Increased
Receptor Consuming
Binding of Low-Density Lipoprotein From Individuals a High-carbohydrate, Low-Saturated-Fat Diet
Hitoshi Hara, William G.H. Abbott, Lidia Patti, Giacomo Ruotolo, Boyd A. Swinburn, Rose M. Fields, Shinkuro Kataoka, and Barbara V. Howard The substitution of saturated fat by complex carbohydrate, according to current dietary recommendations, results in a decrease of plasma and low-density lipoprotein (LDL) cholesterol levels. To determine whether this decrease might result from structural and thus functional changes in LDL particles, the binding internalization and degradation of 1251-LDLwere measured using TR715-19 cells, a mutant CHO line into which has been transfected the human LDL receptor, and in which measurements of binding are highly reproducible. Eleven nondiabetic subjects (35 + 4 years, 27% 2 3% body fat) were studied after they had completed 3 to 5 weeks on each of two isocaloric diets, with one diet containing 42% fat (21% saturated) 43% carbohydrate, 15% protein, and 560 mg cholesterol/d and the other containing 21% fat (6% saturated), 65% carbohydrate, 14% protein, and 524 mg cholesterol/d. LDL cholesterol levels decreased from 125 f 6 to 106 f 5 mg/dL (P < .Ol) on the high-carbohydrate diet. There was an increase in the binding affinity of LDL (K,,, 6.6 -C 2.6~7.3 2 2.7 cg/mL f SD; P < .02), and internalization (P < .lO) and degradation (P c .05) were also higher. The data suggest that decreasing dietary saturated fat may cause alterations in LDL composition that result in increased receptor clearance; this may partially explain the LDL-decreasing effect of this dietary change. Copyright 0 1992 by W.B. Saunders Company
S
EVERAL LINES OF EVIDENCE1-6 have established that diets in which complex carbohydrate is substituted for saturated fat lead to decreased concentrations of plasma low-density lipoprotein (LDL), and this dietary alteration is now the cornerstone of the recommendations of many health-oriented groups, including the National Cholesterol Education Program,’ The American Heart Associatioqa and The American Diabetes Association.9 Although there is considerable evidence that this dietary manipulation leads to decreased LDL levels, there is still uncertainty concerning which aspect of this dietary manipulation (decrease in saturated fat, increase in carbohydrate, or increase in fiber) and what metabolic mechanisms are responsible for the decreased LDL levels. LDL concentrations are controlled primarily by the activity of the LDL receptor, which is present in the liver and peripheral cells and initiates the removal of LDL from plasma.*O One possible mechanism suggested by animal studies is that a decrease in dietary saturated fat content leads to an increase in receptor activity. Shepherd et al” showed that reducing the content of dietary saturated fat leads to an increased clearance rate of LDL in normal From the Clinical Diabetes and Nutrition Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Phoenix, AZ; and Medlantic Research Foundation, Washington, DC. Supported in part by a grant from the Arizona Disease Control Commission. Present addresses: H.H., Second Department of Internal Medicine, Hiroshima University School of Medicine, l-2-3 Kasumi, Minami-Ku Hiroshima, 734 Japan; WG.H.A., Center for Human Nutrition, University of Texas, Health Science Center, 5323 Harry Hines Blvd, Dallas, TX 75235; L.P., Universita Di Napoli-II, Facolta Di Medicina E Chirurgia, Istituto Di Medicina Intema E, Malattie Dismetaboliche, 80131 Napoli, Italy. Address reprint requests to Barbara K Howard, PhD, Medlantic Research Institute, George Hyman Memorial Research Building, Room 242, 108 Irving St NW Washington, DC 20010. Copyright 0 1992 by W.B. Saunders Company 00260495/92/41111-0002$03.00/O 1154
humans, and studie@ have suggested that there is increased direct removal of very-low-density lipoprotein (VLDL) and increased clearance of LDL in both diabetics and nondiabetics consuming high-carbohydrate diets. However, it is likely that the LDL-decreasing effect of these diets is more complex, and may involve mechanisms in addition to effects on receptor activity. Saturated fat may influence cholesterol absorption, or it may decrease the synthesis of LDL through a reduction in hepatic output of apolipoprotein (apo) B-containing particles. It is also possible that alterations in LDL composition induced by changes in diet may affect its rate of clearance independent of the amount of receptor present. Some evidence already exists indicating that diets high in saturated fat are associated with altered LDL composition; three types of alterations have been described. One is in the fatty acid distribution of triglycerides and phospholipids.13-16Another change in LDL composition induced by a high-saturated-fat diet is a decrease in triglyceride levels.17J*A third possibility is that the ratio of lipid to protein and thus the density of LDL may change with dietary manipulation”J6 However, detailed chemical analysis of LDL structure does not indicate whether differences in structure are reflected in changes in function. B/E receptor function has been studied extensively in vitro ever since the advent of the classic work of Goldstein and Brown19 demonstrating the presence of LDL receptors on cultured fibroblasts. This system has been used to detect altered LDL in only a few situations: uncontrolled diabetes,2n after extensive glycosylation,z’ and in a family with defective apo B-100.22 There have also been some suggestions that dietary manipulation may result in LDL with altered binding properties. LDL from dietary-induced hypercholesterolemic primatesz3 show altered binding in vitro, and Baudet et alI6 showed that a pool of LDL from six subjects consuming a diet high in dairy products for 3 months had decreased LDL metabolism in cultured fibroblasts. The assessment of altered LDL function in vitro has been limited to date, because of the relative imprecision19x24 of Metabolism, Vol41, No 11 (November), 1992: pp 1154-1160
1155
DIET AND LDL BINDING
the binding assay when primary cultures such as fibroblasts and smooth muscle cells are used. We have recently developed a sensitive and reproducible assay systemzs for the in-vitro evaluation of LDL binding, using a mutant CHO cell line26 into which the human LDL receptor has been transfected.27.2x The system detects differences in binding between subfractions of a normal pool of LDL isolated by density-gradient centrifugation.25 The present study presents data using this TR715-19 cell system to evaluate LDL binding in 11 individuals who underwent a diet regimen in which complex carbohydrate was substituted for saturated fat in the diet. MATERIALS
AND
Nutrients
Carbohydrate (%) Total (g)
After giving informed consent, each subject was hospitalized on the research metabolic ward of the Clinical Diabetes and Nutrition Section for the duration of the study. Subjects were fed two different diets in random order (Table 2). In some cases, they were fed during a single admission, but some patients were discharged after completion of one dietary period and then readmitted for the second diet. During both dietary periods, caloric intake was estimated from body weight and adjusted on the basis of fasting posturination weight measured each morning. The high-saturatedfat diet, of which a large proportion was derived from dairy products, was fed for at least 3 weeks. The high-carbohydrate, low-fat diet. in which complex carbohydrate was substituted for saturated fat, was fed for at least 5 weeks. We had established previously that these feeding periods resulted in stable LDL concentrations in this population.6 Both diets consisted of natural foods with recipes geared for the dietary preferences of Southwestern Indians. Calories were distributed as 20% breakfast, 30% lunch. 30% dinner, and 20% evening snack. A set of seven daily menus was prepared for each diet and used on a rotating basis. Diet composition was calculated from the sum of all ingredients in all of the menus, using US Department of Agriculture hand-
Table 1. Age, Weight, and Body Composition of Subjects High-Fat
High-Carbohydrate
912
(M/F)
Age (vr)
32.5 2 12.4
‘Neight (kg)
91.0 + 25.5
90.9 + 25.5
(64-139)
(64-135)
(22-57)
Body mass index (weight [kgl/height [cm]*) Percent fat’
31.3 ~fr8.6
31.1 + 8.4
(20.6-46.3)
(20.4 + 46.1)
27*
10
(13-42)
272
11
(13-45)
NOTE. Values are the mean 2 SD with the range in parentheses. by underwater weighing.
High-Fat
High-Carbohydrate
43
65
322
498
Simple (g)
102
148
220
350
42
21
6
6
Fat 1%) Polyunsaturated Monounsaturated
12
8
Saturated
21
6
Protein (%)
15
14
Fiber(g)
Legume
Experimentul Design
Diets
Complex (g)
Vegetable
Ten Southwestern American Indians (eight men, two women) and one white male were studied (Table 1). Subjects were in excellent health as indicated by a full medical history and physical examination; routine hematology, biochemistry, and urinalyses were normal. Two subjects had impaired glucose tolerance, and all others had normal glucose tolerance as diagnosed by World Health Organization Criteria; none were receiving medication at the time of study.
*Measured
Carbohydrate
Cereal
METHODS
Subjects
Sex
Table 2. Nutrient Composition of High-Fat and High-
Cholesterol (mg)
8
12
12
22
9
23
560
524
NOTE. Weights are for a 3,000-calorie diet.
books.2y The increase in dietary complex carbohydrate content resulted in some increase in predominantly vegetable fiber and a change in the ratio of animal protein to vegetable protein. However, cholesterol content was similar between the two diets. At the end of each dietary period, 20 mL blood was drawn on 2 different days to measure fasting lipoprotein concentrations, and 400 mL blood was obtained by plasmapheresis for isolation of LDL. Lipoprotein Concentrations Venous blood samples were collected in EDTA after subjects had fasted overnight. Plasma was separated by centrifugation at approximately 700 x g for 15 minutes at 10°C. A sample was removed for measurement of total plasma cholesterol and triglyceride levels. Lipoproteins were then isolated by ultracentrifugation.‘O Briefly, 5 mL plasma was overlaid with 2 mL 0.15 mol/L NaCl and 1 mmol/L EDTA (EDTA-saline), d = 1.006 g/mL, and VLDL was isolated by ultracentrifugation for 16 hours at 40.000 x g in a Beckman Ultracentrifuge with a type 40 rotor (Beckman, Fullerton, CA). After VLDL was removed, the plasma was adjusted to d = 1.063 g/mL with NaBr (d = 1.35 g/mL), and LDL was isolated by ultracentrifugation for 20 hours at 40,000 x x in a type 40.3 rotor. The high-density lipoprotein (HDL) fraction was the infranatant after removal of LDL by tube slicing. Recovery of cholesterol in lipoprotein fractions averaged 94%. We have reported previously30 that LDL concentrations determined by this method were comparable to those computed by the difference between LDL plus HDL and HDL precipitated by heparin manganese. Isolation of LDL for Binding Assay All procedures were performed under sterile conditions. LDL (d = 1.019 to 1.063) was isolated as previously described.” and lipoproteins were sterilized and stored at 4°C until use. Pooled LDL was isolated from five healthy, normolipidemic volunteers, and frozen aliquots of the pool were used as radiotracer and for assay controls. We have previously shown that LDL prepared and stored in this manner is indistinguishable from a fresh preparation in the same assay system.‘5 For radiotracer, LDL was iodinated by a modification of the iodine monochloride method of McFarlane,” as described by Bilheimer et a1.j” The specific radioactivities were 420 to 580 cpming LDL protein, and more than 97% of the radioactivity was precipitable by incubation with 10% trichloroacetic acid (TCA) at 4°C. LDL from each patient was isolated with the same method,
1156
HARA ET AL
within 5 days of obtaining the plasma sample. To assess binding of each LDL sample, a competition assay was performed using 1251-LDLas tracer and LDL from the patient at concentrations of 1 to 59 ug/mL. Nonspecific binding was determined using 500 ug/mL of a control pool.
Other Assays Protein concentrations in cells and lipoprotein fractions were determined as described by Markwell et al.34 Lipoprotein cholesterol level was measured by the solvent extraction method of Rush et al.35 and triglyceride level was determined by the enzymatic method of Bucolo and David.36 Phospholipids in LDL subfractions were measured using the method of Fiske and Subbarow.37 LDL Subfractions Six subfractions from LDL with d = 1.019 to 1.063 were isolated by a modification of the density-gradient ultracentrifugation procedure described by Shen et aI:* as reported previously.25 This method consisted of isolating six subfractions of increasing density after a 40-hour centrifugation on a discontinuous gradient. RESULTS
high-carbohydrate in total plasma,
diet
was
associated
High-Carbohydrate High-Fat
VLDL cholesterol
TR715-19, a mutant CHO cell line that expresses a transfected human LDL receptor, was used in this study. The line was maintained in culture medium (Ham’s F-12 nutrient mixture supplemented with 20 mmol/L HEPES, PH 7.4, and 2 mmol/L glutamine), 20 umol/L mevinolin, 200 ~mol/L mevalonate, 4% (vol/vol) newborn-calf, lipoprotein-deficient serum and 1% (vol/ vol) fetal calf serum. For binding experiments, cells were seeded at a concentration of 4 to 6 x lo4 cells/dish into petri dishes (60 x 15 mm) containing culture medium supplemented with 10% fetal calf serum. On day 2, cells were refed with medium of identical composition. On day 3. cells received culture medium supplemented with 5% newborn-calf, lipoprotein-deficient serum, 0.1 ug/mL 25-hydroxycholesterol, and 10 ug/mL cholesterol to suppress expression of the endogenous, defective hamster LDL receptors. On day 4, cells were transferred to assay medium (Eagle’s Minimum Essential Medium, 10 mmol/L HEPES, pH 7.4, 24 mmol/L bicarbonate, 1% nonessential amino acids, and 10% human lipoprotein-deficient serum). The assay medium contained 1 ug/mL WLDLprotein and 0, 1.5,3.0,6.0,9.0, 19.0,39.0, or 59.0 ug/mL nonlabeled LDL protein from each subject in triplicate or duplicate dishes. Dishes were incubated at 37°C in a CO2 incubator for 2 hours, a duration previously found to be optimal to assess binding in this system. 25The binding degradation and internalization assays were performed essentially as described by Goldstein et al,24 with modifications as described previously.25 To determine nonspecific binding, 500 ug/mL nonlabeled LDL protein was added to the assay mixture. Apparent dissociation constants (&) were computed from Scatchard analysis33 of the binding data using a linear regression method. Details of the TR715-19 cell line binding assay have been described previously.25
with
de-
LDL, and HDL cholesterol levels 3). There were no significant changes in total, VLDL, or LDL triglyceride concentrations. The ratio of triglyceride to cholesterol in LDL (d = 1.006 to 1.063) was significantly higher when individuals were consuming the high-carbohydrate diet. The composition of LDL (d = 1.019 to 1.063) was as-
of
Plasma and Lipoprotein Fractions on High-Fat and
Plasma Cholesterol
Determination of LDL Binding, Internalization, and Degradation
The creases (Table
Table 3. Mean Cholesterol and Triglyceride Concentrations
LDL
cholesterol
High-Carbohydrate
(N = 11)
(N = 11)
184 2 22
164 + 23t
(156-235)
(135-205)
19 -‘8
20 2 7
(8-37)
(1 l-36)
125 +- 20
108 -c 18t
(100-1671 HDL cholesterol
Diets
39 k 9 (27-57)
(81-144) 34 it_6 (25-46)t
Plasma triglyceride
138 + 53 (67-213)
(70-232)
VLDL triglyceride
88 + 42
97 + 50
LDL triglyceride
462
(22-72)
(26-77)
LDL triglyceride/
.37 -c .12
.45 k .I8
cholesterol
(.16-.60)
(.24-.88)*
(33-173) 16
141 + 53
(36-179) 46 2 18
NOTE. Values are the mean % SD with the range in parentheses *P < .05 by paired tP < .Ol by paired
t test. t test.
sessed by measuring phospholipid, triglyceride, and cholesterol levels in each of six subfractions isolated by densitygradient ultracentrifugation (Fig 1, Table 4). In subjects consuming a low-saturated-fat, high-carbohydrate diet, there was a slight shift toward more dense LDL, as indicated by significantly higher amounts of subfraction no. 4 and significantly lower amounts of fractions no. 1 and 2 (Table 4). When lipid contents were expressed per milligram LDL protein (greater than 95% of the protein was shown to be apo B), 39 the proportion of triglycerides was significantly higher in fractions no. 1, 2, and 3, and appeared to be higher in fraction no. 6 (Fig 1) on the high-carbohydrate diet. There was also an enrichment of cholesterol in the combined fraction no. 1 + 2 (Fig 1). Phospholipid levels tended to be higher in the lighter subfractions (Fig l), but differences were not significant. When fatty acid composition was measured in a subset of LDL preparations, there was a decreased proportion of saturated fatty acids in the high-carbohydrate diet (data not shown). Apparent dissociation constants were computed for binding, degradation, and internalization of LDL isolated from each individual on the two diets (Fig 2). (A typical binding curve for an individual on both diets is shown in Fig 3.) Binding affinity was significantly increased on the lowsaturated-fat, high-carbohydrate diet, and the changes in LDL cholesterol and in the Kd for binding were correlated (r = .5, P < .OS). Only two of the 11 individuals showed increases in K& and one of these was the only subject who
had increased LDL cholesterol concentrations on the high-carbohydrate diet. Eight of the 11 individuals also showed decreases in degradation and internalization, although the mean change in internalization did not reach statistical significance.
1157
DIET AND LDL BINDING
I
.;
Cholesterol
concentration
Inps.02
p.05 -
D’.IO n
200
5
l-
L
150
z::
100
\ g
50
._F g5
._
I
23456
I+2
Triqlyceride
,-
m
concentration
1 HF
1 Phospholipid
I
2
LDL
4
5
subfraction
1+2
6
HF
HC
I
l-
I
L HF
HC
HC
Fig 2. Binding, internalization, and degradation of ‘WLDL to TR715-19 cells. Individual data are shown for 11 subjects who consumed a high-fat (HF) and high-carbohydrate (HC) diet; paired t test was used.
concentration
3
Receptor Consuming
Binding of Low-Density Lipoprotein From Individuals a High-carbohydrate, Low-Saturated-Fat Diet
Hitoshi Hara, William G.H. Abbott, Lidia Patti, Giacomo Ruotolo, Boyd A. Swinburn, Rose M. Fields, Shinkuro Kataoka, and Barbara V. Howard The substitution of saturated fat by complex carbohydrate, according to current dietary recommendations, results in a decrease of plasma and low-density lipoprotein (LDL) cholesterol levels. To determine whether this decrease might result from structural and thus functional changes in LDL particles, the binding internalization and degradation of 1251-LDLwere measured using TR715-19 cells, a mutant CHO line into which has been transfected the human LDL receptor, and in which measurements of binding are highly reproducible. Eleven nondiabetic subjects (35 + 4 years, 27% 2 3% body fat) were studied after they had completed 3 to 5 weeks on each of two isocaloric diets, with one diet containing 42% fat (21% saturated) 43% carbohydrate, 15% protein, and 560 mg cholesterol/d and the other containing 21% fat (6% saturated), 65% carbohydrate, 14% protein, and 524 mg cholesterol/d. LDL cholesterol levels decreased from 125 f 6 to 106 f 5 mg/dL (P < .Ol) on the high-carbohydrate diet. There was an increase in the binding affinity of LDL (K,,, 6.6 -C 2.6~7.3 2 2.7 cg/mL f SD; P < .02), and internalization (P < .lO) and degradation (P c .05) were also higher. The data suggest that decreasing dietary saturated fat may cause alterations in LDL composition that result in increased receptor clearance; this may partially explain the LDL-decreasing effect of this dietary change. Copyright 0 1992 by W.B. Saunders Company
S
EVERAL LINES OF EVIDENCE1-6 have established that diets in which complex carbohydrate is substituted for saturated fat lead to decreased concentrations of plasma low-density lipoprotein (LDL), and this dietary alteration is now the cornerstone of the recommendations of many health-oriented groups, including the National Cholesterol Education Program,’ The American Heart Associatioqa and The American Diabetes Association.9 Although there is considerable evidence that this dietary manipulation leads to decreased LDL levels, there is still uncertainty concerning which aspect of this dietary manipulation (decrease in saturated fat, increase in carbohydrate, or increase in fiber) and what metabolic mechanisms are responsible for the decreased LDL levels. LDL concentrations are controlled primarily by the activity of the LDL receptor, which is present in the liver and peripheral cells and initiates the removal of LDL from plasma.*O One possible mechanism suggested by animal studies is that a decrease in dietary saturated fat content leads to an increase in receptor activity. Shepherd et al” showed that reducing the content of dietary saturated fat leads to an increased clearance rate of LDL in normal From the Clinical Diabetes and Nutrition Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Phoenix, AZ; and Medlantic Research Foundation, Washington, DC. Supported in part by a grant from the Arizona Disease Control Commission. Present addresses: H.H., Second Department of Internal Medicine, Hiroshima University School of Medicine, l-2-3 Kasumi, Minami-Ku Hiroshima, 734 Japan; WG.H.A., Center for Human Nutrition, University of Texas, Health Science Center, 5323 Harry Hines Blvd, Dallas, TX 75235; L.P., Universita Di Napoli-II, Facolta Di Medicina E Chirurgia, Istituto Di Medicina Intema E, Malattie Dismetaboliche, 80131 Napoli, Italy. Address reprint requests to Barbara K Howard, PhD, Medlantic Research Institute, George Hyman Memorial Research Building, Room 242, 108 Irving St NW Washington, DC 20010. Copyright 0 1992 by W.B. Saunders Company 00260495/92/41111-0002$03.00/O 1154
humans, and studie@ have suggested that there is increased direct removal of very-low-density lipoprotein (VLDL) and increased clearance of LDL in both diabetics and nondiabetics consuming high-carbohydrate diets. However, it is likely that the LDL-decreasing effect of these diets is more complex, and may involve mechanisms in addition to effects on receptor activity. Saturated fat may influence cholesterol absorption, or it may decrease the synthesis of LDL through a reduction in hepatic output of apolipoprotein (apo) B-containing particles. It is also possible that alterations in LDL composition induced by changes in diet may affect its rate of clearance independent of the amount of receptor present. Some evidence already exists indicating that diets high in saturated fat are associated with altered LDL composition; three types of alterations have been described. One is in the fatty acid distribution of triglycerides and phospholipids.13-16Another change in LDL composition induced by a high-saturated-fat diet is a decrease in triglyceride levels.17J*A third possibility is that the ratio of lipid to protein and thus the density of LDL may change with dietary manipulation”J6 However, detailed chemical analysis of LDL structure does not indicate whether differences in structure are reflected in changes in function. B/E receptor function has been studied extensively in vitro ever since the advent of the classic work of Goldstein and Brown19 demonstrating the presence of LDL receptors on cultured fibroblasts. This system has been used to detect altered LDL in only a few situations: uncontrolled diabetes,2n after extensive glycosylation,z’ and in a family with defective apo B-100.22 There have also been some suggestions that dietary manipulation may result in LDL with altered binding properties. LDL from dietary-induced hypercholesterolemic primatesz3 show altered binding in vitro, and Baudet et alI6 showed that a pool of LDL from six subjects consuming a diet high in dairy products for 3 months had decreased LDL metabolism in cultured fibroblasts. The assessment of altered LDL function in vitro has been limited to date, because of the relative imprecision19x24 of Metabolism, Vol41, No 11 (November), 1992: pp 1154-1160
1155
DIET AND LDL BINDING
the binding assay when primary cultures such as fibroblasts and smooth muscle cells are used. We have recently developed a sensitive and reproducible assay systemzs for the in-vitro evaluation of LDL binding, using a mutant CHO cell line26 into which the human LDL receptor has been transfected.27.2x The system detects differences in binding between subfractions of a normal pool of LDL isolated by density-gradient centrifugation.25 The present study presents data using this TR715-19 cell system to evaluate LDL binding in 11 individuals who underwent a diet regimen in which complex carbohydrate was substituted for saturated fat in the diet. MATERIALS
AND
Nutrients
Carbohydrate (%) Total (g)
After giving informed consent, each subject was hospitalized on the research metabolic ward of the Clinical Diabetes and Nutrition Section for the duration of the study. Subjects were fed two different diets in random order (Table 2). In some cases, they were fed during a single admission, but some patients were discharged after completion of one dietary period and then readmitted for the second diet. During both dietary periods, caloric intake was estimated from body weight and adjusted on the basis of fasting posturination weight measured each morning. The high-saturatedfat diet, of which a large proportion was derived from dairy products, was fed for at least 3 weeks. The high-carbohydrate, low-fat diet. in which complex carbohydrate was substituted for saturated fat, was fed for at least 5 weeks. We had established previously that these feeding periods resulted in stable LDL concentrations in this population.6 Both diets consisted of natural foods with recipes geared for the dietary preferences of Southwestern Indians. Calories were distributed as 20% breakfast, 30% lunch. 30% dinner, and 20% evening snack. A set of seven daily menus was prepared for each diet and used on a rotating basis. Diet composition was calculated from the sum of all ingredients in all of the menus, using US Department of Agriculture hand-
Table 1. Age, Weight, and Body Composition of Subjects High-Fat
High-Carbohydrate
912
(M/F)
Age (vr)
32.5 2 12.4
‘Neight (kg)
91.0 + 25.5
90.9 + 25.5
(64-139)
(64-135)
(22-57)
Body mass index (weight [kgl/height [cm]*) Percent fat’
31.3 ~fr8.6
31.1 + 8.4
(20.6-46.3)
(20.4 + 46.1)
27*
10
(13-42)
272
11
(13-45)
NOTE. Values are the mean 2 SD with the range in parentheses. by underwater weighing.
High-Fat
High-Carbohydrate
43
65
322
498
Simple (g)
102
148
220
350
42
21
6
6
Fat 1%) Polyunsaturated Monounsaturated
12
8
Saturated
21
6
Protein (%)
15
14
Fiber(g)
Legume
Experimentul Design
Diets
Complex (g)
Vegetable
Ten Southwestern American Indians (eight men, two women) and one white male were studied (Table 1). Subjects were in excellent health as indicated by a full medical history and physical examination; routine hematology, biochemistry, and urinalyses were normal. Two subjects had impaired glucose tolerance, and all others had normal glucose tolerance as diagnosed by World Health Organization Criteria; none were receiving medication at the time of study.
*Measured
Carbohydrate
Cereal
METHODS
Subjects
Sex
Table 2. Nutrient Composition of High-Fat and High-
Cholesterol (mg)
8
12
12
22
9
23
560
524
NOTE. Weights are for a 3,000-calorie diet.
books.2y The increase in dietary complex carbohydrate content resulted in some increase in predominantly vegetable fiber and a change in the ratio of animal protein to vegetable protein. However, cholesterol content was similar between the two diets. At the end of each dietary period, 20 mL blood was drawn on 2 different days to measure fasting lipoprotein concentrations, and 400 mL blood was obtained by plasmapheresis for isolation of LDL. Lipoprotein Concentrations Venous blood samples were collected in EDTA after subjects had fasted overnight. Plasma was separated by centrifugation at approximately 700 x g for 15 minutes at 10°C. A sample was removed for measurement of total plasma cholesterol and triglyceride levels. Lipoproteins were then isolated by ultracentrifugation.‘O Briefly, 5 mL plasma was overlaid with 2 mL 0.15 mol/L NaCl and 1 mmol/L EDTA (EDTA-saline), d = 1.006 g/mL, and VLDL was isolated by ultracentrifugation for 16 hours at 40.000 x g in a Beckman Ultracentrifuge with a type 40 rotor (Beckman, Fullerton, CA). After VLDL was removed, the plasma was adjusted to d = 1.063 g/mL with NaBr (d = 1.35 g/mL), and LDL was isolated by ultracentrifugation for 20 hours at 40,000 x x in a type 40.3 rotor. The high-density lipoprotein (HDL) fraction was the infranatant after removal of LDL by tube slicing. Recovery of cholesterol in lipoprotein fractions averaged 94%. We have reported previously30 that LDL concentrations determined by this method were comparable to those computed by the difference between LDL plus HDL and HDL precipitated by heparin manganese. Isolation of LDL for Binding Assay All procedures were performed under sterile conditions. LDL (d = 1.019 to 1.063) was isolated as previously described.” and lipoproteins were sterilized and stored at 4°C until use. Pooled LDL was isolated from five healthy, normolipidemic volunteers, and frozen aliquots of the pool were used as radiotracer and for assay controls. We have previously shown that LDL prepared and stored in this manner is indistinguishable from a fresh preparation in the same assay system.‘5 For radiotracer, LDL was iodinated by a modification of the iodine monochloride method of McFarlane,” as described by Bilheimer et a1.j” The specific radioactivities were 420 to 580 cpming LDL protein, and more than 97% of the radioactivity was precipitable by incubation with 10% trichloroacetic acid (TCA) at 4°C. LDL from each patient was isolated with the same method,
1156
HARA ET AL
within 5 days of obtaining the plasma sample. To assess binding of each LDL sample, a competition assay was performed using 1251-LDLas tracer and LDL from the patient at concentrations of 1 to 59 ug/mL. Nonspecific binding was determined using 500 ug/mL of a control pool.
Other Assays Protein concentrations in cells and lipoprotein fractions were determined as described by Markwell et al.34 Lipoprotein cholesterol level was measured by the solvent extraction method of Rush et al.35 and triglyceride level was determined by the enzymatic method of Bucolo and David.36 Phospholipids in LDL subfractions were measured using the method of Fiske and Subbarow.37 LDL Subfractions Six subfractions from LDL with d = 1.019 to 1.063 were isolated by a modification of the density-gradient ultracentrifugation procedure described by Shen et aI:* as reported previously.25 This method consisted of isolating six subfractions of increasing density after a 40-hour centrifugation on a discontinuous gradient. RESULTS
high-carbohydrate in total plasma,
diet
was
associated
High-Carbohydrate High-Fat
VLDL cholesterol
TR715-19, a mutant CHO cell line that expresses a transfected human LDL receptor, was used in this study. The line was maintained in culture medium (Ham’s F-12 nutrient mixture supplemented with 20 mmol/L HEPES, PH 7.4, and 2 mmol/L glutamine), 20 umol/L mevinolin, 200 ~mol/L mevalonate, 4% (vol/vol) newborn-calf, lipoprotein-deficient serum and 1% (vol/ vol) fetal calf serum. For binding experiments, cells were seeded at a concentration of 4 to 6 x lo4 cells/dish into petri dishes (60 x 15 mm) containing culture medium supplemented with 10% fetal calf serum. On day 2, cells were refed with medium of identical composition. On day 3. cells received culture medium supplemented with 5% newborn-calf, lipoprotein-deficient serum, 0.1 ug/mL 25-hydroxycholesterol, and 10 ug/mL cholesterol to suppress expression of the endogenous, defective hamster LDL receptors. On day 4, cells were transferred to assay medium (Eagle’s Minimum Essential Medium, 10 mmol/L HEPES, pH 7.4, 24 mmol/L bicarbonate, 1% nonessential amino acids, and 10% human lipoprotein-deficient serum). The assay medium contained 1 ug/mL WLDLprotein and 0, 1.5,3.0,6.0,9.0, 19.0,39.0, or 59.0 ug/mL nonlabeled LDL protein from each subject in triplicate or duplicate dishes. Dishes were incubated at 37°C in a CO2 incubator for 2 hours, a duration previously found to be optimal to assess binding in this system. 25The binding degradation and internalization assays were performed essentially as described by Goldstein et al,24 with modifications as described previously.25 To determine nonspecific binding, 500 ug/mL nonlabeled LDL protein was added to the assay mixture. Apparent dissociation constants (&) were computed from Scatchard analysis33 of the binding data using a linear regression method. Details of the TR715-19 cell line binding assay have been described previously.25
with
de-
LDL, and HDL cholesterol levels 3). There were no significant changes in total, VLDL, or LDL triglyceride concentrations. The ratio of triglyceride to cholesterol in LDL (d = 1.006 to 1.063) was significantly higher when individuals were consuming the high-carbohydrate diet. The composition of LDL (d = 1.019 to 1.063) was as-
of
Plasma and Lipoprotein Fractions on High-Fat and
Plasma Cholesterol
Determination of LDL Binding, Internalization, and Degradation
The creases (Table
Table 3. Mean Cholesterol and Triglyceride Concentrations
LDL
cholesterol
High-Carbohydrate
(N = 11)
(N = 11)
184 2 22
164 + 23t
(156-235)
(135-205)
19 -‘8
20 2 7
(8-37)
(1 l-36)
125 +- 20
108 -c 18t
(100-1671 HDL cholesterol
Diets
39 k 9 (27-57)
(81-144) 34 it_6 (25-46)t
Plasma triglyceride
138 + 53 (67-213)
(70-232)
VLDL triglyceride
88 + 42
97 + 50
LDL triglyceride
462
(22-72)
(26-77)
LDL triglyceride/
.37 -c .12
.45 k .I8
cholesterol
(.16-.60)
(.24-.88)*
(33-173) 16
141 + 53
(36-179) 46 2 18
NOTE. Values are the mean % SD with the range in parentheses *P < .05 by paired tP < .Ol by paired
t test. t test.
sessed by measuring phospholipid, triglyceride, and cholesterol levels in each of six subfractions isolated by densitygradient ultracentrifugation (Fig 1, Table 4). In subjects consuming a low-saturated-fat, high-carbohydrate diet, there was a slight shift toward more dense LDL, as indicated by significantly higher amounts of subfraction no. 4 and significantly lower amounts of fractions no. 1 and 2 (Table 4). When lipid contents were expressed per milligram LDL protein (greater than 95% of the protein was shown to be apo B), 39 the proportion of triglycerides was significantly higher in fractions no. 1, 2, and 3, and appeared to be higher in fraction no. 6 (Fig 1) on the high-carbohydrate diet. There was also an enrichment of cholesterol in the combined fraction no. 1 + 2 (Fig 1). Phospholipid levels tended to be higher in the lighter subfractions (Fig l), but differences were not significant. When fatty acid composition was measured in a subset of LDL preparations, there was a decreased proportion of saturated fatty acids in the high-carbohydrate diet (data not shown). Apparent dissociation constants were computed for binding, degradation, and internalization of LDL isolated from each individual on the two diets (Fig 2). (A typical binding curve for an individual on both diets is shown in Fig 3.) Binding affinity was significantly increased on the lowsaturated-fat, high-carbohydrate diet, and the changes in LDL cholesterol and in the Kd for binding were correlated (r = .5, P < .OS). Only two of the 11 individuals showed increases in K& and one of these was the only subject who
had increased LDL cholesterol concentrations on the high-carbohydrate diet. Eight of the 11 individuals also showed decreases in degradation and internalization, although the mean change in internalization did not reach statistical significance.
1157
DIET AND LDL BINDING
I
.;
Cholesterol
concentration
Inps.02
p.05 -
D’.IO n
200
5
l-
L
150
z::
100
\ g
50
._F g5
._
I
23456
I+2
Triqlyceride
,-
m
concentration
1 HF
1 Phospholipid
I
2
LDL
4
5
subfraction
1+2
6
HF
HC
I
l-
I
L HF
HC
HC
Fig 2. Binding, internalization, and degradation of ‘WLDL to TR715-19 cells. Individual data are shown for 11 subjects who consumed a high-fat (HF) and high-carbohydrate (HC) diet; paired t test was used.
concentration
3
