470
Biochimica et Biophysics @ Elsevier/North-Holland
Acta, 486 (1977) 470-482 Biomedical Press
BBA 56955
TRIA~YL~LYCEROL AND VERY LOW DENSITY LIPOPROTEIN SECRETION INTO PLASMA OF SQUIRREL MONKEYS
OSCAR W. PORT~AN, ~ANFRED D. ROGER ILLINGWORTH
ALEXANDER,
NAOMI TANAKA
and
Department of Nutrition and Metabolic Diseases, Oregon Regional Primate Research Center, Beaverton, Oreg. 97005 and Department of Biochemistry, University of Oregon Health Science Center, Portland, Oreg. 97201 (U.S.A.) (Received
October
8th, 1976)
Summary We determined the effects of varying the types and level of dietary fat and cholesterol on the increase in plasma total triacylglycerol concentrations after injection of Triton WR-1339, an inhibitor of lipoprotein lipase, into monkeys that had been subjected to an overnight fast. The monkeys that had been treated with Triton WR-1339 were then given a test meal by intragastric intubation. Dietary cholesterol, high levels of fat and saturated fat in the habitual diet reduced the rate of release of triacylglycerol to plasma in the fasted monkey. We also determined the changes in protein and lipid concentrations of the different lipoprotein fractions. The injection of Triton WR-1339 resulted in a linear increase with time in the concentration of protein and triacylglycerol in the very low density (chylomicron-free and d < 1.006) lipoproteins, but there was an increase in the ratio of triacylglycerol to protein in that fraction. Most of the increase (96%) in very low density protein was in the B protein. Regardless of the habitual diet, a test meal accentuated the rate of triacylglycerol appearance in whole plasma and in the very low density lipoproteins of Triton WR-1339-treated monkeys, and the rate of increase of the protein component after feeding was slightly higher. Thus the administration of a meal to the fasted, Triton WR-13394reated squirrel monkey further increased the proportion of triacylglycerol in very low density lipoproteins. Although dietary cholesterol and saturated fat in the habitual diet depressed the rate of increase in very low density triacylgly~erol during fasting, the rate -Abbreviations: VLDL, very low density lipoproteins (d < 1.006, but free of chylomicrons): IDL, intermediate density lipoproteins (d 1.006-1.019); LDL, low density lipoproteins (d l.OlS1.063); HDL, high density lipoproteins (d 1.063-1.21). The B and C apoproteins of Vera low density lipoproteins are designated VLDL-B and VLDL-C.
471
of protein synthesis was not significantly affected. After administration of a test meal the rates of increase in triacylglycerol and protein in the very low density lipopro~ins were similar for monkeys from the different diet groups. Triton WR-1339 administration caused a slight and progressive increase in the intermediate density (d 1.006-1.019) lipoproteins and a marked and progressive decrease in the low density (d 1.019-1.063) lipoproteins, There was an immediate (by 5 min) drop of 70% or more in high density (d 1.063-1.21) lipoprotein protein, but the lipids except triacylglycerol remained unchanged. There was a decrease in both the A (the major fraction) and C proteins. The rates of very low density B protein secretion were comparable to the rates of low density lipoprotein catabolism that had been previously demonstrated for this species.
Introduction When Triton WR-1339, a nonionic detergent, is injected into the bloodstream of various animals, the removal of triacylglycerol from the plasma is almost completely blocked [l-3]. This reagent, therefore, provides a way of accurately determining the rate of secretion of triacylglycerol and its carrier lipoproteins into plasma [ 4-6 1. We previously showed that after the injection of 300 mg of Triton WR-1339 per kg of body weight into fasted squirrel monkeys there was a linear increase for 12 h in the concentration of all lipids in VLDL [7]. This was followed by increases in the concentrations of lipids in the IDL and LDL that were consistent with a precursor product relationship. Further studies [6] demonstra~d the rates of secretion of triacylglycerol to be reduced in fasted monkeys that had elevated LDL levels (as a result *of the chronic ingestion of diets containing cholesterol and high levels of butter). In the previous study [6] we only compared monkeys on two diets, and those diets were different in several respects. The first objective of the present study was to determine the relative importance of the type and level of fat and the level of cholesterol in the habitual diet on the secretion rates of triacylglycerol in both fasted monkeys and those given a test meal after the infusion of Triton WR-1339. The second objective was to measure the quantitative changes of the major constituents, particularly the protein and triacylglycerol, of the different lipoprotein fractions in monkeys from five of the diet groups. We were particularly interested in determining whether the increases in VLDL lipids were associated with proportional increases in the protein moieties. We could thus decide whether hypercholesterolemia induced by cholesterol feeding depressed the secretion of VLDL by the liver or only modified their composition .
Methods Experimental animals and diets Forty-four different Brazilian squirrel monkeys (Saimiri sciureus), 25 males and 19 females, were used for 88 different tests of triacylglycerol and lipopro-
472
tein metabolism after the injection of Triton WR-1339. All monkeys were collected at Leticia, Colombia, and were obtained from the Tarpon Zoo, Tarpon Springs, Fla. They were fed semipurified diets similar to those described previously [8,9]. The basal diet contained (w/w) 21.2% casein, 38.5% sucrose, 20.2% fat, 3.4% salt mix, 1.7% vitamin mix, and 15% synthetic fiber. Variations in the diet compositon are indicated on Table I. The Triton WR-1339 and feeding tests Squirrel monkeys had been on the indicated diets for three months or more when they were injected with Triton WR-1339. The dosage (300 mg/kg) and the times after Triton WR-1339 injection for blood samples were selected on the basis of a previous study [6]. Those earlier experiments had shown that after a brief lag period the triacylglycerol of total plasma and of VLDL increased linearly for lo-12 h. Monkeys were fasted for 12 h prior to withdrawal of the zero blood samples from the femoral vein into syringes treated with EDTA and injection of Triton WR-1339 (100 mg/ml of 0.9% NaCI) into the popliteal vein. Blood samples were then drawn at 1, 3, 4 and 6 h after injection. In some experiments, the effects on plasma lipids and lipoproteins of feeding a test meal were determined. The test meal was prepared by suspending with a Virtis homogenizer 12 g of the habitual semipurified diet in 40 ml of H20. Immediately after the 3 h
TAqLE THE
RATES
THE The to
I OF
INJECTION monkeys
between
of
the 9
TRITON
300
fed
in
the
Diet
The
In Group as mean
vs.
significant
fat also Group
1.21. The level of “‘1 in plasma lipids did not change abruptly. These in vivo studies tend to confirm mechanisms b and c above. These effects on HDL indicate that one must be cautious when assuming that Triton WR-1339 is a simple inhibitor of the plasma enzymes lipoprotein lipase and lecithin cholesterol acyltransferase. This assumption was, of course, the basis for the use of the detergent to study VLDL synthesis. Acknowledgements This work was supported by a grant-in-aid from the National Institutes of Health, Bethesda, Md. (HL 09744). This work is publication No. 889 from the Oregon Regional Primate Research Center supported in part by grant No. RR00163. References 1
Friedman.
2
Schotz,
3
Scanu,
M. and M.C.,
A.
and
4
Otway,
5
Fiser,
S. and
6
Illingworth,
Jr..
7
Illingworth, Portman,
9 10
Osuga.
S.O.
A.
Oriente.
D.S.
D.R.,
Whipple,
D.R.,
Portman.
T. and
Page,
O.W.
O.W.
O.W.
and
Alexander,
13
Lowry,
O.H..
14
Dulley.
J.R.
15
Portman,
16
Fairbanks,
R.J.,
Eder.
H.A.
and
and
O.W., G.,
17
Bartlett,
18
Scanu,
G.R.
19
Eisenberg,
20
Illingworth,
D.R.
and
21
Illingworth,
D.R.
(1975)
22
Portman,
23
Shore,
24
Sigurdsson.
A.M.
(1959) (1965)
S. and
O.W., V.G.,
G.,
R.I.
and
D.R. and
(1974)
(1967)
122-133
10,
158-165
Biophys.
Randall.
Biochem.
D.F.H.
Acta
Invest. R.J.
64,
Biophys.
223-226
325-334 Acta
369,
304-314
260.460474
34.
1345-1353
(1951)
J. Biol.
Chem.
193,
265-275
136-141
P. (1976)
(1971)
104,
22.
91.3546
63.
J. Clin.
J. Nutr.
Biochim. J. Nutr.
Res.
N. and Soltys,
Adv.
O.W.
Hart,
L.E.
(1974)
Atherosclerosis
Biochim.
Biochemistry
10,
Biophys.
Acta
450,
185-196
2606-2616
466468
3, 63-138
(1975)
Biochim.
A.
A.L.
234,
Res.
Portman,
B. and
Nicoll,
(1955)
Anal.
Chem.
Lipid
Illingworth.
Shore.
(1975)
J. Biol.
(1975)
Biochim.
J.P.
399402
321-332
W.R.
O.W.
J. Lipid
and Wallach,
Adv. Levy,
Farr,
M., Tanaka,
T.L.
190,
Beisel,
Gastroenterology (1969)
188,
735-757
Lond.
C.A.
M. (1972)
P.A.
Alexander, Steck.
M.
J. Physiol.
and
Portman,
(1972)
N.J.
Grieve,
R.B.
Maruffo,
97,117-130
113,
J. Physiol.
Bragdon,
Rosebrough.
Med.
and Whipple,
M. and
Alexander,
Portman.
(1967) and
and
Havel,
J. Exp.
L.E.
O.W.
11
Am.
Rindsig.
Portman,
12
Med.
(1957)
J.C.,
Alexander, Portman,
J. Exp.
I.H.
P. (1961)
Denniston.
O.W.,
(1953)
and
Robinson,
R.H.,
8
Byers,
Scanu,
Lipid
(1973)
Biophys. and R.G.
Lewis,
Res.
13,
l-89
Circulation Acta
Alexander, (1974) B. (1975)
48,
IV-111
388.38-51 M. (1975)
Biochemistry J. Clin.
Biochim.
Biophys.
13,1579-1585
Invest.
56,
1481-1490
Acta
398.
55-71
Biochimica et Biophysics @ Elsevier/North-Holland
Acta, 486 (1977) 470-482 Biomedical Press
BBA 56955
TRIA~YL~LYCEROL AND VERY LOW DENSITY LIPOPROTEIN SECRETION INTO PLASMA OF SQUIRREL MONKEYS
OSCAR W. PORT~AN, ~ANFRED D. ROGER ILLINGWORTH
ALEXANDER,
NAOMI TANAKA
and
Department of Nutrition and Metabolic Diseases, Oregon Regional Primate Research Center, Beaverton, Oreg. 97005 and Department of Biochemistry, University of Oregon Health Science Center, Portland, Oreg. 97201 (U.S.A.) (Received
October
8th, 1976)
Summary We determined the effects of varying the types and level of dietary fat and cholesterol on the increase in plasma total triacylglycerol concentrations after injection of Triton WR-1339, an inhibitor of lipoprotein lipase, into monkeys that had been subjected to an overnight fast. The monkeys that had been treated with Triton WR-1339 were then given a test meal by intragastric intubation. Dietary cholesterol, high levels of fat and saturated fat in the habitual diet reduced the rate of release of triacylglycerol to plasma in the fasted monkey. We also determined the changes in protein and lipid concentrations of the different lipoprotein fractions. The injection of Triton WR-1339 resulted in a linear increase with time in the concentration of protein and triacylglycerol in the very low density (chylomicron-free and d < 1.006) lipoproteins, but there was an increase in the ratio of triacylglycerol to protein in that fraction. Most of the increase (96%) in very low density protein was in the B protein. Regardless of the habitual diet, a test meal accentuated the rate of triacylglycerol appearance in whole plasma and in the very low density lipoproteins of Triton WR-1339-treated monkeys, and the rate of increase of the protein component after feeding was slightly higher. Thus the administration of a meal to the fasted, Triton WR-13394reated squirrel monkey further increased the proportion of triacylglycerol in very low density lipoproteins. Although dietary cholesterol and saturated fat in the habitual diet depressed the rate of increase in very low density triacylgly~erol during fasting, the rate -Abbreviations: VLDL, very low density lipoproteins (d < 1.006, but free of chylomicrons): IDL, intermediate density lipoproteins (d 1.006-1.019); LDL, low density lipoproteins (d l.OlS1.063); HDL, high density lipoproteins (d 1.063-1.21). The B and C apoproteins of Vera low density lipoproteins are designated VLDL-B and VLDL-C.
471
of protein synthesis was not significantly affected. After administration of a test meal the rates of increase in triacylglycerol and protein in the very low density lipopro~ins were similar for monkeys from the different diet groups. Triton WR-1339 administration caused a slight and progressive increase in the intermediate density (d 1.006-1.019) lipoproteins and a marked and progressive decrease in the low density (d 1.019-1.063) lipoproteins, There was an immediate (by 5 min) drop of 70% or more in high density (d 1.063-1.21) lipoprotein protein, but the lipids except triacylglycerol remained unchanged. There was a decrease in both the A (the major fraction) and C proteins. The rates of very low density B protein secretion were comparable to the rates of low density lipoprotein catabolism that had been previously demonstrated for this species.
Introduction When Triton WR-1339, a nonionic detergent, is injected into the bloodstream of various animals, the removal of triacylglycerol from the plasma is almost completely blocked [l-3]. This reagent, therefore, provides a way of accurately determining the rate of secretion of triacylglycerol and its carrier lipoproteins into plasma [ 4-6 1. We previously showed that after the injection of 300 mg of Triton WR-1339 per kg of body weight into fasted squirrel monkeys there was a linear increase for 12 h in the concentration of all lipids in VLDL [7]. This was followed by increases in the concentrations of lipids in the IDL and LDL that were consistent with a precursor product relationship. Further studies [6] demonstra~d the rates of secretion of triacylglycerol to be reduced in fasted monkeys that had elevated LDL levels (as a result *of the chronic ingestion of diets containing cholesterol and high levels of butter). In the previous study [6] we only compared monkeys on two diets, and those diets were different in several respects. The first objective of the present study was to determine the relative importance of the type and level of fat and the level of cholesterol in the habitual diet on the secretion rates of triacylglycerol in both fasted monkeys and those given a test meal after the infusion of Triton WR-1339. The second objective was to measure the quantitative changes of the major constituents, particularly the protein and triacylglycerol, of the different lipoprotein fractions in monkeys from five of the diet groups. We were particularly interested in determining whether the increases in VLDL lipids were associated with proportional increases in the protein moieties. We could thus decide whether hypercholesterolemia induced by cholesterol feeding depressed the secretion of VLDL by the liver or only modified their composition .
Methods Experimental animals and diets Forty-four different Brazilian squirrel monkeys (Saimiri sciureus), 25 males and 19 females, were used for 88 different tests of triacylglycerol and lipopro-
472
tein metabolism after the injection of Triton WR-1339. All monkeys were collected at Leticia, Colombia, and were obtained from the Tarpon Zoo, Tarpon Springs, Fla. They were fed semipurified diets similar to those described previously [8,9]. The basal diet contained (w/w) 21.2% casein, 38.5% sucrose, 20.2% fat, 3.4% salt mix, 1.7% vitamin mix, and 15% synthetic fiber. Variations in the diet compositon are indicated on Table I. The Triton WR-1339 and feeding tests Squirrel monkeys had been on the indicated diets for three months or more when they were injected with Triton WR-1339. The dosage (300 mg/kg) and the times after Triton WR-1339 injection for blood samples were selected on the basis of a previous study [6]. Those earlier experiments had shown that after a brief lag period the triacylglycerol of total plasma and of VLDL increased linearly for lo-12 h. Monkeys were fasted for 12 h prior to withdrawal of the zero blood samples from the femoral vein into syringes treated with EDTA and injection of Triton WR-1339 (100 mg/ml of 0.9% NaCI) into the popliteal vein. Blood samples were then drawn at 1, 3, 4 and 6 h after injection. In some experiments, the effects on plasma lipids and lipoproteins of feeding a test meal were determined. The test meal was prepared by suspending with a Virtis homogenizer 12 g of the habitual semipurified diet in 40 ml of H20. Immediately after the 3 h
TAqLE THE
RATES
THE The to
I OF
INJECTION monkeys
between
of
the 9
TRITON
300
fed
in
the
Diet
The
In Group as mean
vs.
significant
fat also Group
1.21. The level of “‘1 in plasma lipids did not change abruptly. These in vivo studies tend to confirm mechanisms b and c above. These effects on HDL indicate that one must be cautious when assuming that Triton WR-1339 is a simple inhibitor of the plasma enzymes lipoprotein lipase and lecithin cholesterol acyltransferase. This assumption was, of course, the basis for the use of the detergent to study VLDL synthesis. Acknowledgements This work was supported by a grant-in-aid from the National Institutes of Health, Bethesda, Md. (HL 09744). This work is publication No. 889 from the Oregon Regional Primate Research Center supported in part by grant No. RR00163. References 1
Friedman.
2
Schotz,
3
Scanu,
M. and M.C.,
A.
and
4
Otway,
5
Fiser,
S. and
6
Illingworth,
Jr..
7
Illingworth, Portman,
9 10
Osuga.
S.O.
A.
Oriente.
D.S.
D.R.,
Whipple,
D.R.,
Portman.
T. and
Page,
O.W.
O.W.
O.W.
and
Alexander,
13
Lowry,
O.H..
14
Dulley.
J.R.
15
Portman,
16
Fairbanks,
R.J.,
Eder.
H.A.
and
and
O.W., G.,
17
Bartlett,
18
Scanu,
G.R.
19
Eisenberg,
20
Illingworth,
D.R.
and
21
Illingworth,
D.R.
(1975)
22
Portman,
23
Shore,
24
Sigurdsson.
A.M.
(1959) (1965)
S. and
O.W., V.G.,
G.,
R.I.
and
D.R. and
(1974)
(1967)
122-133
10,
158-165
Biophys.
Randall.
Biochem.
D.F.H.
Acta
Invest. R.J.
64,
Biophys.
223-226
325-334 Acta
369,
304-314
260.460474
34.
1345-1353
(1951)
J. Biol.
Chem.
193,
265-275
136-141
P. (1976)
(1971)
104,
22.
91.3546
63.
J. Clin.
J. Nutr.
Biochim. J. Nutr.
Res.
N. and Soltys,
Adv.
O.W.
Hart,
L.E.
(1974)
Atherosclerosis
Biochim.
Biochemistry
10,
Biophys.
Acta
450,
185-196
2606-2616
466468
3, 63-138
(1975)
Biochim.
A.
A.L.
234,
Res.
Portman,
B. and
Nicoll,
(1955)
Anal.
Chem.
Lipid
Illingworth.
Shore.
(1975)
J. Biol.
(1975)
Biochim.
J.P.
399402
321-332
W.R.
O.W.
J. Lipid
and Wallach,
Adv. Levy,
Farr,
M., Tanaka,
T.L.
190,
Beisel,
Gastroenterology (1969)
188,
735-757
Lond.
C.A.
M. (1972)
P.A.
Alexander, Steck.
M.
J. Physiol.
and
Portman,
(1972)
N.J.
Grieve,
R.B.
Maruffo,
97,117-130
113,
J. Physiol.
Bragdon,
Rosebrough.
Med.
and Whipple,
M. and
Alexander,
Portman.
(1967) and
and
Havel,
J. Exp.
L.E.
O.W.
11
Am.
Rindsig.
Portman,
12
Med.
(1957)
J.C.,
Alexander, Portman,
J. Exp.
I.H.
P. (1961)
Denniston.
O.W.,
(1953)
and
Robinson,
R.H.,
8
Byers,
Scanu,
Lipid
(1973)
Biophys. and R.G.
Lewis,
Res.
13,
l-89
Circulation Acta
Alexander, (1974) B. (1975)
48,
IV-111
388.38-51 M. (1975)
Biochemistry J. Clin.
Biochim.
Biophys.
13,1579-1585
Invest.
56,
1481-1490
Acta
398.
55-71
