Am J Physiol Endocrinol Metab 305: E573–E584, 2013.
First published July 2, 2013; doi:10.1152/ajpendo.00122.2013.
Distinct metabolic and vascular effects of dietary triglycerides and cholesterol
in atherosclerotic and diabetic mouse models
Marc-André Laplante,1 Alexandre Charbonneau,1 Rita Kohen Avramoglu,1 Patricia Pelletier,1
Xiangping Fang,4 Hélène Bachelard,2 Seppo Ylä-Herttuala,3 Markku Laakso,3 Jean-Pierre Després,1
Yves Deshaies,1 Gary Sweeney,4 Patrick Mathieu,1 and André Marette1
1
Centre de recherche de l’Institut Universitaire de Cardiologie et Pneumologie de Québec, Hôpital Laval, Québec, Canada;
Centre de Recherche du CHUQ, Endocrinologie et Néphrologie, Québec,Canada; 3University of Eastern Finland, Institute of
Clinical Science, Internal Medicine, Kuopio and Kuopio University Hospital, Kuopio, Finland; and 4York University, Toronto,
Ontario, Canada
2
Submitted 7 March 2013; accepted in final form 26 June 2013
diabetes; obesity; atherosclerosis; endothelial function
rich in saturated triglycerides and cholesterol is commonly used as a reference of poor dietary habits
leading to the development of obesity, type 2 diabetes (T2D), and
vascular diseases. This diet has been adapted for mouse studies on
obesity and is used to worsen the development of atherosclerosis
in genetically altered murine models such as the low-density
lipoprotein (LDL) receptor-deficient mouse (14, 15).
While dietary triglycerides or dietary cholesterol are often
used in combination, each one has its own distinctive effects on
vascular and metabolic disorders. Current evidence suggests
that dietary cholesterol is more important than triglycerides for
plaque formation. Indeed, cholesterol at ⬎0.15% in a low-fat
THE WESTERN-STYLE DIET
Address for reprint requests and other correspondence: A. Marette, Institut
Universitaire de Cardiologie et de Pneumologie de Québec (Hôpital Laval),
Québec, QC, Canada, G1V 4G5 (e-mail: [email protected]).
http://www.ajpendo.org
diet could worsen lesion size (27). Dietary triglycerides, despite promoting insulin resistance, also made little difference in
aortic root lesion size and only in very old mice (31). On the
other hand, the diabetogenic potential of dietary cholesterol is
less clear. A cholesterol-rich diet did not affect glucose tolerance in a LDL receptor-deficient mouse (11). However, studies
in pancreatic cell lines and isolated pancreatic islets showed
that hypercholesterolemia may impair ␤-cell function and insulin response to glucose (2, 5, 10). In humans, the link
between atherosclerosis and metabolic disorders is of particular
interest. T2D morbidity and mortality strongly correlates with
coronary artery disease. However, the development of atherosclerosis associated with familial and nonfamilial hypercholesterolemia has been suggested to be independent from insulin
resistance (1, 7, 21).
The rationale of the present study was to clarify the metabolic
effects of both dietary triglycerides and cholesterol and assess
whether or not diabetes could worsen atherosclerosis in three
different murine models: wild-type C57BL6 (C57), atherosclerotic LDLr⫺/⫺ ApoB100/100 (LRKOB100), and atherosclerotic/
diabetic IGF-II ⫻ LDLr⫺/⫺ ApoB100/100 (LRKOB100/IGF)
mice. The LRKOB100 mouse is a model of hypercholesterolemia and atherosclerosis that exclusively produces apolipoprotein B100 (ApoB100) and was shown to exhibit a lipoprotein
profile resembling that seen in human atherosclerosis characterized by a shift toward elevated very low density lipoprotein
and LDL fractions at the expense of the high-density lipoprotein (HDL) fraction normally prominent in mice (29). When
combined with the overexpression of insulin-like growth factor-II (IGF-II) in pancreatic ␤-cells, the resulting transgenic
LRKOB100/IGF mouse is more prone to develop T2D upon
feeding a high-fat diet, while retaining a plasma lipoprotein
profile more similar to that seen in humans, thus providing the
only mouse model available for studying the impact of true
diabetes in a proatherosclerotic milieu. There is the added
benefit that the disease phenotype in these models results
directly from the genetic modifications, thus avoiding dietassociated variability or the confounding effect of factors
found in other genetic models (e.g., leptin deficient) that may
indirectly produce similar phenotypes (30).
Our results show distinctive effects of dietary triglycerides
and cholesterol on insulin resistance, endothelial function, and
plaque formation in the LRKOB100 and LRKOB100/IGF
models. Strikingly, we found that dietary cholesterol reduces
the insulin-resistant effects of a high-fat diet in all mouse
models. Our data also demonstrate that insulin resistance and
0193-1849/13 Copyright © 2013 the American Physiological Society
E573
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.4 on June 16, 2017
Laplante MA, Charbonneau A, Kohen Avramoglu R, Pelletier P, Fang X, Bachelard H, Ylä-Herttuala S, Laakso M,
Després J, Deshaies Y, Sweeney G, Mathieu P, Marette A.
Distinct metabolic and vascular effects of dietary triglycerides and
cholesterol in atherosclerotic and diabetic mouse models. Am J
Physiol Endocrinol Metab 305: E573–E584, 2013. First published
July 2, 2013; doi:10.1152/ajpendo.00122.2013.—Cholesterol and
triglyceride-rich Western diets are typically associated with an increased occurrence of type 2 diabetes and vascular diseases. This
study aimed to assess the relative impact of dietary cholesterol and
triglycerides on glucose tolerance, insulin sensitivity, atherosclerotic
plaque formation, and endothelial function. C57BL6 wild-type (C57)
mice were compared with atherosclerotic LDLr⫺/⫺ ApoB100/100
(LRKOB100) and atherosclerotic/diabetic IGF-II ⫻ LDLr⫺/⫺
ApoB100/100 (LRKOB100/IGF) mice. Each group was fed either a
standard chow diet, a 0.2% cholesterol diet, a high-fat diet (HFD), or
a high-fat 0.2% cholesterol diet for 6 mo. The triglyceride-rich HFD
increased body weight, glucose intolerance, and insulin resistance but
did not alter endothelial function or atherosclerotic plaque formation.
Dietary cholesterol, however, increased plaque formation in
LRKOB100 and LRKOB100/IGF animals and decreased endothelial
function regardless of genotype. However, cholesterol was not associated with an increase of insulin resistance in LRKOB100 and
LRKOB100/IGF mice and, unexpectedly, was even found to reduce
the insulin-resistant effect of dietary triglycerides in these animals.
Our data indicate that dietary triglycerides and cholesterol have
distinct metabolic and vascular effects in obese atherogenic mouse
models resulting in dissociation between the impairment of glucose
homeostasis and the development of atherosclerosis.
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INSULIN RESISTANCE AND VASCULAR COMPLICATIONS
T2D per se are not primary factors causing atherosclerosis and
endothelial dysfunction in obese hypercholesterolemic mice.
MATERIALS AND METHODS
Table 1. Metabolic phenotype of C57 mice following 6 mo on different diets
C57
SD
CD
HFD
HFCD
Body wt, g*,†
Body wt change, g*,†
Energy intake, kcal/day*,†
Liver wt, g*,†
eWAT wt, g*,†,¶
Gastrocnemius wt, mg*
Pancreas wt, mg*,†
Fasting glycemia, mM*,†,¶
Fasting insulin, ng/ml*,†
34.0 ⫾ 0.6d,e
15.9 ⫾ 0.5e
11.70 ⫾ 0.1c
1.30 ⫾ 0.05c
1.24 ⫾ 0.15b,c,d,e
188 ⫾ 9a,b
184 ⫾ 9a,b,c
9.65 ⫾ 0.47b
0.84 ⫾ 0.13b
38.6 ⫾ 1.1b,c,d
20.4 ⫾ 1.1b,c
11.50 ⫾ 0.269c
1.85 ⫾ 0.10b,c
1.87 ⫾ 0.22b
200 ⫾ 6a
197 ⫾ 11a,b
11.12 ⫾ 0.38b
1.34 ⫾ 0.32a,b
42.9 ⫾ 1.2a,b
24.6 ⫾ 1.2a,b
14.79 ⫾ 0.22b
2.40 ⫾ 0.23b
3.25 ⫾ 0.27a
181 ⫾ 5a,b
215 ⫾ 9a
11.28 ⫾ 0.37b
3.00 ⫾ 0.58a
43.9 ⫾ 1.3a
25.6 ⫾ 1.3a
15.48 ⫾ 0.18a
3.25 ⫾ 0.28a
3.27 ⫾ 0.19a
199 ⫾ 1a
207 ⫾ 7a,b
10.40 ⫾ 0.41b
2.38 ⫾ 0.72a,b
Values are means ⫾ SE, n ⫽ 10 –22 mice/group. SD, standard chow diet; CD, 0.2% cholesterol diet; HFD, high-fat diet; HFCD, high-fat 0.2% cholesterol
diet; eWAT, epididymal adipose tissue. Body weights were recorded before death. Plasma for glucose or insulin measurement was isolated from blood drawn
after a 5-h fast. Organ weights were taken at death following a 5-h fast. Food intake represents an average of kcal/day measured over the course of 6 mo. Groups
not sharing the same letter are considered significantly different when comparing effects of both diet ⫻ genotype among the different groups. Symbols represent
differences between diet groups (*), differences between genotypes (†), and differences between diet ⫻ genotype (¶).
AJP-Endocrinol Metab • doi:10.1152/ajpendo.00122.2013 • www.ajpendo.org
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Animals. Animal handling was conducted according to the Laval
University Hospital Research Centre Animal Committee guidelines.
Studies were performed in accordance with the Canadian Guide for
the Care and Use of Laboratory Animals and were approved by the
Laval University Animal Care Committee. Male mice were housed in
a pathogen-free, temperature-controlled environment under a 12:12-h
light-dark cycle and fed ad libitum either a standard chow diet (SD;
Harlan Teklad T-2018), a cholesterol diet (CD, 0.2% cholesterol; Harlan Teklad TD-07798), a high-fat diet (HFD, 55% kcal from fat;
Harlan Teklad TD-93075), or a high-fat 0.2% cholesterol diet (HFCD;
Harlan Teklad TD-07799) starting at 6 wk of age. Both LRKOB100
and LRKOB100/IGF mice were generated from original founders
kindly provided by Drs. Seppo Ylä-Herttuala and Markku Laakso
(Kuopio University, Kuopio, Finland) and backcrossed on a C57B1/6J
background from Jackson Laboratories (Bar Harbor, ME). Agematched C57B1/6J mice were used as a control. The addition of
cholesterol did not substantially change the caloric content of the diets
(see Supplemental Tables 1 and 2 for diet compositions. Supplemental
data for this article may be found at http://andremarette.com/projects/
ccd/diets-for-ajp-paper-2013-laplante-et-al).
Lipid and lipoprotein measurement. Plasma lipids were measured
following a 6-h fast with a 2-h refeeding period between 4:00 A.M.
and 6:00 A.M. Enzymatic kits were used for measuring triglyceride,
cholesterol (Randox Laboratories, Kearneysville, WV), and nonesterified fatty acid (Wako Diagnostics, Richmond, VA). Apolipoprotein
B and apolipoprotein AI were quantified from fasting plasma samples
by immunoblot (antibodies from Midland Bioproducts, Boone, IA).
Plasma lipoprotein fast protein liquid chromatography. Equal
volumes of plasma from three to four mice of the same genotype and
diet were filtered through a 0.45-␮m membrane, and 200 ␮l were
injected on a Superose 6 100/300 GL column. Lipoproteins were
eluted using Tris buffer (10 mM Tris, 150 mM NaCl, 2 mM CaCl2,
100 ␮M diethylenetriaminepentaacetic acid, and 0.02% NaN3; pH ⫽
7.4) at a flow rate of 0.5 ml/min. Triglyceride and cholesterol content
in each fraction was measured enzymatically.
Fasting insulin and intraperitoneal glucose tolerance tests. Plasma
insulin levels were measured by radioimmunoassay following a 6-h fast
(Linco Research, St. Charles, MO). For intraperitoneal glucose tolerance
tests (IPGTT), mice were fasted 5 h before experiment. Mice were
injected intraperitoneally with 10 ␮l/g of 10% dextrose solution. Blood
was sampled from the saphenous vein in conscious mice and analyzed
using a glucometer (MediSense; Abbott Laboratories).
Hyperinsulinemic-isoglycemic clamp. Hyperinsulinemic-isoglycemic clamps (HIIC) were performed on conscious, unrestrained mice
as previously described (4). After a 6-h fast, a 5-␮Ci bolus of
[3-3H]glucose (Perkin Elmer, Boston, MA) was given at t ⫽ ⫺90 min
followed by a 0.05 ␮Ci/min infusion for 90 min. The insulin clamp
began at t ⫽ 0 min with a primed-continuous infusion of human
insulin (16 mU/kg bolus followed by 4 mU·kg⫺1·min⫺1 infusion;
Humulin R; Eli Lilly, Indianapolis, IN). The [3-3H]glucose infusion
was increased to 0.07 ␮Ci/min for the remainder of the experiment to
minimize changes in specific activity from the equilibration period.
During the clamp, isoglycemia was maintained at the basal glycemia
concentration (average of glycemias at time ⫺90, ⫺30, ⫺20, ⫺10,
and 0 min). Blood samples (60 –200 ␮l) were taken every 10 min from
t ⫽ 80 to 120 min and processed to determine glucose specific
activity. Mice received saline-washed erythrocytes from donor mice
throughout the experiment (5– 6 ␮l/min).
Histology and cytokines measurements in tissues. Epididymal adipose tissue (eWAT) were fixed in phosphate-buffered paraformaldehyde 4% solution and embedded in paraffin for immunohistology with
F4/80 rat antibody (MCA497RT; AbD Serotec, Raleigh, NC). Detection was performed by 3,3=-diaminobenzidine staining with hematoxylin counterstaining (Vector Laboratories, Burlingame, CA). Cytokine
measurements were performed with Milliplex kits (Millipore, Billerica, MA) on tissue extracts using Luminex technology. Proteins
were extracted from flash-frozen tissues the day of measurement using
1% Nonidet P-40 in PBS.
Vascular reactivity. Mice were anesthetized with ketamine/xylazine (0.1 ml/10 g ip), and thoracic aortas were excised into cold Krebs
buffer (118 mM NaCl, 4.7 mM KCl, 3.3 mM CaCl2, 1.17 mM
MgSO4·7H2O, 1.17 mM KH2PO4, 1, 25 mM NaHCO3, and 1%
dextrose, pH ⫽ 7.4). Aortic cross sections 2–3 mm were cut, cleaned,
and set on two vessel holders (Harvard Apparatus, Montreal, QC) in
an oxygenated bath at 37°C on a force transducer with an initial
tension of 1.5 g. A 1-h resting period was applied with changes of
buffer each 15 min. Three different cumulative dose-response curves
were tested: phenylephrine, phenylephrine precontraction at 10⫺6 M
with carbacholine, and phenylephrine precontraction with sodium
nitroprusside (SNP).
En face lesion analysis. En face preparation was performed following the protocol published by the Animal Models of Diabetic
Complications Consortium (www.diacomp.org). Images of vessels
were taken, and the percentage atherosclerotic lesions area positive for
Sudan IV was quantified using ImagePro software (Media Cybernetics, Bethesda, MD).
Data and statistical analysis. Hepatic glucose production and Rd
during the clamp were determined using Mari’s non-steady-state
equations for a two-compartmental model (4). Data are presented as
means ⫾ SE. A P ⬍ 0.05 was considered significant. Two-way
ANOVA followed by Tukey-Kramer post hoc test was performed
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INSULIN RESISTANCE AND VASCULAR COMPLICATIONS
Table 2. Metabolic phenotype of LRKOB100 mice following 6 mo on different diets
LRKOB100
SD
,†
33.6 ⫾ 0.8
15.3 ⫾ 0.7d,e
11.54 ⫾ 0.07c
1.21 ⫾ 0.03d
0.83 ⫾ 0.07d,e
181 ⫾ 10a,b
152 ⫾ 6c
9.48 ⫾ 0.35b
1.10 ⫾ 0.15a,b
Body wt, g*
Body wt change, g*,†
Energy intake, kcal/day*,†
Liver wt, g*,†
eWAT wt, g*,†,¶
Gastrocnemius wt, mg*
Pancreas wt, mg*,†
Fasting glycemia, mM*,†,¶
Fasting insulin, ng/ml*,†
CD
d,e
HFD
35.6 ⫾ 1.0
17.3 ⫾ 0.9c,d,e
11.58 ⫾ 0.07c
1.65 ⫾ 0.09c
1.06 ⫾ 0.12c,d,e
188 ⫾ 9a,b
180 ⫾ 10a,b,c
10.38 ⫾ 0.37b
1.30 ⫾ 0.21a,b
c,d,e
HFCD
39.3 ⫾ 0.9
21.0 ⫾ 0.8b,c
14.83 ⫾ 0.10a,b
1.82 ⫾ 0.10b,c
1.62 ⫾ 0.13b,c
163 ⫾ 5b
201 ⫾ 8a,b
10.41 ⫾ 0.49b
1.61 ⫾ 0.35a,b
b,c
39.2 ⫾ 0.8b,c
21.0 ⫾ 0.7b,c
14.88 ⫾ 0.14a,b
2.45 ⫾ 0.15b
1.86 ⫾ 0.09b
167 ⫾ 1a,b
187 ⫾ 8a,b,c
10.34 ⫾ 0.35b
1.87 ⫾ 0.37a,b
Values are means ⫾ SE, n ⫽ 10 –22 mice/group. Body weights were recorded before death. Plasma for glucose or insulin measurement was isolated from
blood drawn following a 5-h fast. Organ weights were taken at death following a 5-h fast. Food intake represents an average of kcal/day measured over the course
of 6 mo. Groups not sharing the same letter are considered significantly different when comparing effects of both diet ⫻ genotype among the different groups.
Symbols represent differences between diet groups (*), differences between genotypes (†), and differences between diet ⫻ genotype (¶).
RESULTS
Metabolic phenotypes of LRKOB100 and LRKOB100/IGF
mice. To better understand the effect of obesity-linked insulin
resistance and diabetes on the development of cardiovascular
pathologies, 6-wk-old LRKOB100 and LRKOB100/IGF mice
were fed either low-fat SD, atherogenic CD, obesogenic HFD,
or atherogenic/obesogenic HFCD for 24 wk after which their
phenotypes were assessed (Tables 1–3). Regardless of genotype, mice fed the HFD or HFCD consumed more calories and
gained more weight compared with mice of the same genotype
fed a SD diet. Within each genotype, liver mass was also
increased by CD, HFD, or HFCD, whereas there were no
relevant changes in gastrocnemius muscle or pancreas mass.
C57 mice also gained a significant amount of weight on the CD
compared with C57 mice on the SD despite these two diets
being isocaloric. Even though weight gain was seen in all
groups, both LRKOB100 and LRKOB100/IGF groups fed CD,
HFD, or HFCD weighed slightly less compared with the C57
group fed the same diet.
In C57 mice, fasting insulinemia was increased in response
to HFD and HFCD. These dietary interventions had a lesser
impact on the glycemia and insulinemia of LRKOB100 animals, which is coherent with the lessened weight gain observed
in these groups. LRKOB100/IGF animals were hyperinsulinemic regardless of diet, and a HFD resulted in a significant
increase in fasting glycemia. This hyperglycemic state failed to
promote further insulin production, suggesting a lack of ␤-cell
pancreatic response, an expected outcome of this model of
T2D (6). Interestingly, fasting hyperglycemia was not observed
in HFCD, suggesting that adding cholesterol in the diet prevented the development of diabetes independent from changes
in insulinemia.
As expected, deletion of the LDL receptor increased both
plasma cholesterol and apoB100 in LRKOB100 and
LRKOB100/IGF mice compared with C57 controls, an effect
that was observed even in animals fed the SD (Fig. 1, B and E).
In addition to the genotype effect, there was an additive and
significant diet effect on plasma cholesterol. High-fat feeding
(HFD or HFCD) increased circulating cholesterol in both
LRKOB100 and LRKOB100/IGF mice. We were able to
confirm by fast protein liquid chromatography that the majority
of circulating cholesterol in LRKOB100 and LRKOB100/IGF
mice was found in the LDL fraction while the majority of
cholesterol in C57 mice was found in the HDL fraction (Fig.
1F). On the other hand, there were no significant differences in
circulating apoAI levels between any of the groups (Fig. 1D).
HFD and HFCD increased plasma triglycerides significantly in
the LRKOB100 and LRKOB100/IGF mice (Fig. 1C). HFD and
HFCD also induced an accumulation of triglyceride in the LDL
fraction that was also shifted over a lower-density range for
both LRKOB100 and LRKOB100/IGF mice (Fig. 1G).
Impact of diets on glucose intolerance in genetic mouse
models. We performed IPGTT to assess the impact of the genotype and diets on glucose homeostasis (Fig. 2A). As expected, C57
mice on CD, HFD, or HFCD were more glucose intolerant than
Table 3. Metabolic phenotype of LRKOB100/IGF mice following 6 mo on different diets
LRKOB100/IGF
SD
CD
HFD
HFCD
Body wt, g*,†
Body wt change, g*,†
Energy intake, kcal/day*,†
Liver wt, g*,†
eWAT wt, g*,†,¶
Gastrocnemius wt, mg*
Pancreas wt, mg*,†
Fasting glycemia, mM*,†,¶
Fasting insulin, ng/ml*,†
33.1 ⫾ 0.9e
15.0 ⫾ 0.9e
11.46 ⫾ 0.14c
1.32 ⫾ 0.05c
0.76 ⫾ 0.09e
178 ⫾ 3a,b
172 ⫾ 6b,c
10.17 ⫾ 0.42b
1.92 ⫾ 0.25a,b
37.3 ⫾ 1.0c,d,e
19.2 ⫾ 1.1c,d,e
11.38 ⫾ 0.02c
1.89 ⫾ 0.13b,c
1.21 ⫾ 0.12b,c,d,e
167 ⫾ 6a,b
178 ⫾ 9a,b,c
11.44 ⫾ 0.67b
2.42 ⫾ 0.4a,b
40.0 ⫾ 1.6a,b,c
21.8 ⫾ 1.5a,b,c
14.86 ⫾ 0.04a,b
1.90 ⫾ 0.14b,c
1.65 ⫾ 0.19b,c,d
170 ⫾ 6a,b
182 ⫾ 13a,b,c
15.23 ⫾ 0.8a
2.42 ⫾ 0.41a,b
38.8 ⫾ 0.7b,c,d
20.4 ⫾ 0.8b,c,d
14.69 ⫾ 0.12b
2.44 ⫾ 0.17b
1.62 ⫾ 0.08b,c,d
168 ⫾ 1a,b
210 ⫾ 9a,b
9.62 ⫾ 0.66b
2.12 ⫾ 0.39a,b
Values are means ⫾ SE, n ⫽ 10 –22 mice/group. Body weights were recorded before death. Plasma for glucose or insulin measurement was isolated from
blood drawn following a 5-h fast. Organ weights were taken at death following a 5-h fast. Food intake represents an average of kcal/day measured over the course
of 6 mo. Groups not sharing the same letter are considered significantly different when comparing effects of both diet ⫻ genotype among the different groups.
Symbols represent differences between diet groups (*), differences between genotypes (†), and differences between diet ⫻ genotype (¶).
AJP-Endocrinol Metab • doi:10.1152/ajpendo.00122.2013 • www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.4 on June 16, 2017
using JMP 9.0 software (SAS Institute, Cary, NC). Groups not sharing
the same letter are considered statistically different.
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INSULIN RESISTANCE AND VASCULAR COMPLICATIONS
SD
a
a
a
a
a
a
a
a
a
a
Plasma ApoAI
(relative units)
100
a
a
a
a,b,c
a,b,c
a,b,c
a,b
a,b,c
b,c
c
120
80
60
40
20
e,f
a
a
150
100
50
0
LRKOB100/IGF
b
C57
LRKOB100
LRKOB100/IGF
a,b
a
Prob(F)
< 0.0001
< 0.0001
< 0.0001
diet
genotype
diet*genotype
c,d
c,d
d
d
3
c,d
4
d
d
c,d
b,c
5
b,c
6
2
1
C57
VLDL
11
16
IDL/LDL
21
26
31
HDL
36
41
LRKOB100/IGF
3
2.5
2
1.5
1
0.5
0
LRKOB100
11
4
C57
3
2
1
0
11
16
21
26
31
36
Fraction number
16
21
26
31
36
41
3
2.5
2
1.5
1
0.5
0
LRKOB100/IGF
SD
CD
HFD
HFCD
11
Fraction number
41
Triglyceride (mM)
Triglyceride (mM)
Fraction number
Cholesterol (mM)
Cholesterol (mM)
3
2.5
2
1.5
1
0.5
0
LRKOB100
4
LRKOB100
3
2
1
0
11
16
21
26
31
36
Fraction number
16
21
26
31
36
41
Fraction number
41
Triglyceride (mM)
C57
Cholesterol (mM)
0
a
a
a
200
a
a
b,c
b,c
LRKOB100
9
7
LRKOB100/IGF
Prob(F)
< 0.0001
genotype
b
C57
8
LRKOB100
b
b
5
c,d,e
10
d,e,f
c,d
15
b
b
20
C57
250
Plasma ApoB100
(relative units)
25
E
Prob(F)
< 0.0001
< 0.0001
< 0.001
a
diet
genotype
diet*genotype
0
LRKOB100/IGF
a
LRKOB100
a
C57
f
Plasma cholesterol (mM)
140
0.1
30
Plasma triglyceride (mM)
Prob(F)
< 0.05
180
160
0.2
F
G
HFCD
c
0.3
0
C
D
HFD
0.4
0.0
B
a,b,c
0.5
a,b,c
a,b,c
0.6
diet*genotype
4
LRKOB100/IGF
3
2
1
0
11
16
21
26
31
36
Fraction number
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Plasma fatty acids (mM)
0.7
CD
f
A
Prob(F)
< 0.0001
diet
< 0.005
genotype
diet*genotype < 0.005
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INSULIN RESISTANCE AND VASCULAR COMPLICATIONS
A
SD
CD
LRKOB100
35
30
HFCD
30
30
25
20
15
10
5
Glucose (mM)
35
25
20
15
10
5
0
0
0
15
30
45
60
75
90
diet
genotype
diet*genotype
15
30
45
60
75
90
0
15
30
45
60
75
90
Time (minutes)
HFD
b
a,c
a
LRKOB100
LRKOB100/IGF
a,d
a
a,c
a,c
a,c
5
CD
HFCD
a,c
10
SD
Prob(F)
< 0.0001
< 0.0001
< 0.005
d
1500
d
d
2000
15
Time (minutes)
3000
2500
20
1000
500
0
C57
Fig. 2. Intraperitoneal glucose tolerance tests performed after 23 wk on C57, LRKOB100, and LRKOB100/IGF mice on SD, CD, HFD, or HFCD. A: C57,
LRKOB100, and LRKOB100/IGF mice. B: the area under the curve (AUC) of each individual intraperitoneal glucose tolerance test was calculated for statistical
analysis, n ⫽ 10 –15 animals/group. Groups not sharing the same letter are considered statistically different when comparing effects of both diet ⫻ genotype
among the different groups.
their SD-fed counterparts. The diets did not exacerbate glucose
intolerance to the same extent in LRKOB100 mice as the C57
genotype. The relative protection from glucose intolerance in
the LRKOB100 mice may be explained by the lesser weight
gain in this genotype (Table 1). However, LRKOB100/IGF
mice under HFD displayed glucose intolerance (Fig. 2A).
Worthy of note, the addition of cholesterol to the HFD (HFCD)
reduced, to a great extent, glucose intolerance (Fig. 2, A and B),
which is consistent with the prevention of fasting hyperglycemia in this group (Table 1).
HFD but not CD or HFCD decreases insulin sensitivity in
LRKOB100 and LRKOB100/IGF mice. To exclude the influence of defective pancreatic responses to glucose challenge, we
evaluated whole body insulin sensitivity by performing HIIC
studies. In C57 mice, CD decreased the glucose infusion rate
(GIR) compared with SD, whereas HFD severely impaired
insulin sensitivity as seen by a fourfold decrease in GIR
relative to SD (Fig. 3A). Comparing across genotypes fed SD,
the LRKOB100 and LRKOB100/IGF mice had a lower GIR
compared with C57 mice, suggesting that dyslipidemic mice
already have compromised insulin sensitivity even in the absence of dietary intervention. Surprisingly, the CD and the
HFCD improved insulin sensitivity in both LRKOB100 and
LRKOB100/IGF mice. In C57 mice, the insulin resistance
caused by HFD was also partly prevented by a concomitant
cholesterol input in the HFCD, but the mice remained insulinresistant compared with SD-fed controls. This effect of dietary
cholesterol on insulin sensitivity was explained by a lower
Fig. 1. Plasma lipids and apoproteins measured in C57, LRKOB100, and LRKOB100/IGF after 24 wk on standard chow diet (SD), 0.2% cholesterol diet (CD),
high-fat diet (HFD), or high-fat 0.2% cholesterol diet (HFCD). Nonesterified fatty acids (A), cholesterol (B), and triglyceride (C) were measured in freshly isolated
plasma from mice following a 6-h fasting and 3-h refeeding period. Apolipoprotein (Apo) AI (D) and apoB (E) from fasting plasma were quantified by
immunoblot and compared with a reference standard loaded on each immunoblot; n ⫽ 3–10 experiments. Plasma lipoproteins were resolved by fast protein liquid
chromatography using a Superose 6 100/300 GL column. Fractions 11 through 45 containing the spectrum of lipoproteins from very low density lipoprotein
(VLDL) to high-density lipoprotein (HDL) were used to quantify cholesterol (F) and triglyceride (G) content, representative of n ⫽ 3 experiments with each
experiment containing pooled plasma from 2– 4 animals. Groups not sharing the same letter are considered statistically different when comparing effects of diet ⫻
genotype among the different groups.
AJP-Endocrinol Metab • doi:10.1152/ajpendo.00122.2013 • www.ajpendo.org
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B
25
0
0
Time (minutes)
AUC (relative units)
LRKOB100/IGF
HFD
Glucose (mM)
Glucose (mM)
C57
35
E578
INSULIN RESISTANCE AND VASCULAR COMPLICATIONS
SD
diet
genotype
diet*genotype
70
HFD
HFCD
a
d,e,f
f
e,f
d
40
d
d,e
c
50
b
b,c
60
30
20
g
GIR (mg/kg/min)
CD
a,b
A
Prob(F)
< 0.0001
< 0.0001
< 0.0001
10
0
b
b,c
b
b
a,b
b,c
a,b
a,b,c
a,b
Prob(F)
< 0.0001
< 0.0001
< 0.0001
b
f
e,f
d
b
40
d,e,f
c
50
30
g
c,d
b,c
b,c
a,b
c,d,e
d,e
5
d,e
10
LRKOB100/IGF
b,c
60
LRKOB100
diet
genotype
diet*genotype
70
c,d,e
b,c
15
C57
F
Prob(F)
< 0.0001
< 0.0001
< 0.0005
a
20
10
a
diet
genotype
diet*genotype
e
Ra (mg/kg/min)
25
20
d,e
C
30
0
LRKOB100/IGF
Rd (mg/kg/min)
LRKOB100
a
C57
40
a,b
5
50
b,c
c
10
60
a
a
Basal Rd (mg/kg/min)
d
b,c,d
15
Prob(F)
< 0.0001
< 0.0001
diet
diet*genotype
70
a,b,c,d
b,c,d
b,c,d
a,b
a,b,c,d
20
b,c,d
Basal Ra (mg/kg/min)
25
E
c,d
B
LRKOB100/IGF
a,b,c
LRKOB100
Prob(F)
diet
< 0.0001
genotype
< 0.05
diet*genotype < 0.01
a,b,c
C57
20
10
0
f
30
10
Prob(F)
< 0.0001
< 0.05
20
10
5
a
c,d
a,b,c
a,b
a,b,c,d
c,d
6
a,b,c,d
7
a,b,c
8
d
4
d
c,d
c,d
c,d
e,f
40
d,e
c,d
50
diet
diet*genotype
LRKOB100/IGF
9
b,c,d
a,b,c
b
60
LRKOB100
c,d
G
80
70
C57
b,c,d
90
a
100
0
LRKOB100/IGF
Prob(F)
diet
< 0.0001
genotype
<0.0001
diet*genotype <0.0001
a
Suppression Ra (%)
D
LRKOB100
Fold increase Rd
C57
3
2
1
0
C57
LRKOB100
LRKOB100/IGF
0
C57
LRKOB100
AJP-Endocrinol Metab • doi:10.1152/ajpendo.00122.2013 • www.ajpendo.org
LRKOB100/IGF
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0
INSULIN RESISTANCE AND VASCULAR COMPLICATIONS
only when combined with dyslipidemia in the LRKOB100 genetic background. SNP responses were not significantly different
among groups, showing that the response to nitric oxide or the
mechanical properties of the vessel wall were not altered by the
genotypes or diets (Fig. 6).
DISCUSSION
We have explored the contributions of dietary cholesterol,
triglycerides, and of insulin resistance/diabetes on the development of atherosclerosis using genetic and dietary approaches. Our
work discriminated between the effects of either triglycerides
or cholesterol and is the first to establish a detailed glucose
homeostasis and insulin sensitivity profile in atherogenic
mouse models fed these diets. Of interest, we documented that,
whereas a HFD is accompanied by insulin resistance/diabetes,
it does not exacerbate the level of plaque area in atherogenic
mouse models. Conversely, a cholesterol-rich diet reduced
insulin resistance in HFD-fed animals and exacerbated atherosclerosis. Hence, these findings highlight a differential effect of
the cholesterol vs. the triglyceride-rich diet on insulin resistance and atherosclerosis. Furthermore, this study has brought
to light a novel and intriguing finding. The development of
lipid-rich atherosclerotic plaques within the aorta is not related
to the insulin-resistant state in atherosclerosis-prone mouse
models.
The LDLr⫺/⫺ mouse presents a resistance to diet-induced
metabolic alterations. In this regard, CD, HFD, and HFCD
caused glucose intolerance in C57 and LRKOB100/IGF, but
this was less pronounced in the LRKOB100 mice. These mice
also gained less weight in response to dietary intervention.
Metabolic differences such as lower epididymal adipose tissue
weight, lower body weight, and improved glucose tolerance
under a HFD were previously reported in LDLr⫺/⫺ mice and
could at least be partially explained by an enhanced thermogenesis, but this observation on weight gain was not reproduced in one study with diets richer in carbohydrates, suggesting complex genetic-dietary interactions (20, 24). Despite the
lack of increased plasma glucose in LDLr⫺/⫺ mice following
prolonged high-cholesterol feeding, a study showed increased
plasma insulin hinting at the existence of insulin resistance in
these animals independent of weight gain (11). Genetic deletions of the LDL receptor and ApoB48 therefore appear to have
an overall metabolic impact, preventing weight gain but also
increasing insulin resistance, an effect that was observed
through our clamp studies.
To the best of our knowledge, this is the first study to
document insulin resistance in the LRKOB100 model relative
to C57 mice. This insulin resistance could be a consequence of
elevated circulating triglycerides since we previously have
shown a strong correlation between elevated levels of circulating lipids and insulin resistance (4). Previous studies also
found that circulating cholesterol had deleterious hepatic effects in LDLr⫺/⫺ mice and may increase endoplasmic reticulum stress that could explain the occurrence of insulin resis-
Fig. 3. Hyperinsulinemic-isoglycemic clamps (HIIC) were performed in C57, LRKOB100, and LRKOB100/IGF mice following 24 wk on SD, CD, HFD, or
HFCD. Glucose infusion rate (A), basal glucose production (B), clamp glucose production (C), percentage of glucose production suppression (D), basal glucose
uptake (E), glucose uptake during clamp (F), and fold increase in glucose uptake (G) are shown; n ⫽ 3–15 animals/group. Groups not sharing the same letter
are considered statistically different when comparing effects of both diet ⫻ genotype among the different groups.
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basal (Fig. 3B) and insulin-mediated suppression of hepatic
glucose production (Fig. 3, C and D) and an increase in
insulin-stimulated glucose uptake by peripheral tissues (Fig. 3,
F and G). Conversely, the HFD was found to reduce GIR (Fig.
3A), increase hepatic glucose production (Fig. 3C), and decrease the rate of peripheral glucose uptake (Fig. 3F) compared
with the CD and HFCD in all genotypes. Overall, these data
indicate that dietary cholesterol can exert some protective
effects on hepatic and peripheral insulin resistance but only
when administered in mice with compromised insulin sensitivity.
Adipose tissue inflammation. Mice from all genotypes fed HFD
or HFCD showed increased macrophage infiltration in their adipose tissues compared with mice fed SD or CD, as shown by the
presence of crown-like structures (CLS) (Fig. 4, A and B). Feeding
CD, HFD, or HFCD increased several chemokines/cytokines
[monocyte chemoattractant protein-1, regulated on activation,
normal T cell expressed and secreted (RANTES), IL-1␤, IL-6,
interferon-␥ inducible protein 10 (IP-10)] in eWAT of C57 mice,
suggesting an effect on adipose tissue inflammation (Fig. 4, C–G).
We also observed a further increase in the diabetic LRKOB100/
IGF mouse model independent of obesity or CLS number. In
LRKOB100/IGF mice on CD and HFCD, there was a tendency
toward a decrease in RANTES, IL-1␤, and IP-10 compared with
the HFD group of the same genotype; however, this failed to
attain statistical significance. This may be related to the improvement of insulin resistance previously observed (Fig. 3).
Dietary cholesterol exacerbates aortic plaque formation and
impairs endothelial function. We then assessed atherosclerosis
to examine the influence of the insulin-resistant/diabetic state
on vascular complications. As expected, C57 mice failed to
develop atherosclerosis regardless of the diet (Fig. 5, A and C).
The LRKOB100 and LRKOB100/IGF groups developed atherosclerotic plaques on SD, and there was a trend of increased
lesion area with the CD and HFCD in LRKOB100 mice. In
contrast, relative to SD, the HFD did not exacerbate plaque
area in either LRKOB100 or LRKOB100/IGF mice despite
their overt glucose intolerance. Therefore, a dyslipidemic
genotype and dietary cholesterol additively promoted atherogenesis in these mouse models, whereas a triglyceride-rich diet
led to glucose intolerance but did not alone or in combination
with cholesterol exacerbate the development of atherosclerotic
plaques.
With regard to vascular reactivity, following an initial contraction with phenylephrine, there was a lessened maximal
vasodilatory effect of carbacholine on aortic rings from mice
fed the CD or HFCD compared with HFD or SD. Genotype
had no significant influence on endothelial function. C57 mice
fed CD and HFCD developed endothelial dysfunction but
without concomitant plaque formation, suggesting that the two
phenomena are not necessarily linked together (compare Fig. 5,
C and D). Indeed, endothelial dysfunction is often described as
an early event that can develop in the absence of structural
vascular or microvascular modification (3). In our models, endothelial dysfunction was associated with an increased plaque size
E579
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INSULIN RESISTANCE AND VASCULAR COMPLICATIONS
A
C57
LRKOB100
a
a
3
2
1
0
a
a
a,b
a,b
a,b
a,b
a,b
50
40
30
20
10
a
10
5
0
30
20
a
a,b,c
b,c
c,d
40
a,b,c,d
a,b
50
c,d
a
a
a
a
a
a
a
20
Prob(F)
< 0.0005
< 0.0001
b,c,d
25
LRKOB100/IGF
60
b,c,d
a
30
diet
genotype
70
b,c,d
a
a
a
< 0.05
LRKOB100
d
F
Prob(F)
genotype
C57
a,b,c
LRKOB100/IGF
pg IP-10/50 µg protein
LRKOB100
C 35
10
0
20
0
C57
LRKOB100
LRKOB100/IGF
50
40
30
a
a,b
a,b,c
a,b,c
60
a,b,c
70
b,c
a,b
40
80
LRKOB100/IGF
b,c
b
b
b
60
b
80
b
100
a,b
a,b
120
a,b
a,b
a,b
140
a,b,c
160
Prob(F)
< 0.005
genotype
diet*genotype < 0.05
90
a,b,c
a
diet
< 0.01
genotype
< 0.005
diet*genotype < 0.05
LRKOB100
a,b,c
G
Prob(F)
180
C57
a,b,c
D
LRKOB100/IGF
c
LRKOB100
pg RANTES/50 µg protein
C57
pg MCP-1/50 µg protein
60
0
C57
15
70
< 0.0001
b
a
a
4
a
a
a
a
HFCD
b
a
a
5
Prob(F)
genotype
80
a,b
a
HFD
6
HFCD
E 90
CD
< 0.0001
a
Crown-like structures/100
adipocytes (%)
diet
pg IL-1β/50 µg protein
Prob(F)
7
HFD
a,b
SD
B
pg IL-6/50 µg protein
CD
a,b
SD
20
10
0
C57
LRKOB100
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LRKOB100/IGF
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LRKOB100/IGF
INSULIN RESISTANCE AND VASCULAR COMPLICATIONS
Merat et al. (19) who showed that a normoinsulinemic fructose-fed LDLr⫺/⫺ mouse developed significantly greater atherosclerosis than a hyperinsulinemic and glucose-intolerant
Western diet-fed LDLr⫺/⫺ mouse. They are also consistent
with the report that dietary fat cannot alter plaque formation
unless the lipoprotein profile was made more atherogenic (31).
A recent study has also shown that hyperinsulinemia did not
affect plaque formation in ApoE⫺/⫺ mice, another genetic
model of atherosclerosis (22). However, previous studies could
not exclude the possibility that hyperglycemia and T2D were
actually required to further increase plaque formation, vascular
function, and atherosclerosis pathology. Our data using diabetic LRKOB100/IGF mice address this limitation of previous
studies and argue against the role of insulin resistance and
diabetes in promoting the development of lipid-rich atherosclerotic plaques. However, we should be cautious in inferring that
diabetes does not impact the development of atherosclerosis.
The present study investigated the presence of lipid-rich atherosclerotic plaques by the en face technique and did not
investigate plaque biology or stability. In this regard, other
effects of diabetes on plaque composition were not measured
and cannot be excluded. For instance, increased mineralization
of blood vessels is a hallmark of insulin resistance/diabetes in
both humans and animal models (12).
One intriguing observation is that, even though circulating
cholesterol levels were barely affected by dietary cholesterol,
increased endothelial dysfunction was still evident in mice fed
cholesterol regardless of genotype. Surprisingly, this was true
even in C57 mice in the absence of plaque formation. Zilversmit (32) extensively discussed the possibility that chylomicrons loaded with cholesterol from the diet may have important atherogenic properties. Chylomicrons are the main
carrier of cholesterol postprandially, and the first stage of their
catabolism takes place on endothelial cells where uptake by the
arterial wall can occur (25). The interactions between oxidized
chylomicron remnants and endothelial cells were reported to be
deleterious to endothelial cell function measured by carbacholine-mediated vasorelaxation (8). At least one other recent
study confirmed that addition of cholesterol to the diet only
contributes marginally to the total circulating amount of cholesterol but has important effects for atherosclerotic plaque
formation and that blocking intestinal cholesterol absorption
could alleviate these effects (11, 28). This could explain why
cholesterol derived specifically from dietary origin can have
such important vascular consequences, even before plaque
formation is visible. Finally, it cannot be ruled out that the
distinct metabolic impact of the HFD and HFCD are somewhat
linked to the overwhelming genetic effects on the lipid profiles
in the LDLRKOB100 models. Future work is also needed to
determine whether the metabolic effect of cholesterol in those
models can be linked to regulation at the level of the central
nervous system.
In summary, dietary triglycerides promote insulin resistance
and glucose tolerance but have marginal effect on atheroscle-
Fig. 4. Assessment of inflammation in adipose tissue of C57, LRKOB100, and LRKOB100/IGF mice following 24 wk on SD, CD, HFD, or HFCD. A and
B: F4/80 immunohistological staining was performed on epididymal adipose tissue (eWAT) for crown-like structure (CLS) numbering. Over 300 adipocytes on
5 different random fields were counted to establish the number of CLS/100 adipocytes. C–G: cytokines measured from eWAT lysates by Multiplex. Values are
adjusted by the protein content of the samples; n ⫽ 6 – 8 animals/group. Groups not sharing the same letter are considered statistically different when comparing
effects of both diet ⫻ genotype among the different groups.
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tance even in the face of a low-fat diet (9, 26). This is
especially important considering that the LDLr⫺/⫺ is a model
often used in metabolic studies.
Our clamp studies further documented that dietary cholesterol promotes insulin resistance in C57 mice. However, we
also made the surprising observation that insulin sensitivity
was improved by dietary cholesterol in both LRKOB100 and
LRKOB100/IGF mice even upon feeding a HFD. The molecular mechanisms underlying this effect of dietary cholesterol in
mice lacking the LDL receptor remains to be clarified, but our
clamp studies indicate that both a decreased hepatic glucose
output and an increased peripheral tissue glucose uptake contribute to this effect. Cholesterol derivatives have important
signaling properties in the liver through the liver X receptor
receptor family and are known to improve glucose metabolism
and may be involved in this unexpected improvement of
insulin sensitivity (13). The suppression of the lecithin cholesterol acyltransferase gene in LDLr⫺/⫺ animals led to increased
circulating free cholesterol and improved hepatic insulin signaling (18). A lack of functional LDL receptors has previously
been reported to protect peripheral tissues against ectopic
accumulation of cholesterol and the lipotoxicity resulting from
cholesterol accumulation (17). A similar peripheral tissue protection may have occurred in LRKOB100 and LRKOB100/
IGF groups. However, some insulin-sensitizing effect of
dietary cholesterol was also seen in high-fat-fed C57 mice,
suggesting that this protective effect is not simply related to
the lack of LDL receptor in the atherogenic models. In this
regard, our finding of reduced adipose tissue inflammation
in LRKOB100/IGF mice fed CD and HFCD suggests that
dietary cholesterol may also improve insulin sensitivity by
dampening obesity-linked inflammation.
Previous studies have reported that human familial hypercholesterolemia is not associated with significant alterations in
whole body glucose uptake, glucose oxidation, or lipid oxidation, arguing against a deleterious effect of cholesterol on
glucose metabolism (7, 16). Our observations should also be
considered in the context of recent evidence from the JUPITER
trial that showed that statins increased the occurrence of T2D
despite improvements in cholesterolemia and atherosclerosis
(23). In fine, if dietary cholesterol is known to promote arterial
lesions in mice, its metabolic effects are likely to be more
complex and even perhaps beneficial in certain circumstances
of already compromised insulin resistance.
Another interesting finding of our study is that we could
clearly dissociate the level of insulin resistance (as measured
by clamp studies) from the development of atherosclerosis in
an atherosclerosis-prone model. Indeed, dietary cholesterol
clearly impaired endothelial function and promoted plaque
formation despite its metabolic benefits in LRKOB100 and
LRKOB100/IGF mice. Moreover, LRKOB100/IGF mice showed
no alteration in plaque formation or endothelial function above
that of LRKOB100 regardless of the impact of diet on glucose
intolerance and insulin resistance. These data are in line with
E581
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INSULIN RESISTANCE AND VASCULAR COMPLICATIONS
A
C57
LRKOA100
SD
CD
HFD
HFCD
B
HFCD
80
CD
60
40
20
0
-8
-6
-4
80
40
20
0
-2
-8
C
-6
-4
CD
HFCD
80
60
40
20
0
-2
-8
Carbachol concentration (log M)
Prob(F)
< 0.005
SD
HFD
100
60
Carbachol concentration (log M)
diet
LRKOA100/IGF
SD
HFD
HFCD
CD
100
% relaxation
% relaxation
LRKOA100
SD
HFD
% relaxation
C57
100
-6
-4
-2
Carbachol concentration (log M)
D
Prob(F)
< 0.0001
diet
a
SD
b
CD
non detectable
C57
C57
a
HFD
b
HFCD
a,b
a,b
a,b
a,b
LRKOA100
LRKOA100
a,b
a,b
a,b
a
a
b
a,b
LRKOA100/IGF
b
LRKOA100/IGF
a,b
a,b
a,b
0
10
20
% plaque area
b
30
40
0
20
40
60
80
100
120
% maximum relaxation
Fig. 5. Assessment of vascular pathologies in C57, LRKOB100, and LRKOB100/IGF mice following 24 wk on SD, CD, HFD, or HFCD. A: quantification of
atherosclerotic plaque by the en face method. Representative aortas for atherosclerotic plaque formation were stained with Sudan IV, n ⫽ 5–7. B and C: carbacholine vasorelaxation for aortic rings (B) and quantification of atherosclerotic plaques in percent surface area (C). D: the maximal vasorelaxations are
displayed in a separate graphic for comparison, n ⫽ 7 animals/group. Groups not sharing the same letter are considered statistically different when comparing
effects of both diet ⫻ genotype among the different groups.
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LRKOA100/IGF
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INSULIN RESISTANCE AND VASCULAR COMPLICATIONS
A
SD
HFD
HFCD
CD
120
100
80
60
40
20
0
-10
-8
-6
-4
% relaxation
% relaxation
LRKOA100
140
140
SD
CD
HFCD
HFD
120
100
80
60
40
20
0
-10
SNP concentration (log M)
-8
-6
-4
% relaxation
C57
140
LRKOA100/IGF
HFD
SD
HFCD
CD
120
100
80
60
40
20
0
-10
SNP concentration (log M)
-8
-6
-4
SNP concentration (log M)
B
CD
a
C57
HFD
a
HFCD
a
a
a
LRKOA100
a
a
a
a
LRKOA100/IGF
a
a
0
20
40
60
80
100
120
140
% maximum relaxation
Fig. 6. A: effect of a nitric oxide donor (SNP) on aortic vasorelaxation. B: the maximal vasorelaxations are displayed in a separate graphic for comparison,
n ⫽ 7 animals/group. Groups not sharing the same letter are considered statistically different when comparing effects of both diet x genotype among the different
groups.
rosis. Dietary cholesterol also induces metabolic impairments
in C57 mice but remarkably improves insulin resistance in
high-fat-fed insulin-resistant animals. This dual action of cholesterol on metabolism may be linked to adipose tissue inflammation, but further studies will be required to determine
whether such inflammation promotes endothelial dysfunction
before the onset of measurable atherosclerosis or whether these
two events proceed concurrently. We conclude that dietary
triglycerides and cholesterol have distinct metabolic and vascular consequences in obese atherogenic mouse models. We
also found a clear dissociation between the impairment of
glucose homeostasis and the development of atherosclerosis
and endothelial function in these mouse models, suggesting
that diabetes per se does not play a dominant role in the
development of lipid-rich atherosclerotic plaques.
ACKNOWLEDGMENTS
We thank Christine Dion and Kim Denault (Centre de Recherche de
l’Institut Universitaire de Cardiologie et Pneumologie de Québec) for help
with animal procedures and Dr. Dominic Ng (St. Michael’s Hospital/Research
Institute, University of Toronto) for critical reading of this manuscript.
GRANTS
This work was supported by a grant (no. 161971 to A. Marette) from the
Canadian Institutes of Health Research (CIHR) and a CIHR/Pfizer Research
Chair to A. Marette in the pathogenesis of insulin resistance and cardiovascular
diseases. M.-A. Laplante was supported by the Heart and Stroke Foundation of
Canada.
DISCLOSURES
André Marette is the guarantor of this work and, as such, had full access to
all the data in the study and takes responsibility for the integrity of the data and
the accuracy of the data analysis. The authors declare no conflict of interest.
AUTHOR CONTRIBUTIONS
Author contributions: M.-A.L., A.C., R.K.A., and A.M. conception and
design of research; M.-A.L., A.C., R.K.A., P.P., and X.F. performed
experiments; M.-A.L., A.C., R.K.A., Y.D., and A.M. analyzed data; M.A.L., A.C., R.K.A., H.B., S.Y.-H., M.L., J.-P.D., Y.D., G.S., P.M., and
A.M. interpreted results of experiments; M.-A.L., A.C., R.K.A., and X.F.
prepared figures; M.-A.L., R.K.A., and A.M. drafted manuscript; M.-A.L.,
A.C., R.K.A., P.P., X.F., H.B., S.Y.-H., M.L., J.-P.D., Y.D., G.S., P.M.,
and A.M. edited and revised manuscript; M.-A.L., A.C., R.K.A., P.P., X.F.,
H.B., S.Y.-H., M.L., J.-P.D., Y.D., G.S., P.M., and A.M. approved final
version of manuscript.
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INSULIN RESISTANCE AND VASCULAR COMPLICATIONS
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