Pfizer Global R&D, Alameda, California 94502
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ABSTRACT |
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The effects of high-fat feeding on
the development of obesity were evaluated in intercellular adhesion
molecule-1 (ICAM-1) knockout and C57BL/6J (B6) male mice fed a high-fat
diet for 50 days. Serum and tissues were collected at baseline and
after 1, 11, and 50 days on the diet. After 11 days on the diet,
ICAM-1-deficient, but not B6, mice developed fatty livers and showed a
significant increase in inguinal fat pad weight. At day 50,
ICAM-1-deficient mice weighed less, and their adiposity index and
circulating leptin levels were significantly lower than those of B6
controls. To better understand the early differential response to the
diet, liver gene expression was analyzed at three time points by use of
Affymetrix GeneChips. In both strains, a similar pattern of gene
expression was detected in response to the high-fat diet. However,
sterol regulatory element-binding protein-1, apolipoprotein A4, and
adipsin mRNAs were significantly induced in ICAM-1-deficient livers,
suggesting that these genes and their associated pathways may be
involved in the acute diet response observed in the knockout mice.
intercellular adhesion molecule-1 knockout mice; obesity; dyslipidemia; steatosis; microarray
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INTRODUCTION |
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THE PHYSIOLOGICAL EFFECTS of murine diet composition have been the focus of much study, particularly regarding obesity, diabetes, and atherosclerosis (11, 26, 29, 31, 37, 41, 44). The studies of these human pathologies have been greatly furthered by the use of the C57BL/6J inbred strain because of its susceptibility to these disorders, thus serving as a model for the human diseases. Liver steatosis, or fatty liver, is characterized by excessive lipid accumulation in the hepatocytes and is a related pathology associated with obesity and type 2 diabetes (23, 36). The excess liver lipid accumulation may be comprised of cholesterol or triglycerides or both. Although hepatic lipid deposition may result from a number of causes, few genes have been positively correlated with this pathology (12, 19, 23, 33, 36).
Intercellular adhesion molecule-1 (ICAM-1) is a cytokine-responsive
integral membrane receptor having five immunoglobulin (Ig)-like
extracellular domains, a transmembrane domain, and a short cytoplasmic
tail. ICAM-1-deficient mice were previously reported to spontaneously
develop maturity-onset obesity and fatty livers without an increase in
food intake, and they had an increased susceptibility to obesity
induced by a high-fat diet (6). Adhesion molecules such as
ICAM-1 are known to play an important role in the development of
atherosclerosis (1, 40) and may also contribute to the
atherosclerosis pathology in diabetic individuals (3, 14).
There has been no reported characterization of the molecular mechanism
involved in ICAM-1-deficient obesity, and the role of leukocytes in the
regulation of both lipid metabolism and energy expenditure remains
obscure. A possible role for the peroxisome proliferator-activating
receptor- (PPAR
) transcription factor pathway was suggested
(6), but this hypothesis has not been validated. Although
it is not known whether repression of leukocyte adhesion receptors is
associated with obesity in humans, ICAM-1 (
/
) mice represent an
interesting animal model for studying potentially novel mechanisms that
regulate adiposity without significant appetite modification.
In this report, we have characterized the response of C57BL/6J
(B6) and ICAM-1 (/
) mice to high-fat feeding at both the physiological and molecular levels. With short-term high-fat feeding (11 days), the ICAM-1 (
/
) mice accumulated significantly more adipose and liver lipid than B6 controls. However, after 50 days of
high-fat feeding, the B6 mice weighed more than ICAM-1 (
/
) mice,
and the liver lipid accumulation was similar in the two strains. By
assessing the expression levels of thousands of genes by use of an
Affymetrix GeneChip array, we sought to gain insights into the
molecular events that are altered in the livers during the development
of dietary obesity. Microarray technology has been successfully
used to identify differences in gene expression in various altered
metabolic conditions, including aging and caloric restriction, genetic
obesity, diabetes, and cancer (5, 9, 21, 25, 38). However,
this is the first report to describe global changes in liver gene
expression upon high-fat feeding.
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MATERIALS AND METHODS |
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Animals.
ICAM-1-deficient mice [ICAM-1 (/
); stock no. 002867] and C57BL/6J
mice (B6; stock no. 000664) were obtained from the Jackson Laboratory
(Bar Harbor, ME). The ICAM-1 (
/
) mice were originally generated in
a mixed 129-B6-DBA background (43) and then backcrossed to
the C57BL/6J background for 8 generations. The homozygous knockouts are
viable and fertile, allowing ICAM-1 (
/
) breeding pairs to be
intercrossed to maintain the line. The experimental colony described
here were bred and maintained on a mouse chow diet (Laboratory Rodent
Diet 5001, PMI Feeds, St. Louis, MO). Male mice were individually housed at 6 wk of age. At 7 wk of age, these mice were switched to the
Western type of high-fat diet that contained 42% of calories as fat
(Adjusted Calories Diet no. 88137, Harlan Teklad, Madison, WI). Body
weights and food intakes were measured twice a week from the start of
high-fat feeding. Mice were maintained on a 12:12-h light-dark cycle,
with water ad libitum. All reported investigations were
carried out in accordance with the Guidelines of the National
Institutes of Health and Medical Research (NIH publication No.
87-848, 1987). To generate ICAM-1 (
/
) and control mice of the
proper background strain, ICAM-1 (
/
) males were mated to C57BL/6J
females. The progeny of this backcross were intercrossed to produce
F2-generation littermates of the three genotypes (
/
, +/
, and
+/+). Genotypes of the wild type and knockout ICAM-1 alleles of all of
the experimental mice were confirmed by PCR. The wild-type allele was
amplified by PCR primer pairs IC1-1 (5'
TGCCTCTGAAGCTCGGATATACC-3') and IC1-2 (5'
CTGTAGACTGTTAAGGTCCTCTGCG-3'). The knockout allele was amplified with
primers IC1-1 and PGK-1 (5'-TGAGCCCAGAAAGCGAAGGAA-3'). PCR was
performed in a 15-µl volume containing 0.8 µM of pairs of either
primer and 25 ng of genomic DNA. During amplification, a 5-min
denaturing step at 95°C was followed by 30 cycles of 30 s at
94°C, 30 s at 57°C, and 2 min at 72°C.
Serum chemistry and tissue collection.
Mice from all experimental groups were killed at baseline (day
0) or after 1, 11, or 50 days on the diet. The animals were fasted
for 4-5 h before blood and tissue collection. After a drop of
blood was obtained from the tail vein for glucose measurements, each
mouse was anesthetized with isoflurane and weighed. Blood was collected
by cardiac puncture and subsequently assayed for insulin, leptin, and
lipid levels. Circulating insulin and leptin levels were determined by
radioimmunoassays (Linco Research, St. Charles, MO). Serum triglyceride
levels were determined by a colorimetric assay kit (Wako Chemicals USA,
Richmond, VA). Serum lipoproteins were separated by HPLC (Varian,
Walnut Creek, CA) by use of a Superose 6 column (Amersham Pharmacia
Biotech, Piscataway, NJ), and the cholesterol content in each
lipoprotein fraction was determined by use of a cholesterol esterase
kit (Roche Diagnostic Systems, Somerville, NJ). Livers and individual
fat pads were dissected, weighed, and snap-frozen in liquid nitrogen.
The adiposity index for individual animals was calculated by dividing
the sum of the weights of the dissected fat depots by the body weight
(minus the fat depot weight). Although this method is not as accurate as carcass analysis, it gives a more rapid and reproducible evaluation of body fat content in mice (40). Liver histology was
evaluated on either paraffin or frozen sections. For paraffin sections, the livers from three B6 and five ICAM-1 (/
) males at day
11 of high-fat feeding were dissected, fixed in 10% formalin, and embedded in paraffin. Sections (5 µm) were stained with hematoxylin and eosin. For frozen sections, livers from five male ICAM-1 (
/
) and B6 mice fed the high-fat diet for 11 days were snap-frozen. Sections of 10-15 µm were cut, fixed in formalin, and stained with Oil Red O.
Expression profiling.
Total RNA was isolated from the livers of seven mice in each group at
days 0 and 11 and of four ICAM-1 (/
) and six
B6 livers at day 11 with Trizol reagent (Life Technologies,
Gaithersburg, MD). To prepare samples for Affymetrix GeneChip analysis,
cDNA was generated from 15 µg of pooled total RNA (50 µg RNA per
liver per time point and genotype, as indicated above) by use of a
modified oligo-dT primer and a 5' T7 RNA polymerase promoter oligo
primer with the Superscript Choice System for cDNA Synthesis (Life
Technologies). After phenol-chloroform extraction and ethanol
precipitation, one-half of the cDNA reaction (0.5-1.0 µg) was
used as a template for an in vitro transcription reaction (BioArray
High Yield kit, Enzo Biochem, Farmingdale, NY) by following the
manufacturer's protocol. The resulting cRNA was purified on an
affinity resin column (RNeasy, Qiagen, Valencia, CA) and quantified by
ultraviolet (UV) absorbance. For each reaction, 15 µg of biotinylated
cRNA were randomly fragmented to an average size of 50 nucleotides by
incubating them at 94°C for 35 min in 40 mM Tris-acetate, pH 8.1, 1,000 mM potassium acetate, and 30 mM magnesium acetate. The fragmented
cRNA was divided into two aliquots that were each used for
hybridization to an MU6500 Affymetrix GeneChip according to the
manufacturer's protocol (Affymetrix, Santa Clara, CA), with a
duplicate data set generated for all samples. Data were analyzed with
the Affymetrix software algorithm to generate "average difference"
and/or degree of difference. When the degree of difference was
calculated, each average difference <10 was brought up to 10 to get
meaningful fold difference value. Variation between duplicate samples
did not exceed 30%. Therefore, the mean of the average difference
values obtained for the duplicates was used to generate Table 3. For
Northern blot analyses, duplicate samples of 20 µg of pooled total
liver RNA were separated on 1% agarose gels containing 6.7%
formaldehyde in 1× MOPS buffer and blotted onto Duralon-UV membranes
(Stratagene, La Jolla, CA). 32P-labeled cDNA probes were
hybridized to the Northern blots by use of the ExpressHyb solution
(Clontech, Palo Alto, CA), and radioactive signals were analyzed by the
Storm System phosphoimager with ImageQuant 5.0 software (Molecular
Dynamics, Sunnyvale, CA). The 36B4 message was used as a control for
gene expression levels (20). The cDNA probes were
generated either by RT-PCR by use of gene-specific primers and liver
total RNA as template (adipsin probe only, forward primer:
5'-TCCGCCCCTGAACCCTACAAG-3'; reverse primer:
5'-CTTTTTGCCATTGCCACAGACG-3'; product size 447bps) or by PCR of IMAGE
clones obtained from Research Genetics (Huntsville, AL) as
described below. The IMAGE clone numbers are for ICAM-1, 315622; malic
enzyme (ME), 876123; sterol regulatory element-binding protein
(SREBP)-1, 747669; fatty acid synthase (FAS), 851908; apolipoprotein A (apoA)-4, 481110; keratinocyte fatty acid binding protein (mal 1), 1196423; squalene synthase, 1347569; PPAR
, 677502; PPAR
, 317536; carnitine o-palmitoyltransferase 1 (CPT-1),
335258; and 36B4, 331627. Esherichia coli harboring the
desired IMAGE clones were grown in a 96-well plate overnight in LB
medium containing the appropriate antibiotic for plasmid selection. To
amplify the plasmid inserts, PCR was performed on 1 µl of liquid
culture in a 50-µl reaction using T3 and T7 primers (category no.
302001, Stratagene) and the following PCR conditions: denaturation at 96°C for 3 min followed by 35 cycles of 30 s at 94°C, 60 s at 55°C, and 90 s at 72°C, finishing with 5 min at 72°C
for a final elongation. All amplified inserts generated by PCR were
sequence-verified, as previously described (10).
Data and statistical analysis. The data were analyzed by two-way ANOVA (strain × diet) with SAS (Morrisville, NC) version 8 and the ANALYST program set. Pair-wise statistical significance was established using a post hoc Student t-test. In some cases, statistical outliers were removed and the analysis was revisited; however, no effect was found on the overall significance levels. Data are presented as means ± SE. Statistical signifiance is defined as P < 0.05. Affymetrix GeneChip studies considered those gene expression changes >2-fold and those <0.5-fold to be significantly changed, as recommended by Affymetrix.
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RESULTS |
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ICAM-1 (/
) and C57BL/6J (B6) mice were fed a high-fat diet,
containing 42% of calories as fat, beginning at 7 wk of age. Body
weights and food consumption were measured twice a week beginning at
day 0 on the diet and ending at day 50. Contrary
to a published report (6), ICAM-1-deficient male mice did
not gain more weight than control mice upon high fat feeding. Although
both strains were diet responsive, B6 male mice were on average 4 g heavier than ICAM-1 (
/
) animals by day 50 (Table
1). Moreover, no significant differences
in body weight and adiposity were found between high fat-fed B6 and
ICAM-1 (
/
) females, and for both sexes, maturity-onset obesity was
not observed in animals on the chow diet (not shown). In all cases, no
significant difference in food intake was detected between strains (not
shown). Overall, independent of the gender, no obvious obesity
phenotype was detected in ICAM-deficient mice. Therefore, a detailed
phenotyping analysis was performed on male mice only, and the following
parameters were measured: body mass index (BMI); liver weight; fat pad
weights; adiposity index (Table 1); leptin, glucose, and insulin levels
(Table 1); and blood lipid levels (Table
2).
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Individual fat pad weights from ICAM-1 (/
) male mice were compared
with those from B6 male mice. Before high-fat diet feeding, there were
no significant differences in the relative weights of the examined fat
pads, except for the relatively smaller epididymal fat in ICAM-1
(
/
) (Table 1). At day 11, inguinal and brown fat pad
weights were significantly greater in ICAM-1 (
/
) mice than in B6
mice. However, the difference in weight of these fat pads disappeared
by day 50 (Table 1). The intra-abdominal mesenteric, retroperitoneal, and epididymal fat depots showed a slightly different profile but were all significantly heavier in B6 than in ICAM-1 (
/
)
mice by day 50 (Table 1). As a result, the adiposity indexes for the two strains were the same at days 0 and
11 but were significantly higher for B6 mice at day
50 (Table 1). Serum leptin levels were the same at day
0, high at day 11, and low at day 50 in
ICAM-1 (
/
) males compared with the B6 males (Table 1).
Eleven days after initiation of the high-fat feeding, a number of
differences were apparent in livers taken from ICAM-1 (/
) male
mice. Upon gross observation, ICAM-1-deficient livers were paler,
distended, and heavier than B6-derived livers, suggesting hepatic
steatosis (not shown and Table 1). The histological analyses revealed
major hepatic abnormalities in ICAM-1-deficient mice that corresponded
to excessive lipid accumulation, as assessed by the lipid-specific Oil
Red O-positive staining of frozen liver sections (Fig.
1). After 50 days of high-fat feeding,
there were no more significant differences between B6 and ICAM-1
(
/
) liver appearance, with both strains exhibiting equally fatty
livers (not shown).
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To characterize the effects of high-fat feeding on glucose metabolism, blood glucose and insulin levels were measured in both strains. Serum insulin levels were higher in ICAM-1-deficient mice than in B6 mice before and also after 11 days of high-fat feeding (Table 1). The difference in blood insulin levels became statistically insignificant between the two strains after 50 days of high-fat feeding. Despite elevated insulin levels in the knockouts at days 0 and 11, the blood glucose levels were not significantly different at any time point in the two strains (Table 1). Glucose tolerance and insulin sensitivity were also evaluated after 6 wk of high-fat diet feeding but did not differ between strains (not shown).
The analysis of blood lipid profiles of B6 and ICAM-1 (/
) mice
showed that the knockout mice had higher levels of serum triglycerides
and very low density lipoprotein (VLDL) cholesterol than the B6 mice
when fed a high-fat diet for 11 days (Table 2). Similar results were
obtained from mice fed a chow diet, suggesting that the observed
differences are not dependent on the dietary fat quantity or
composition. In contrast, no significant differences were found in
low-density lipoprotein (LDL), high-density lipoprotein (HDL), and
total cholesterol levels between these two strains (Table 2).
To determine whether the discrepancies in our results compared with
those of Dong et al. (6) were due to a strain background effect rather than ICAM-1 deficiency, ICAM-1 (/
) males were backcrossed to C57BL/6J females. The F1 progeny was intercrossed to
obtain F2-generation animals of all genotypes at the ICAM-1 locus. Male
knockout or wild-type F2 animals were individually housed and fed the
high-fat diet, as described previously. At days 0,
11, and 50, animals were killed and fat pads were
dissected and weighed as described. Figure
2 depicts body weight gain, liver, inguinal fat, and epididymal fat weights over time for all genotypes. Figure 2A shows the weight gain over time in B6 male mice
and ICAM-1 knockout males (left) and the weight gain over
time of F2-generation ICAM-1 (+/+) male mice compared with that of
their ICAM-1 (
/
) male littermates (right). Body weight
gain over time was significantly higher for B6 control mice compared
with ICAM-1 (
/
) mice (Fig. 2A, left). In
contrast, no significant difference in weight gain was found between
ICAM-1 (+/+) and ICAM-1 (
/
) F2 littermates (Fig. 2A,
right). In neither case did ICAM-1-deficient mice gain
weight as rapidly as wild-type animals. After 11 days of high-fat
feeding, the livers and the inguinal fat pads derived from ICAM-1
(
/
) males weighed more, as a percentage of body weight, than those
of B6 mice (Fig. 2B, left) or than those of their
ICAM-1 (+/+) littermates (Fig. 2B, right). In
contrast, at every time point analyzed, a distinct picture emerged for
the epididymal fat pad weight in the F2 generation. The significant differences that were detected between B6 controls and ICAM-deficient mice (Fig. 2B, left) were abolished between (+/+)
and (
/
) F2 littermates (Fig. 2B, right).
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Because ICAM-1 (/
) mice developed fattier livers, heavier fat pads,
and hyperlipidemia by day 11 of high-fat feeding, specific mRNA levels were compared from livers of ICAM-1 (
/
) and B6 male mice at this time point by use of Affymetrix MU6500 GeneChips. RNAs
harvested from B6 livers obtained at day 0 were used as
baseline controls. To address potential immediate responses to high-fat feeding, day 1 liver total RNA samples were also obtained
for expression analysis. To generate pair-wise comparisons, the gene expression data were analyzed with the Affymetrix software. First, pair-wise comparisons were performed in a time-dependent fashion: for
each strain, gene expression levels at day 1 and day
11 were compared with the day 0 levels. Table 3, which
is available as Supplementary Material at the AJP-Endocrinology and
Metabolism web site, lists the genes that were differentially regulated
at day 1 and/or day 11 in B6 controls and/or
ICAM-1-deficient livers. In addition, pair-wise comparisons were also
performed in a strain-dependent way: for each time point, the gene
expression levels were compared between B6 control and ICAM-deficient
mice. Overall, there were fewer expression changes greater than twofold
between the two strains at any of the time points examined than there
were within a strain at the three time points of high-fat feeding (data
not shown).
Pair-wise comparisons of mRNA levels between days 1 or
11 and day 0 indicated that several genes
involved in lipogenesis and lipid transport were similarly induced at
day 1 but returned to baseline levels by day 11 in both ICAM-1 (/
) and B6 male mice (Table 3). In both strains,
genes involved in cholesterol synthesis, including hydroxymethyl
glutaryl-coenzyme A (HMG-CoA) reductase, squalene synthase, and
squalene epoxidase, were repressed at day 11. Interestingly,
SREBP-1, an important liver transcription factor controlling a large
number of genes involved in the metabolism of cholesterol and other
lipids, was induced at day 1 and day 11 in
ICAM-1-deficient mice only. Among the secreted proteins, the pattern of
expression of adipsin markedly differed between the two strains: a
dramatic induction of the adipsin message was observed in
ICAM-deficient livers (Table 3 and Fig.
3). In addition, significant differences
in apoA-4 mRNA levels were also detected between strains. Although the
difference in apoA-4 expression was minimal when the pair-wise
comparisons were performed in a time-dependent fashion for each strain
(day 1 vs. 0 and day 11 vs. day
11, Supplementary Table 3), this was not the case when the
pair-wise comparisons were performed in a strain-dependent fashion
(ICAM-deficient vs. B6 control) for a chosen time point. At day
11, the average signal was about fivefold higher in ICAM-deficient livers compared with B6 control livers (average signal 2164 for ICAM-deficient vs. 439 for B6 control).
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To validate the GeneChip results, a few genes involved in lipid
metabolism were arbitrarily selected, and Northern blot analysis was
used to confirm differential expression. Lack of ICAM-1 message in the
knockout mice was also verified by this method (Fig. 3). As shown in
Fig. 3, the transcriptional alterations observed by GeneChip analysis
for ME, FAS, mal 1, SREBP-1, apoA-4, and adipsin were confirmed. A
comparison of the degree of differences in hybridization intensity over
time from the Northern blot analysis and Affymetrix data is shown in
Fig. 4. Overall, this graphic comparison
of the data shows that the results obtained by the two methods are very similar. The only exception is squalene synthase, for which the message
levels were method dependent, both in degree of change and in overall
trend (Fig. 4).
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The PPAR transcription factor pathway has been suggested to be
involved in ICAM-1 (
/
) obesity (6). As shown in Fig. 5, no difference in message levels of
either PPAR
or CPT-1, a gene transcriptionally controlled by
PPAR
, was found between ICAM-1-deficient and B6 control livers at
any of the time points examined. Figure 5A shows the
Northern blot, and Fig. 5B shows the relative expression
level, expressed as degrees of difference over time, for both Northern
blot and GeneChip analysis. Although PPAR
and CPT-1 mRNA levels did
not differ between strains, the Northern blot results revealed that
both genes were induced by approximately twofold after 11 days of
high-fat feeding (Fig. 5A). In contrast, PPAR
mRNA levels
were slightly elevated only at day 11 and only in the
knockout mice (Fig. 5, A and B).
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DISCUSSION |
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ICAM-1-deficient mice were previously reported to develop
maturity-onset obesity spontaneously without an increase in food intake
and also to demonstrate increased susceptibility to obesity when fed a
Western type of high-fat diet (6). These authors suggested
that leukocytes can suppress excess triglyceride deposition and may be
involved in modulating lipid transport and storage. We sought to
understand the underlying molecular mechanism responsible for these
reported differences in fat deposition in ICAM-1 (/
) animals
compared with control animals.
Interestingly, our findings did not confirm those of Dong et al.
(6). We did not observe spontaneous obesity in either gender, even when ICAM-1 (/
) animals were fed chow diet for over a
year (data not shown). Moreover, after 50 days of high-fat feeding, no
significant increase in body weight, adiposity index, or fat pad
weights was observed in male ICAM-1 (
/
) mice over that of the B6
controls (Table 1).
Although we cannot exclude a role of environmental differences such as batch of diet, source of water, type of cage, ambient temperature, and/or the immune environment, we believe that discrepancies in results between the two studies are due to differences in strain background. The animals that Dong et al. (6) used in their work were originally established on a mixed 129/B6/DBA2 background and were backcrossed four times (N4) to the B6 background. The animals used in this study were similar except that they were backcrossed either eight (N8) or nine (N9 for the F2 generation) times to B6 mice. Therefore, our animals had a significantly higher percentage of B6 genome than the mice used in the study conducted by Dong et al. The contribution of 129/DBA2 genomes at the N4 is statistically expected to be ~4.7%, whereas it should be <0.3% at N8-N9. A genome-wide scan for allelic contribution would be necessary to assess the fundamental differences between the strains used in both studies. Our results reemphasize the often-overlooked power of gene interactions and their influence on the phenotype of knockout animals.
In our study, the comparison between either B6 and ICAM-1-deficient
mice or wild-type and ICAM-1 (/
) F2 littermates revealed that
significant differences were present at day 11 for inguinal fat and liver tissue weights, expressed as a percentage of body weight.
Although these differences were smaller or had vanished by day
50, they may indicate that ICAM deficiency alters the early metabolic response to high-fat feeding. Therefore, we used DNA microarrays to assess the changes in liver gene expression associated with high-fat feeding and hepatic lipid deposition in B6 vs.
ICAM-1-deficient mice. In addition to comparing the two mouse strains,
we also analyzed different time points to better dissect the metabolic response to high-fat feeding. A total of 83 genes were differentially regulated either between strains or within a strain in a time-dependent fashion. These genes were distributed in nine different classes, including cell growth, arrest, and death, transcription factors, and
carbohydrate and lipid metabolism enzymes. Initially we were struck by
the low number of expression differences between strains at the three
time points analyzed. However, upon examination of the data, we found
that most of the expression changes were in a similar direction in both
strains, but the magnitude of the change differed. As a result, few of
the expression changes were greater than twofold when the two strains
were compared with one another at the specific time points. The
expression differences between strains are a measure of the
compensatory changes resulting from the ICAM-1 (
/
) mice and also of
the differences in strain background. Because we were primarily
interested in the fatty liver phenotype, several genes falling into the
"lipid metabolism" category were chosen for the follow-up studies.
Most of these genes were acutely regulated, because they were markedly
induced after 1 day and repressed after 11 days of high-fat feeding.
This reemphasized the necessity of including multiple time points in gene expression profiling studies, because a single time point allows
only the detection of too much (day 1) or too little
(day 11) change in mRNA levels.
Previous research on hepatic steatosis in mice has examined the control of metabolic pathways involved in this disease, but so far only a few genes have been positively correlated with this pathology (23, 36). SREBP-1 protein and FAS mRNA levels were found to be elevated in the fatty livers of ob/ob mice, implicating increased cholesterol and fatty acid biosynthesis as potential causes of the lipid deposition (36). SREBP-1 is an important liver transcription factor controlling a large number of genes involved in the metabolism of cholesterol and other lipids. There are two splice variants of the SREBP-1 protein, known as SREBP-1a and SREBP-1c. Elevated SREBP-1 gene expression is observed at both day 1 and day 11 in ICAM-deficient livers, suggesting that the SREBP-1 pathway is involved in the rapid hepatic lipid accumulation observed in the knockout animals. However, neither the GeneChip nor the Northern blot analysis can differentiate between the splice variants, impairing accurate identification of the regulated isoform. SREBP-1c is a likely candidate, because circulating insulin levels are elevated in ICAM-deficient mice at day 11. Insulin was indeed shown to selectively increase SREBP-1c mRNA in both cultured hepatocytes and diabetic rat liver (8, 35, 36). In contrast, the expression of several SREBP-1 downstream genes, including FAS, ME, ATP-citrate lyase , HMG-CoA reductase, and squalene synthase (34), did not follow a similar pattern and was generally similar in both strains. Transcriptional activation is a very complex phenomenon, and these genes are most likely coregulated by other transcription factors. These additional players may not be differentially regulated by high-fat feeding or may not differ between the two mouse strains. In addition, it should be kept in mind that the mRNA level may not appropriately reflect the protein level and/or the transcriptional activity.
Among other genes that were differentially expressed but not selected for confirmatory studies, SPOT14 would deserve further examination. Although SPOT14 expression was repressed after 11 days of high-fat feeding in both strains, its mRNA level was still significantly elevated in ICAM-1 knockout mice compared with B6 control mice. Interestingly, SREBP-1 was shown to regulate SPOT14 gene transcription positively (12, 24). Therefore, the elevated SREBP-1 expression detected in ICAM-deficient mice at day 11 may help to sustain SPOT14 mRNA. Although the function of SPOT14 is still poorly understood, it was reported to play a role in lipid synthesis (17), indicating that it may play a key role in the rapid development of the fatty liver phenotype observed in ICAM-deficient mice.
At all time points examined that were independent of the diet, apoA-4
message levels were higher in ICAM-1 (/
) animals than in B6 mice,
and the differences in message levels were particularly pronounced
after 11 days of high-fat feeding. In the knockout mice, both SREBP-1
and PPAR-
were induced at day 11, suggesting that perhaps
these two transcription factors may act in a coordinate fashion to
increase apoA-4 gene expression. Other apolipoproteins, such as apoA-2,
are indeed known to be transcriptionally regulated by both SREBP-1 and
PPAR
(27, 28), and a similar mechanism may be taking
place for apoA-4 in ICAM-1-deficient liver. The differential apoA-4
expression observed between ICAM-1 knockout and control mice may also
be due to the genetic variation between these strains rather than to
the lack of ICAM-1. Striking genetic variations in the levels of apoA-4
mRNA in the liver have previously been reported among inbred mouse
strains (30), and, as stated above, the genetic background
of ICAM-1-deficient and control mice used in this study significantly
differed. The determination of apoA-4 expression levels in (
/
) and
(+/+) F2 littermates would be necessary to clarify this issue.
Overexpression of apoA-4 was shown to result in high plasma
triglyceride, free fatty acid, total cholesterol, and HDL cholesterol
levels when mice were fed an atherogenic diet (4).
Therefore, the elevated apoA-4 expression may contribute to the
elevated circulating triglyceride levels observed in ICAM-1 (
/
)
males fed either the chow or high-fat diet. However, triglyceride and
cholesterol levels did not correlate well with the pronounced elevation
in apoA-4 message detected in high fat-fed knockout mice. Further
investigations will be necessary to support this hypothesis. In
rodents, the intestine accounts for the major proportion of circulating
apoA-4 (15). Therefore, it will be of interest to
determine whether the message levels of apoA-4 are also elevated in the
intestine of ICAM-1 (
/
) mice, and to determine whether these mice
have elevated levels of plasma apoA-4.
The most unexpected and novel finding of this study concerns the
dramatic induction of adipsin mRNA in high fat-fed ICAM-deficient livers. In humans and in rodents, the major site of synthesis of
adipsin, also known as factor D, is adipose tissue (42), and this is the first study to report adipsin expression in rodent liver. Our data also demonstrate that liver adipsin mRNA levels are
diet regulated and indicate that this regulation is tissue specific,
because adipose tissue adipsin mRNA levels are not changed by
overfeeding or high-fat feeding (7, 32). It is very likely that adipsin synthesis occurs in the hepatocyte rather than in any
other liver cell type, because adipsin/factor D was recently reported
to be constitutively synthesized by normal cultured human hepatocytes
(18). Adipsin/factor D, together with factor B and factor
C3, are components of the alternative complement pathway circulating in
plasma. Interaction among these three factors leads to production of
C3a, which is almost immediately converted to C3adesarg, also known as
acylation stimulation protein or ASP (2). ASP is a major
determinant of the rate of triglyceride synthesis in the adipocyte and
therefore may also play a role in hepatic lipid storage. However,
assessing ASP production in liver and in adipose tissues would be
necessary to determine whether the increase in adipsin mRNA levels
correlates with increased level of ASP. Although adipsin gene induction
was much more dramatic in ICAM-1-deficient livers, adipsin message was
also significantly increased in control mice after 11 days of high-fat
feeding. Interestingly, both strains had equally fatty liver by
day 50, supporting the hypothesis that elevation of adipsin
message in liver may contribute to hepatic lipid accumulation. In light
of our findings, it would be interesting to determine whether the
plasma levels of adipsin and/or ASP are altered in ICAM-1 (/
) and
control mice.
The PPAR pathway is also involved in the development of fatty liver:
PPAR
deficiency led to massive hepatic accumulation in response to
short-term starvation or a high-fat diet (16, 22). In
addition, the PPAR
pathway was suggested to be involved in the
development of obesity in ICAM-1 (
/
) mice (6).
However, neither our GeneChip nor our Northern blot data support this
hypothesis. No significant difference in expression levels of PPAR
or CPT-1, a PPAR
downstream gene, was observed between the two
strains at any of the three time points. Moreover, both PPAR
and
CPT-1 mRNA levels were elevated in both strains at day 11,
suggesting that fatty acid
-oxidation was increased in response to
high-fat feeding.
Overall, our results clearly indicate that ICAM-1 deficiency does not result in an obesity phenotype, and they question the function of this adhesion molecule in the regulation of body weight and adipose tissue mass. In contrast, the physiological analysis has revealed a differential response to feeding a high-fat diet, mostly in terms of lipid deposition in the liver and in specific fat pads. Additionally, the hepatic mRNA expression analysis has provided a number of tantalizing targets and possible novel regulatory pathways involved in liver lipid metabolism that deserve further study. Confirming these changes at the protein level would be the first necessary step in assessing their biological relevance.
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ACKNOWLEDGEMENTS |
---|
We thank Steve Madore and Tim Jatkoe for assistance with the Affymetrix studies, Judy Udove and Eric Kaldjian for histological preparations, and Rong Ni and Arnold Essenburg for excellent technical help.
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FOOTNOTES |
---|
* These authors contributed equally.
Address for reprint requests and other correspondence: F. M. Gregoire, Metabolex, Inc., 3876 Bay Center Place, Hayward, CA 94545 (E-mail: fmgregoire{at}metabolex.com).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpendo.00072.2001
Received 28 February 2001; accepted in final form 26 October 2001.
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