contained 20, 57.6, 4.0, 6, 0.2, 0.2, 0.02 and 12% by weight of casein, cornstarch, alphacel AIN 76 mineral mix, choline, methionine, BHT and fat (containing I0 IU vitamin E acetate/g oil), respectively. The control diet contained 2 and 10% safflower oil and hydrogenated coconut oil while the experimental diet contained 2 and 10% safflower oil and menhaden oil aS sources of dietary fatty acids. Safflower oil was used to provide essential fatty acids. The fatty acid composition of dietary fats are summarized in Table i. Fresh diets were fed to the animals everyday, and uneaten food was discarded. TABLE l Fatty Acid Composition of Dietary Fat Fatty Acid
10% Menhaden Oil Enriched Diet
Control wt%
12:0 38.9 14:0 16.0 16:0 i0.6 16:1 O.1 18:0 9.8 18:1 7.8 18:2 15.4 18:3 18:4 20:4 20:5 22:5 22:6 ~The fatty acids are abbreviated as number of carbon atoms : bonds.
0.6 6.2 14.0 9.6 4.2 16.7 14.5 3.2 4.3 l.O 14.3 1.8 6.1 number of double
Lipid peroxidation was routinely monitored by malondialdehyde formation using thiobarbituric acid reagent (17). None of the diets used showed significant increase in malondialdehyde formation during the course of the experiment. Animals were maintained on control and menhaden oil supplemented diet for 3 weeks before they were sacrificed. Food consumption and body weight gain of animals during the course of experiments were similar. Isolation of Plasma Membranes and Lipid Analysis Liver plasma membranes were prepared on a discontinuous sucrose gradient according to the method of Emmelot et al. (18). The yield of plasma membranes was approximately 0.32 mg protein/g wet weight of the liver. Glucose 6-phosphatase and 5'-nucleotidase were assayed according to the method of Aronson and Touster (19) by measuring the release of inorganic phosphate from glucose 6 phosphate and 5'-adenosine monophosphate, respectively. Protein was determined according to Lowry et al. (20) using bovine serum albumin as reference standard. Lipids were extracted by the method of Bligh and Dyer (21). Total phospholipids were determined as described by Stewart (22). Cholesterol was measured by the o-phthalaldehyde method (23). Phospholipids were separated from neutral lipids on silica gel G (Merck) plates in the solvent system chloroform : methanol (8:iv/v). Lipids were eluted from the silica gel with choloroform : methanol (2:Iv/v). Lipids were saponified with 0.5N NaOH in methanol and fatty acids were methylated as described by Bowyer et al. (24). The fatty acid methyl esters were quantified by gas liquid chromatography (Hewlett Packard 5880) using pentadecanoic acid as the internal standard (25).
5' NUCLEOTIDASE
279
RESULTS AND DISCUSSION Purity of Plasma Membrane Initial studies with the liver plasma membranes isolated from rats maintained on regular chow diet indicated that the plasma membranes were enriched in 5'-nucleotidase (12 fold) and Na+/K + ATPase (23 fold), activities compared to the values for liver homogeaates. There was no enrichment of either glucose 6-phosphatase or succinate dehydrogenase activities in plasma membranes indicating a minimal contamination (<5%) from microsomes and mitochondrial fractions. Electron micrographs of plasma membranes indicated that the membranes were isolated mostly in the form of sheets and some in the form of vesicles (Fig. i). No mitochondrial contamination was observed in electron micrograph pictures. Lipid Analysis The fatty acid composition of total phospholipids in plasma membranes were significantly altered in the rats maintained for three weeks on menhaden oil enriched diets (Table 2). Eicosapentaenoic acid and docosahexaenoic acid TABLE 2 Concentration of Phospholipids, Cholesterol and Fatty Acid Composition of Total Phospholipids in Liver Plasma Membranes Menhaden Oil Control Diet Enriched Diet (n=4) (n=4) [nmoles/mg protein (Mean• 14:0 iO • 3 8 16:0 113 • 27 139 16:1 12 • 2 15 18:0 142 • 38 129 18:1 45 • 8 60 18:2 (n-6) 60 • 25 82 20:4 (n-6) 125 • 46 81 20:5 (n-3) 6 • 2 24 22:5 (n-3) Nd 9 22:6 (n-3) 28 • 7 53 Phospholipid (~g/mg protein) 257 • 83 243 Cholesterol (~g/mg protein) 17.3 • 1.5 20.4 Plasma membranes from 3 animals are pooled in each set. Nd: Not detected : *p
• • • • • • • • • • + +
7 24 4 22 27 16 16 8* 3 13" 27 2.8
increased four-fold and two-fold, respectively, whereas there was a concomitant 35% decrease in the arachidonic acid content of membrane phospholipids. No significant differences in the oleic and linoleic acid contents were observed in membrane lipids suggesting that n-3 fatty acid enrichment did not affect the ~-9 and A-6 desaturase enzyme activities. There was a small but significant increase in docosapentaenoic acid levels after feeding the rats with the menhaden oil enriched diet. Saturated fatty acid levels were not affected by the experimental diet. Total phospholipid or cholesterol contents of the plasma membrane were not affected by the dietary fatty acids. These data clearly show that plasma membrane fatty acids can be easily altered by dietary n-3 polyunsaturated fatty acids and thus are consistent with changes in total fatty acids reported earlier (5). Earlier studies have shown that these fatty acid changes in membrane phospholipids are easily detectable after three days feeding of fatty acid enriched diets (4,26,27). Our ongoing studies are in agreement with these observations.
280
J. FLIER et al.
~
i 84)~
~ ~
U~84
/
FIG. I Electron micrograph of liver plasma membrane isolated from on control diets as described in methods [~ 28,000 x , Imm = 357 A]
rats
5' NUCLEOTIDASE
28l
Enzyme Analysis Glucose 6-phosphatase was not enriched in the plasma membrane following purification indicating that the membrane isolated had minimal contamination from microsomes (Table 3). The isolated membranes were significantly enriched in 5'-nucleotidase, an important enzyme (28) frequently used as a marker for the enrichment of plasma membranes. The specific activity of 5'-nucleotidase in n-3 fatty acid enriched plasma membranes was twice that observed in control plasma membranes. The reason for this increased 5'-nucleotidase activity could be the consequence of the changes in the membrane fluidity induced by dietary n-3 fatty acid or due to the specific effect of n-3 fatty acids on the enzyme itself (29-31).
Enzyme
TABLE 3 Enzyme Activity in Liver Plasma Membranes Isolated from Rats Receiving Menhaden Oil Control Menhaden Oil EnrichedDiet Plasma Plasma Homo~enate Membrane Homogenate Membrane ~(nmoles Pi liberated/mg protein/min)
5'-nucleotidase glucose 6-phosphatase ~Mean•
35.7 • 3.3 16.3 • 4.7
313.3 • 70 15.7 • 4.3
34.6 • 5.4 17.2 • 6.8
650.0 • 106.7 16.1 • 5.6
n = 7 for 5'-nucleotidase and n = 4 for glucose 6-phosphatase
This observation is significant since it may indicate that n-3 polyunsaturated fatty acids may affect other membrane bound enzymes such as phospholipase (14) by altering membrane fluidity thereby affecting the release of arachidonic acid and eicosanoid production. The fluidity may also affect adenylcyclase which is important in mediating hormonal responses (32) and Na+/K + ATPase which is critical in maintaining salt balance (33). Preliminary data indicate a decrease in membrane bound ATPase in rats on fish oil diets (unpublished observations, 1984). Further studies to monitor the effects of n-3 fatty acids on arachidonic acid release from plasma membrane are warranted. Acknowledgement: Support Sea Grant Program.
in part by USDA Grant #82-CRCR-I-1036 and New
York
REFERENCES i. TAHIN, Qo S., BLUM, M. and CARAFOLI, E. The fatty acid composition of subcellular membranes of rat liver, heart and brain: Diet induced modifications. Eur. J. Biochem 1981; 121: 5-13. 2. HARRIS, W. S., CONNOR, W. E. AND Mc MURRY, M. P. The comparative reduction of the plasma lipids and lipoproteins by dietary polyunsaturated fats: Salmon oil versus vegetable oil. Metabolism 1983; 32: 179-184. 3. MARSHALL, L. A. and JOHNSTON, P . V . The effect of dietary -linolenic acid in the rat on fatty acid profiles of immunocompetent cell populations. Lipids 1983; 18: 737-742. 4. STUBBS, C. D. and SMITH, A. D. The modification of mammalian membrane polyunsaturated fatty acid composition in relation to membrane fluidity and function. Biochim. Bio~hys. Acta 1984; 779: 89-137. 5. BRUCKNER, G. G., LOKESH, B., GERMAN, B. AND KINSELLA, J. E. Biosynthesis of prostanoids, tissue fatty acid composition and thrombotic parameters in
282
J. FLIER et al. rats fed diets enriched with docosahexaenoic or Thrombosis Res. 1984; 34: 479-497.
eicosapentaenoic
acids.
6. LOKESH, B. R., BRUCKNER, G. and KINSELLA, J. E. Reduction in thromboxane formation by n-3 fatty acids enriched lung microsomes from rat and guinea pig following the ingestion of dietary menhaden oil. Prostaglandins Med. 1984; 15:337-348. 7. COREY, E. J., SHIN, C. and CASHMAN, J. R. Docosahexaenoic acid is a strong inhibitor of prostaglandin but not leukotriene biosynthesis. Proc. Natl. Acad. Sci. 1983; 80: 3581-3585. 8.
MAGRUM, L. J. and JOHNSTON, P. V. Modulation of prostaglandin synthesis in rat peritoneal macrophages with w-3 fatty acids. Lipids 1983; 18: 514-521.
9. STORCH, J. and SCHACHTER, D. Dietary induction of acyl chain desaturases alters the lipid composition and fluidity of rat hepatocyte plasma membranes. Biochemistry ; 23: 1165-1170. i0. DAVE, J. R., BROWN, N. V. and KNAZEK, R. A. Prolactin modifies the prostaglandin synthesis, prolactin binding and fluidity of mouse liver membranes. Biochim. Biophys. Res. Commun. ; 108: 193-199. ii. OPMEER, F. A., ADOLFS, M. J. P. and BONTA, I. L. prostaglandin E 2 receptors in vivo by dietary fatty acids macrophages from rats. J_~.Lipid Res. 1984; 25: 262-268. 12. LIU, M. S., GHOSH, S. and YANG, Y. induced by phospholipase A activation: Life Sci. 1983; 33: 1995-2002.
Regulation of in peritoneal
Change in membrane lipid fluidity A mechanism of endotoxic shock.
13, BORMANN, B. J., HUANG, C. K., MACKIN, W. M. and BECKER, E. L. Receptor mediated activation of phospholipase A 2 in rabbit neutrophil plasma membrane. Proc. Natl. Acad. Sci. 1984; 81: 767-770. 14. IRVINE, R. F. mammalian cells.
How is the level of free arachidonic acid controlled Biochem. J. 1982; 204: 3-16.
15. VAN DEN BOSCH, H. Intracellular phospholipase A. 1980; 604: 191-246.
in
Biochim. Biophys. Acta
16. CHERN, J., BRUCKNER, G. and KINSELLA, J. E. Effects of linoelaidate on fatty acid composition and phospholipase activity liver microsomes. Nutr. Res. 1983; 3: 571-581.
dietary in rat
17. HAMMER, C. T. and WILLS, E. D. The role of lipid components of the diet in the regulation of the fatty acid composition of the rat liver endoplasmic reticulum and lipid peroxidation. Biochem. J. 1978; 174: 585-598. 18. EMMELOT, P., BOS, C. J., VAN HOEVEN, R. P. and VAN BLITTERSWIJK, W. J. Isolation of plasma membranes from rat and mouse livers and hepatomas. Methods Enzymol. 1974; XXXI: 75-90. 19. ARONSON, N. N. and TOUSTER, fragments in isotonic sucrose.
O. Isolation of rat liver plasma membrane Methods Enzymol. 1974; XXXI: 90-102.
20. LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L. and RANDALL, R. J. Protein Measurement with folin-phenol reagent. J. Biol. Chem 1951; 193: 265-275.
5' NUCLEOTIDASE
283
21. BLIGH, E. G. and DYER, W. J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 1959; 37: 911-917. 22. STEWART, J. C. M. Colorimetric determinationof phospholipids with ammonium ferrothiocynate. Anal. Biochem. 1980; 104: 10-14. 23. RUDEL, L. L. phthalaldehyde.
and MORRIS, M. D. Determination of cholesterol using 0J__~.Lipid Res. 1973; 14: 364-366.
24. BOWYER, D. E., LEAT, W. M. F., HOWARD, A. N. and GRESHAM, G. A. The determination of the fatty acid composition of serum lipids separated by thin layer chromatography; and a comparison with column chromatography. Biochim. Biophys. Acta 1963; 70: 423-431. 25. YU, P., MAI, J. and KINSELLA, J. E. The effects of dietary trans, trans methyl octadecadienoate acid on composition and fatty acids of rat heart. Am. J. Clin. Nutrition 1980; 33: 598-605. 26. LOKESH, B. R., MATHUR, S. N. and SPECTOR, A. A. Effect of fatty acid saturation on NADPH-dependent lipid peroxidation in rat liver microsomes. J__LLipid Res. 1981; 22: 905-915. 27. VAN GOLDE, L. M. B., PIETERSON, W. A. and VAN DEENEN, L. L. M. Alterations in the molecular species of rat liver lecithin by corn oil feeding to essential fatty acid deficient rat as a function of time. Biochim. Biophys. Acta 1968; 152: 84-95. 28. SUN, A. S., HOLLAND, J. F., OHRUMA, T. and CHAHINION, M. S. 5'-nucleotidase activity in permanent human lymphoid cell lines. Implication for cell proliferation and aging in vitro. Biochim. Biophys. Acta 1982; 714: 530-535. 29. DIPPLE, I. and HOUSLAY, M. D. The activity of glucagon stimulated adenylate cyclase from rat liver plasma membranes is modulated by the fluidity of its lipid environment. Biochem. J. 1978; 174: 179-190. 30. AWAD, A. B. and CHATTOPADHYAY, J. P. Effect of dietary fats on the lipid composition and enzyme activities of rat cardiac sarcolemma. J. Nutr. : 1983; 113: 1878-1884. 31. LOUIS, S. L., BRIVIO-HAUGLAND, R. P. and WILLIAMS, M. A. Effect on essential fatty acid deficiency on activity of liver plasma membrane enzymes in the rat. J__L Suyf. Molecular. Str. 1976; 4: 487-496. 32. COLARD, O., KERVABON, A. and ROY, C. Effects on adenylate cyclase activities of unsaturated fatty acid incorporation into rat liver plasma membrane phospholipids. Specific modulation by linoleate. Biochim. Biophys. Res. Commun. 1980; 95: 97-102. 33. KEEFE, E. B., SCHARSCHMIDT, B. F., BLANKENSHIP, N. M. and OCKNER, R. K. Studies of relationships smong bile flow, liver plasma membrane Na+/K +ATPase and membrane microviscosity in the rat. J__~.Clin. Invest 1979; 64: 1590-1598.
Accepted for publication January 16, 1985.