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3
*
Minneapolis Veterans Affairs Medical Center, Minneapolis, MN 55417 and
Department of Medicine, University of Minnesota Medical School, Minneapolis MN 55455
3To whom correspondence should be addressed at Medicine Service 111M, VAMC, One Veterans Drive, Minneapolis, MN 55417.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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KEY WORDS: • monounsaturated • carbohydrate • big VLDL • diabetes • humans
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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). This holds in the presence of a normal fasting lipid
profile in the majority of the patients (Howard 1987
).
However, other abnormalities are present in type 1 diabetes; namely,
the composition of triglyceride
(TG4
)-rich lipoproteins is abnormal, with the diabetic particles being
cholesterol enriched and phospholipid poor compared with normal
particles (Georgopoulos and Rosengard 1989
). It is
possible that these changes can increase the atherogenic potential of
the diabetic particles (Georgopoulos et al. 1994
).
The hallmark of early atherosclerotic lesions is foam cell formation
through lipid accumulation in macrophages, after the uptake of
apolipoprotein (apo)B-containing lipoproteins (Bierman 1992
). In addition to LDL and lipoprotein(a), VLDL were
recently isolated from human atherosclerotic lesions (Rapp et al. 1994
).
Esterified cholesterol (EC) accumulation in macrophages is
characteristic of foam cell formation. We therefore used EC
accumulation in THP-1 macrophages incubated with VLDL subfractions as
an in vitro model of foam cell formation. THP-1 macrophages, a human
monocytic cell line, has been used by us (Georgopoulos et al. 1994
) and others (Auwerx et al. 1988
,
Hara et al. 1987
) as an in vitro model. In previous
studies, we (Georgopoulos et al. 1994
) and others
(Klein et al. 1989
) have reported that EC accumulation
by macrophages is increased in a dose-dependent manner when the
cells are incubated with increasing amounts of TG-rich lipoproteins
isolated from the plasma of patients in the fasting and postprandial
state who have type 1 diabetes versus lipoproteins isolated from age-
and sex-matched normal subjects.
Because the particle number and composition and the amount of EC accumulated in macrophages could affect the VLDL-related atherosclerotic risk in type 1 diabetes, we measured all of these variables in the present study to determine whether there is a difference between a high monounsaturated fatty acid (Mono) diet and a high carbohydrate (CHO) diet on the atherogenic potential of VLDL subfractions isolated from fasting patients with type 1 diabetes.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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We studied 19 subjects (11 men and 8 women) with type 1 diabetes
(based on accepted criteria of the American Diabetes Association).
Their clinical characteristics were as follows. the body mass index was
normal (mean ± SD 24.5 ± 2.1 kg/m2
body surface); similarly, they had a normal lipid profile and normal
liver, kidney, thyroid and hematologic variables and proteinuria of
<300 mg/24 h. Their mean age was 29 ± 8.6 y (age range
22–47 y), and their duration of diabetes was 13 ± 7.2 y
(range 2–28 y). None of the subjects were taking medications that
affect lipoprotein metabolism other than insulin and, in 2 women, oral
contraceptives. The lipoprotein measurements in all women were made at
the same time of their menstrual cycle during both dietary periods.
Patients remained on the same dose of medications other than insulin
throughout the study. None of the study participants used alcohol on a
regular basis. All participating patients signed the consent form,
which complied with the revised Helsinki Declaration and was approved
by the institutional review board. The patients maintained steady
levels of exercise throughout the study and were asked to keep records
of their blood glucose and insulin requirements. A randomized crossover
design was used, with each dietary period lasting 4 wk. All food was
prepared in the metabolic kitchen of the Minneapolis VA Clinical
Research Center and provided to the participants. A 3-d rotating menu
plan was used. Prepared extra snacks were provided for periods of
exercise or episodes of hypoglycemia. The high Mono diet contained 40%
total fatty acids (25% Mono, 6% polyunsaturated and 9% saturated),
45% CHO and 15% protein. The high CHO diet contained 24% total fatty
acids (9% Mono, 6% polyunsaturated and 9% saturated), 61% CHO and
15% protein. The polyunsaturated/saturated fatty acid ratio of both
diets was 0.67. All other nutrients, including cholesterol (300 mg/d)
and fiber (28–30 g/d), were the same in both diets. Nutritional
analysis of the menus used in the two diets was carried out with the VA
nutrition software program and verified by the National Nutrition
Coordinating Center (Minneapolis, MN). Glycemic control was evaluated
by measurements of fructosamine [RoTAG; Roche Diagnostic Systems
(Johnson et al. 1982
)] and hemoglobin A1c
measurements (Glyc-Affin; Isolab Inc., Akron, OH). Blood glucose
records were reviewed once or twice per week, and adjustments were made
in insulin dose or time and energy distribution of meals to avoid
frequent hypoglycemia or sustained blood glucose levels of >13.8
mmol/L. Patients visited the center two or threes times per week to be
weighed and pick up their meals. Subjects were asked to abstain from
alcohol during the last 10 d of each diet period. During the last
week of each dietary period, three blood samples for determination of
fasting (12 h) blood glucose and plasma lipid (total, LDL and HDL
cholesterol, TG) and apoA-I and apoB levels were obtained from each
subject. The clinical, lipoprotein and apoB data for 11 of 19 subjects
in this study have also been included in another report in which the
differences were compared between the high Mono and the high CHO diets
on the metabolism of TG-rich lipoproteins isolated from the plasma
in the postprandial and fasting state in patients with type 1 diabetes
(Georgopoulos et al. 1998
).
Handling of blood samples.
To avoid lipoprotein degradation and lipid oxidation, blood samples for
lipoprotein analysis were collected in tubes containing EDTA (1 g/L)
and placed on ice. Plasma was separated by centrifugation at 10°C,
and a 1:100 dilution of a solution containing 1 g/L DTPA, 120 g/L
-aminocaproic acid, 50 g/L glutathione, 10 g/L thimerosal and 10 g/L
butylated hydroxytoluene was added. DTPA has been shown to be a more
potent antioxidant than EDTA (Heinecke et al. 1986
).
VLDL subfractions were analyzed within 1–2 wk.
Determination of fasting lipid and lipoprotein variables.
Plasma TG levels were measured enzymatically with a commercially available kit (catalogue no. 816370; Boehringer-Mannheim Diagnostics, Indianapolis, IN). The coefficient of variation of these assays in our laboratory is 4%. Plasma total and HDL cholesterol levels were measured enzymatically, the latter after heparin-manganese precipitation according to the Lipid Research Clinics protocol. Both apoB and apoA-I were determined nephelometrically with commercially available kits (Catalogue no. 86071 and 86070 for apoB and apoA-I, respectively; Incstar, Stillwater, MN). These measurements were performed in the VA laboratory and were made under quality control testing with the Northwest Lipid Research Laboratory in Seattle, WA.
Isolation of TG-rich lipoproteins.
All isolations were performed under aseptic conditions within
48–72 h from harvesting of plasma by salt density gradient
ultracentrifugation with an SW 28 rotor according to the method of
Lindgren as modified by Redgrave and Carlson (1979)
and
as described previously (Georgopoulos et al. 1994
). Two
subfractions were isolated: Sf 100–400
(31.2 x 106 g · min-1) containing
big VLDL and Sf 20–100 (152.0 x 106 g · min-1) containing mostly small VLDL.
Each subfraction was collected under aseptic conditions, dialyzed
against Tris-EDTA (or Tris-DTPA) buffer at 4°C, concentrated with
Aquacide, filtered and stored at 4°C for use in tissue culture
experiments within 1–2 wk.
The apoB determinations in the TG-rich lipoprotein
subfractions were performed by electroimmunoassay recognizing both
apoB-100 and apoB-48 (Koren and Alaupovic 1991
) in Dr.
Alaupovic’s laboratory (Oklahoma Medical Research Foundation, Oklahoma
City, OK). The coefficient of variation of the assay was 5%.
Chemical composition of the isolated TG-rich lipoproteins.
The total protein concentration of the lipoproteins was determined
according to a modification of the Lowry method (Lowry et al. 1951
). Free cholesterol and EC were measured by gas-liquid
chromatography after lipid extraction as described later
(Ishikawa et al. 1974
). The EC values were multiplied by
1.68 to account for the fatty acid mass. Phospholipid phosphorous was
determined according to the method of Bartlett (1979)
and multiplied by 25 to obtain phospholipid mass.
Cell culture.
THP-1 monocyte/macrophage cells were obtained from American Type
Culture Collection (Rockville, MD). The cells were grown in suspension
in RPMI-1640 containing 100 mL/L fetal calf serum, 100 mg/L
streptomycin, 105 U/L penicillin and 250 mg/L
fungizone-containing amphotericin as described previously
(Georgopoulos et al. 1994
). Cells were maintained at
0.5–1 x 109 cells/L by pelleting the cells twice
weekly and completely changing the medium. Forty-eight hours before
use, the cells were seeded (1–2 x 106 cells/35-mm
culture dish) and induced to differentiate into macrophages by the
addition of 1 x 10-7 mol/L
phorbol-12-myristate-13-acetate as described by others (Auwerx et al. 1988
, Hara et al. 1987
). The cells were
washed twice with phosphate-buffered saline before incubation with
the lipoproteins for 48 h. The apoB concentration used was 50 mg/L
for both VLDL. The incubation medium consisted of lipoproteins in RPMI
without fetal calf serum or albumin.
Mass measurement of cellular cholesterol.
The masses of free cholesterol and EC were determined by
gas-liquid chromatography according to the method of
Ishikawa et al. (1974)
. The lipid extracts were dried
under a nitrogen stream, resolubilized and injected into the gas
chromatograph at 270°C using an HP-17 cross-linked 50%
phenylmethylsilicone capillary column and a helium flow of 30 mL/min.
Stigmasterol was used as an internal standard. The areas under the
curves were calculated, and the mass of EC was derived from the
subtraction of free cholesterol from total cholesterol after
saponification.
Statistical analysis.
The effects of the diets on lipoproteins and clinical variables,
including glycemic control, and on apoB in VLDL subfractions were
compared by paired t test (Snedecor and Cochran 1989
).To compare the effects of lipoprotein subfractions on
free cholesterol and EC mass accumulation in macrophages, each
lipoprotein subfraction was analyzed in triplicate in every experiment;
control cells (without lipoproteins) were also included. The triplicate
results were averaged and were analyzed by an analysis of covariance
with the BMDP statistical package (1992 BMDP; Dynamic Statistical
Software, Los Angeles, CA). EC accumulation in the control wells was
used as a covariate to account for the variability of the cells within
experiments. Finally, to evaluate differences in the composition of
diabetic and normal lipoproteins, a log ratio analysis of compositions
was performed as described previously (Georgopoulos et al. 1994
).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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) and by others (Klein et al. 1989
, Lindqvist et al. 1983
), that the
degree of cellular EC accumulation after the uptake and degradation of
a TG-rich lipoprotein depends on the concentration of the
lipoprotein incubated with the cells. Moreover, at the same
concentration, TG-rich lipoproteins from diabetics compared with
those from control subjects result in a higher EC accumulation in
macrophages (Georgopoulos et al. 1994
, Klein et al. 1989
, Kraemer et al. 1985
). Therefore, the
atherogenic potential of a given intervention (i.e., diet) depends on
the concentration and corresponding number of the lipoprotein particles
and on the degree of cellular EC accumulation that they cause for a
given concentration, which is possibly related to particle composition.
In the present study, we assessed these variables by comparing
the effect of a high CHO versus a high Mono diet on the atherogenic
potential of VLDL subfractions from fasting subjects. Our results show
that the particle number, estimated by measuring apoB concentration in
the big VLDL particles (Sf 100–400),
was higher during the high Mono diet than during the high CHO diet
(P = 0.02) (Fig. 1
). This was not the case for the small VLDL particles
(Sf 20–100). We contend that the
difference in the big VLDL particle number between the two diet periods
is the result of dietary differences, because other confounding
factors, like weight, insulin dose, glycemic control and fasting lipid
profiles (including LDL, HDL and apoB and apoA-I), that could account
for the observed differences were similar at the end of the two dietary
periods (Table 1
). This finding is consistent with our previous study
(Georgopoulos et al. 1998
), in which compared with the
high Mono diet, the high CHO diet was associated with fewer
postprandial lipoprotein particles of both hepatic and intestinal
origin. The mechanism for the observed difference in big VLDL particle
number is unclear. It could be due to decreased production or increased
clearance of big VLDL during the high CHO diet versus the high Mono
diet. As reviewed by Krauss (1998)
, in addition to IDL
and very small VLDL (Sf 12–60), big
VLDL particles are considered to be atherogenic. Animal studies have
also shown that the retention of lipoprotein particles is greater for
bigger VLDL particles (Nordestgaard 1996
). Human
turnover studies have supported the hypothesis that accumulation in the
circulation of bigger VLDL particles
(Sf 100–400) can lead to the
generation of a series of atherogenic lipoproteins, including IDL and
dense LDL (Packard and Shepherd 1997
). Moreover, two
recent studies in which nuclear magnetic resonance spectroscopy was
used have shown that large VLDL particles are associated with more
angiographically verified atherosclerosis (Freedman 1998
) and a higher atherosclerotic risk (Framingham Offspring
Study; Otvos 1999
).
|
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) as a covariate in the analysis of the results. We found that this
covariate had a significant effect on cellular accumulated EC. After
accounting for its effect by analysis of covariance, there was no
difference in the effect of the diets on the amount of cellular
accumulated EC (Table 2)
.
|
). The lack of
difference in the particle composition between the diets could have
accounted for the observed lack of difference in the degree of cellular
EC accumulation. In previous studies, differences in TG-rich
lipoprotein composition were associated with the greater degree of EC
accumulation in macrophages incubated with diabetic versus normal
TG-rich lipoproteins (Georgopoulos et al. 1994
).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by grants from the American Diabetes Association and the Minnesota Medical Foundation.
4 Abbreviations used: apo, apolipoprotein; CHO, carbohydrate; EC, esterified cholesterol; TG, triglycerides.
Manuscript received March 16, 2000. Initial review completed May 18, 2000. Revision accepted June 26, 2000.
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