From the U465 INSERM, Centre de Recherches Biomédicales des
Cordeliers (Université Paris 6), 15 Rue de l'Ecole de
Médecine, 75270 Paris Cedex 06, France and the
Centre de Recherche Smithkline Beecham, 4 Rue du Chesnay
Beauregard, 35260 Saint Grégoire, France
Received for publication, December 5, 2000, and in revised form, February 14, 2001
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ABSTRACT |
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Enlarged fat cells exhibit modified metabolic
capacities, which could be involved in the metabolic complications of
obesity at the whole body level. We show here that sterol regulatory
element-binding protein 2 (SREBP-2) and its target genes are induced in
the adipose tissue of several models of rodent obesity, suggesting
cholesterol imbalance in enlarged adipocytes. Within a particular fat
pad, larger adipocytes have reduced membrane cholesterol concentrations compared with smaller fat cells, demonstrating that altered cholesterol distribution is characteristic of adipocyte hypertrophy per
se. We show that treatment with methyl- Depending upon physiological or pathological conditions, lipid
storage within the adipocyte may vary dramatically. This is particularly obvious in the obese state in which long term
disequilibria in energy balance produces massive fat cell hypertrophy.
Enlarged fat cells isolated from obese animals or humans exhibit
modified metabolic properties and do not respond to hormones as do
adipocytes from lean controls. For example, they develop insulin
resistance (1-4) and produce increased quantities of secreted products
such as leptin (1) or
TNF- Results from previous studies summarized below lead us to propose a
role for cholesterol as a signaling molecule for fat cell hypertrophy.
Cholesterol accumulates in large quantities in a non-esterified form
within the adipocyte lipid droplet (for review see Ref. 6)
proportionally to the triglyceride content (7, 8). Several lines of
evidence also suggest that concomitantly with the enlargement of the
triglyceride stores, adipocyte cholesterol is redistributed from the
plasma membrane to the lipid droplet. First, a decrease in plasma
membrane cholesterol, associated with increased fluidity, has been
reported in hypertrophied adipocytes (9). Second, plasma membrane
cholesterol content decreases during the differentiation of 3T3F442A
preadipocytes to lipid-loaded adipocytes, further suggesting that
massive accumulation of intracellular triglycerides alters the
distribution of intracellular cholesterol (10). Finally, we have shown
that the transcription factor SREBP-2 that is sensitive to membrane
cholesterol depletion is selectively induced in enlarged adipocytes
from obese Zucker rats (11). SREBP-2 is mainly involved in the control
of cholesterol metabolism (for review see Ref. 12). In the case of
membrane cholesterol depletion, the SREBP-2 pathway activates genes
controlling cholesterol synthesis (HMG-CoA reductase and HMG-CoA
synthase genes) and uptake (LDL receptor gene) and autoregulates its
own synthesis (13). Thus an increased content of active SREBP-2
protein in nuclear extracts from obese rat fat cells is the signature
of a relative cholesterol-depleted state of the adipocyte plasma
membrane, concomitant with ongoing cholesterol accumulation in the
lipid droplet of enlarged fat cells.
It is now becoming obvious that cholesterol can no longer be considered
only as a structural component of plasma membranes; it also
actively participates in the regulation of cell physiology. The
importance of cholesterol as a key component of the regulation of
signal transduction through membrane lipid-ordered micro-domains (14)
and of the regulation of gene expression through cholesterol-activated transcription factors (12, 15) is now well established.
The aim of this work was then to examine whether cholesterol could be a
link between adipocyte cell size and the changes in its metabolic
activity as seen in obese states. As a first approach we manipulated
adipocyte cholesterol status and investigated its consequences
on glucose metabolism, insulin sensitivity, and gene profiling. Our
results indicate that alteration of membrane cholesterol in adipocytes
modifies the phenotype of the cells into one partly resembling that of
an "obese" fat cell, including changes in hormone sensitivity and
secretion of adipocyte-derived products.
Cell Preparation and Culture--
Fat cells were isolated from
subcutaneous inguinal adipose tissue of 1-month-old rats by collagenase
treatment as described (16). For short term cholesterol depletion,
isolated cells were resuspended in Krebs-Ringer solution without
bovine serum albumin, and cyclodextrins (0-5 mM) loaded or
not with cholesterol (40 mg/g) were added. After a 1-h incubation at
37 °C, the medium was aspirated, and cells were used for further
studies. In some experiments, small (35-µm diameter) and large
(50-µm diameter) adipocytes were obtained after collagenase treatment
of epididymal adipose tissue of 2-month-old lean rats followed by
several filtrations through 30-µm Nytex.
3T3-L1 cells (a kind gift of Dr. J. Pairault, Paris, France) were
cultured in high glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Eurobio) and antibiotics. At
confluence, differentiation was induced by adding
methyl-isobutylxanthine (100 µM), dexamethasone (0.25 µM), and insulin (1 µg/µl) for 2 days. Then cells
were allowed to differentiate either in standard conditions (10% serum
and insulin) or in cholesterol-depleted medium. Cholesterol-depleted
medium contained insulin, 10 µM mevastatin (Sigma), and
10% lipoprotein-deficient fetal bovine serum prepared as described
(17) from the batch used for standard conditions. In some experiments,
cholesterol (1 µg/ml) in ethanol solution was added to cells cultured
in depleted conditions. The final ethanol concentration in the culture
medium did not exceed 0.1% v/v. Differentiated cells were harvested at
day 7 following confluence.
Membrane Preparation--
The fat cell suspension was diluted
four times in ice-cold hypotonic buffer (0.1 mM
KHCO3, 0.25 mM MgCl2, 0.2 mM EGTA, 0.2 mM
4-(2-aminoethyl)-benzenesulfonyl fluoride, and 0.15 mM
Tris-HCl, pH 7.5), vigorously shaken, and then centrifuged at 700 × g for 10 min at 4 °C. The supernatant below the fat
cake was collected and centrifuged at 160,000 × g for
45 min at 4 °C. The final pellet was suspended in 1 mM
EDTA, 0.2 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride, 25 mM Tris-HCl, pH 7.5
Glucose Metabolism--
After treatment with cyclodextrins,
0.5-ml triplicate aliquots of isolated adipose cells were incubated in
plastic vials at 37 °C with 2 ml of Krebs bicarbonate buffer
containing 5 mM glucose and 0.25 µCi of
[U-14C]D-glucose (Amersham Pharmacia Biotech)
in the presence or absence of various insulin concentrations. The
flasks were gassed with 95% CO2, 5% O2 and
capped with a stopper equipped with a central well. After 2 h at
37 °C, the incubation was ended by addition of 0.3 ml of hyamine to
the well and 0.5 ml of 6 N sulfuric acid to the medium.
CO2 production was estimated by counting radioactivity in
the central well by liquid scintillation.
Western Blots--
Membrane preparations were used in
Western blots and probed with GLUT 1 and GLUT 4 antibodies as
previously described (18).
Lipid Determination--
Total lipids were extracted as
described by Folch (19) from total cell lysates or membrane
preparations. Dried lipids were resuspended in isopropanol, and
determination of free cholesterol and triglycerides was performed using
F-CHOL (Roche Molecular Biochemicals) and Peridochrom TG GPO-PAP (Roche
Molecular Biochemicals), respectively. Protein concentration in
membrane preparations was assessed as described by Bradford (20).
RNA Preparation and Real-time RT-PCR--
Total RNA was
extracted as described (21) from differentiated 3T3-L1 cells, inguinal
adipose tissue from Zucker rats, epididymal adipose tissue from fat
mice, or isolated fat cells from retroperitoneal adipose tissue of
ob/ob mice. Northern blots were performed as previously described (22).
For RT-PCR purposes, cDNA was synthesized from 5 µg of RQI DNase
I-treated (Promega) total RNA using random hexamers and Moloney murine
leukemia virus reverse transcriptase (Life Technologies, Inc.). Design
of specific primers was done using either the Primer Express
(PerkinElmer Life Sciences) or Oligo (MedProbe)
softwares. The complete list of gene-specific primers is provided as
supplemental data. Real-time quantitative RT-PCR analyses were
performed starting with 50 ng of reverse-transcribed total RNA, with
200 nM sense and antisense primers (Genset) in a final
volume of 25 µl using the Sybr Green PCR core reagents in an ABI
PRISM 7700 sequence detection system instrument (PerkinElmer Life
Sciences). Relative quantitation for a given gene, expressed as fold
variation over control (untreated cells), was calculated after
normalization to 18 S ribosomal RNA and determination of the
difference between CT (cycle threshold) in treated and
control cells using the 2-CT formula (23).
Glyceraldehyde-3-phosphate dehydrogenase expression of control RNA was
used as an interplate calibrator. Individual CT values are
means of triplicate measurements. Experiments were repeated three to
five times.
Statistical Analysis--
Statistical significance was assessed
by paired t test analysis or analysis of variance (ANOVA)
followed by Newman-Keuls comparison tests (Statistica, StatSoft Inc.).
p < 0.05 was considered to be the threshold of
statistical significance.
Altered Cholesterol Distribution Correlates with Fat Cell
Hypertrophy per Se--
We have reported previously (11) that the
mature cleaved form of SREBP-2 is present in higher amounts in the
nucleus of enlarged adipocytes from obese Zucker leptin
receptor-deficient rats, suggesting an alteration in cholesterol
metabolism in those cells. To examine whether such a feature is unique
to adipocytes from Zucker rats or is a general characteristic of the
obese state, we measured the expression of SREBP-2 and its target genes
(LDL receptor and HMG-CoA reductase genes) in two other models of
obesity: the carboxypeptidase E-deficient
fatcpe Short Term Cholesterol Depletion of Isolated Adipose Cells Induces
Insulin Resistance of Glucose Metabolism--
In the next series of
experiments, we examined whether mimicking in normal adipocytes the
membrane cholesterol depletion of enlarged adipocytes modifies the
metabolic activity of the former in a way similar to that found in the
latter. We studied the insulin response of glucose metabolism, one of
the parameters characteristically altered in hypertrophied adipocytes
from obese animals or humans. We used methyl- Cholesterol Depletion Alters Fat Cell Gene Expression--
Another
distinctive feature of the enlarged fat cell from obese animals or
humans is an altered pattern of gene expression, leading to sustained
metabolic changes. In another series of experiments, we thus
investigated whether changes in cholesterol content could influence the
gene expression profile in adipocytes. Because this requires long term
studies, we addressed this question in vitro in
differentiated 3T3-L1 adipocytes. In this system, we used a standard
protocol in which sterol depletion was induced by addition of an
inhibitor of sterol synthesis (compactin) in the culture medium, in the
absence of exogenous cholesterol supply (lipoprotein-deficient fetal
bovine serum). Treatment of differentiated 3T3-L1 cells lasted for 4 days. Under these conditions, cells showed no obvious morphologic
changes. The triglyceride content of cells grown in the depleting
medium showed only a slight and non-significant decrease as compared
with those grown under standard conditions (2153 ± 231 µg versus 3004 ± 730 µg/dish,
respectively). In contrast, as expected, total cell cholesterol was
decreased 3-fold in conditions of sterol depletion versus
standard medium (39 ± 4.8 µg versus 134 ± 26 µg/dish, respectively; p < 0.05). One obvious
consequence of this cholesterol depletion should be an activation of
the SREBP-2 pathway. We then assessed the expression of the three SREBP
isoforms, SREBP-1a, -1c, and -2, in 3T3-L1 adipocytes differentiated in sterol-depleted medium (Fig.
4A). Our results show that in
cells differentiated under standard conditions, SREBP-1a mRNA is
present with the lowest abundance; SREBP-1c and SREBP-2 mRNA are
expressed at 6- and 4-fold higher levels, respectively. Importantly,
when 3T3-L1 adipocytes were cultured in sterol-depleted medium, SREBP-2 mRNA was induced 3-4-fold, with no significant change for SREBP-1a mRNA and a slight decrease in SREBP-1c mRNA. Thus,
sterol depletion resulted in a switch from SREBP-1c to SREBP-2 as the
major SREBP isoform expressed in these cells. As expected, this was
accompanied by an up-regulation (5-10-fold) of SREBP-2 target genes
(Fig. 4B) in cells differentiated in sterol-depleted medium,
which could be reversed by addition of cholesterol. These results
confirm that our cholesterol depletion mimics in these 3T3-L1 cells the depletion observed in enlarged adipocytes.
We then used this system to investigate on a wider scale the effect of
cholesterol depletion on adipocyte gene expression profile. For this
purpose, we examined the expression of 35 adipocyte genes using
quantitative RT-PCR. Table I shows that a
wide range of variations could be observed in sterol-depleted
conditions versus standard medium (from a 10-fold induction
to 5-fold reduction). For five genes (glycerol-3-phosphate
dehydrogenase, FAS, TNF- In this study, our salient results are (i) that enlarged
adipocytes exhibit a relative decrease in membrane cholesterol content, as objectified by the induction of SREBP-2 and its target genes, and
(ii) that it is possible to mimic some characteristic features of
hypertrophied adipocytes from obese rodents by changing fat cell
cholesterol distribution. Marked insulin resistance is a major
characteristic of enlarged adipocytes from obese rodents or humans, but
the reasons for this alteration remain largely unknown. We show that it
is possible to alter the ability of adipocytes to respond to insulin by
changing cholesterol content of fat cells. This is based mainly on our
observation that acute cholesterol depletion of plasma membranes
through cyclodextrin treatment led to a decreased insulin response of
glucose oxidation. The mechanism responsible for decreased insulin
action was not investigated in the present study. However, in a report
published very recently (26) a decreased ability of insulin to induce
IRS-1 phosphorylation and the further downstream protein kinase B was
observed after cyclodextrin treatment of 3T3-L1 cells. In that study,
an impaired insulin response of glucose transport was reported,
indicating that in addition to glucose oxidation, other metabolic
effects of insulin are altered in cyclodextrin-treated adipocytes. It can be postulated that disorganization of cholesterol-rich caveolae, in
which the insulin receptor is located (27), might play an important
role. Indeed, two reports (28, 29) have shown that caveolins, the main
structural proteins of caveolae, via their scaffolding domain, were
strong activators of insulin signaling in cells cotransfected with
components of the insulin-signaling machinery. Because cholesterol
depletion of plasma membranes has been shown to induce the
internalization of caveolins (30), this might provide a potential
explanation of the previously unexplained insulin resistance of
enlarged adipocytes.
Culture of adipocytes in a cholesterol-depleted medium is also shown to
modulate fat cell gene expression. Cholesterol depletion activates the
expression of the cholesterol-regulated transcription factor SREBP-2,
which then becomes the most abundantly expressed SREBP isoform because
SREBP-1a represents a minor form and because the expression of SREBP-1c
is not altered. This fits with recent results showing that the
abundance of SREBP-1c is dependent upon the carbohydrate status rather
than the cholesterol status (31, 32). Induction of SREBP-2 activates in
turn the expression of its known target genes such as the LDL receptor,
HMG-CoA reductase, and HMG-CoA synthase genes that are involved mainly
in the repletion of cell cholesterol concentration either by increasing
its synthesis or its uptake. Among the genes whose expression is
modulated by cholesterol depletion in adipocytes, we found some
proteins involved in cholesterol intracellular trafficking such as
caveolin 2 (the most abundant caveolin in adipocytes), the high density
lipoprotein receptor SR-BI, and ABC1 (which controls cholesterol
efflux). Moreover, the use of quantitative RT-PCR allowed direct
comparison of expression levels between genes and indicated that SR-BI
and ABC1 transcripts were present at significant levels in adipocytes, similar to FAS or CD36 mRNAs, two highly expressed genes in adipose tissue. This observation, in addition to the fact that high amounts of
cholesterol can be stored in adipose tissue (6), strengthens the
potential importance of adipose tissue in whole body cholesterol homeostasis. In cholesterol-depleted adipocytes, SR-BI and caveolin 2 are up-regulated, and ABC1 is down-regulated, suggesting a concerted compensatory mechanism to maintain cholesterol homeostasis in the fat
cell. Up-regulation of the SR-BI gene and down-regulation of the ABC1
gene by cholesterol depletion is also in agreement with the recent
implication of cholesterol-regulated transcription factors as important
components of the expression of these genes in other cell types, SREBPs
for the SR-BI gene (33) and liver X receptor for the ABC1 gene
(34).
However, genes involved in cholesterol metabolism and trafficking are
not the only ones showing differential expression after cholesterol
depletion. Enlarged adipocytes from obese rodents and humans exhibit
altered gene expression profiles. Particularly, some genes encoding
secreted products such as TNF- Whether all these genes are direct SREBP-2 targets or are controlled by
other cholesterol-activated transcription factors remains to be
investigated. The evaluation of their expression in adipose tissue of
transgenic mice overexpressing SREBP-2 (46) would provide some clues to
this question. It would be of interest to investigate whether SREBP-2
transgenic mice are insulin-resistant and develop obesity-like complications.
We did not observe changes in leptin expression in cholesterol-depleted
cells. Given the importance of leptin in signaling from the adipocytes
and the widely documented overexpression of leptin in obesity, induced
leptin expression could have been anticipated. One report (31)
described the transactivation of the leptin promoter by cotransfected
ADD1/SREBP1 in rat1-IR cells, suggesting that leptin might be a SREBP
target. The lack of effect of cholesterol depletion on leptin gene
expression in our experiments might arise from a strict specificity in
the SREBP isoform requirement at the leptin promoter level or from the
use of 3T3-L1 cells. Indeed, although they resemble fat cells in many
aspects of adipocyte metabolism, 3T3-L1 cells express and secrete very
low levels of leptin as compared with adipocytes isolated from tissues
(47). Thus more attention on the regulation of leptin by cholesterol in
another adipose cell system is needed before conclusions can be drawn.
In conclusion, this study shows for the first time that by altering the
cholesterol balance in fat cells in a way that mimics the effect of
adipocyte enlargement, it is possible to reproduce part of the defects
seen in hypertrophied adipocytes, such as insulin resistance and
altered expression of adipocyte-derived gene products, usually
associated with the metabolic complications of obesity. We also show
that the proteins involved in cholesterol metabolism and trafficking
are expressed at a high level in this cell type. Because a specific
decrease in membrane cholesterol from adipocytes is associated with
increased fat cell size, our results argue in favor of the potential
importance of cholesterol as an intracellular signal in the adipocyte,
which might then serve as a link between the level of fat stores and
adipocyte metabolic activity. If these results are confirmed in further studies, they could also form the basis of new therapeutic strategies aimed at uncoupling fat cell enlargement in obesity from severe metabolic complications.
-cyclodextrin, which
mimics the membrane cholesterol reduction of hypertrophied adipocytes, induces insulin resistance. We also produced cholesterol depletion by
mevastatin treatment, which activates SREBP-2 and its target genes. The
analysis of 40 adipocyte genes showed that the response to cholesterol
depletion implicated genes involved in cholesterol traffic (caveolin 2, scavenger receptor BI, and ATP binding cassette 1 genes) but also
adipocyte-derived secretion products (tumor necrosis factor
,
angiotensinogen, and interleukin-6) and proteins involved in energy
metabolism (fatty acid synthase, GLUT 4, and UCP3). These data
demonstrate that altering cholesterol balance profoundly modifies
adipocyte metabolism in a way resembling that seen in hypertrophied fat
cells from obese rodents or humans. This is the first evidence that
intracellular cholesterol might serve as a link between fat cell size
and adipocyte metabolic activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 (5). These
characteristics could be secondary to the hormonal and metabolic
changes linked to obesity but could also be due to cell hypertrophy
per se and thus be part of an adaptative mechanism in
relationship with the status of energy stores in adipose tissue.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
mouse and the leptin-deficient
ob/ob mouse. Fig. 1 shows that the
induction of SREBP-2 itself and of its target genes is clearly present
in Zucker fa/fa rats, fatcpe
/
mice, and
ob/ob mice. It is noteworthy that this alteration is not limited to
particular adipose tissue localization, because subcutaneous inguinal
or abdominal perigonadal sites were examined. Moreover, isolated
adipocytes were used in ob/ob mice, indicating that the adipocyte
component, rather than some other cell types in adipose tissue, was
responsible for the induction of SREBP-2 and its target genes. Thus
activation of the SREBP pathway appears as a common characteristic of
obese fat cells. This raises the possibility that cholesterol
distribution might be altered in enlarged fat cells, independently of
the metabolic and hormonal milieu. To test this possibility, we
measured membrane cholesterol content in two fat cell populations of
different size isolated from the same lean rat fat pad. Epididymal fat
pads of 10-12-week-old rats were chosen because of their intrinsic
large heterogeneity in individual adipose cell size. Using filtration
techniques, we obtained two cell populations with a 10-µm difference
in their diameter (Fig. 2A),
which represents a 1.7-fold difference in cell surface area (5000 ± 195 versus 8500 ± 362 µm2 in small
and large fat cells, respectively). In these two populations, the
larger fat cells exhibit a 42% reduction in membrane
cholesterol:protein ratio (Fig. 2B), a value similar to the
48% decrease reported for plasma membranes from subcutaneous
adipocytes isolated from obese rats compared with those from lean rats
(9). It is noteworthy that the absolute amount of membrane cholesterol
per cell would be similar because of the 1.7-fold increase in cell
surface. Nevertheless, this would correspond in large cells to a
dilution in membrane cholesterol, sensed as true cholesterol depletion.
Indeed, as for the obese versus lean rat adipocyte system,
we observed a 2-fold increase in SREBP-2 mRNA levels in large
versus small adipocytes (expression measured in triplicate
in an RNA pool from small and large adipocytes, respectively: 0.77 ± 0.08 versus 1.86 ± 0.2 arbitrary units after
normalization by 18 S ribosomal RNA). Taken together, these data
indicate that a cholesterol-depleted state of membranes accompanied by
activation of the SREBP-2 transcription factor is a common feature of
hypertrophied adipocytes.
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Fig. 1.
Expression of SREBP-2 and its target genes in
adipose tissue of obese rodents. The expression of SREBP-2,
HMG-CoA reductase, and LDL receptor were determined by fluorescent
RT-PCR in lean (white bars) or obese (black bars)
rats of the Zucker strain (left panel),
fatcpe /
mice (central panel),
and ob/ob mice (right panel). Triplicate amplifications of
total RNA were performed with gene-specific primers, and mRNA
levels were normalized to 18 S ribosomal RNA. Values are means ± S.E. from independent RNA preparations in each genotype. In ob
mice, RNA was prepared from isolated adipocytes obtained from a pool of
30 lean and 8 obese mice. Significant differences are indicated (*,
p < 0.05; **, p < 0.01)
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Fig. 2.
Cholesterol content of membranes from large
and small adipocytes isolated from the same adipose fat pad. Small
and large fat cells were obtained from epididymal adipose tissue of
10-12-week old lean rats by differential flotation. A shows
mean fat cell diameters of the two cell populations obtained after
separation. Each point represents an independent experiment in which
adipose tissues from four to six individuals were pooled. B
shows membrane cholesterol content measured in the two fat cell
populations. Each bar is the mean value ± S.E. calculated from at
least seven independent determinations. Significant statistical
differences (paired t test analysis) were found between
large and small adipocytes (p < 0.01).
-cyclodextrin as the
cholesterol-depleting agent because it induces rapid cholesterol
removal from the plasma membrane by acting as an extra cellular
acceptor, an effect extensively studied in various cell types (for
review see Ref. 24). Fig. 3 shows the
effect of a 1-h treatment of rat fat cells isolated from subcutaneous
adipose tissue with increasing concentrations of
methyl-
-cyclodextrins. The membrane cholesterol concentration of
adipocytes is decreased by cyclodextrin treatment in a
dose-dependent manner (Fig. 3A). A significant
depletion occurs at 1 mM cyclodextrin, and approximately
half of the membrane cholesterol is removed at the highest
concentration tested (5 mM). If cyclodextrins are loaded
with cholesterol prior to incubation, adipocyte membrane cholesterol is
not decreased, but rather increased. This demonstrates that membrane
cholesterol depletion is indeed related to the cholesterol-trapping properties of cyclodextrins. Fig. 3B shows the
insulin response of glucose metabolism in the adipocytes treated for
1 h with cyclodextrins. Maximal insulin stimulation of untreated
control fat cells leads to a 3-4-fold stimulation of glucose oxidation
rate, as previously observed (25). Under basal conditions, glucose
incorporation into CO2 did not change significantly in
cells treated with increasing concentrations of
methyl-
-cyclodextrins (data not shown). However, treatment with
methyl-
-cyclodextrins induced a marked reduction of insulin maximal
stimulation proportional to their concentration. This effect was
clearly related to cholesterol depletion, because a normal insulin
response persisted when adipocytes were treated with
methyl-
-cyclodextrins previously loaded with cholesterol. There is a
clear-cut decrease in the maximal response to insulin of glucose
oxidation in the cyclodextrin-treated adipocytes when compared with the
two other experimental conditions. In contrast, insulin sensitivity is
not different, as seen from Fig. 3C, where results
are expressed as a percent of the maximal insulin effect. The extent of
cholesterol depletion at 1 mM cyclodextrin leading to the
effect on insulin response was in the same range as that described in
enlarged fat cells from obese rodents (40-50%). Taken together, these results strongly suggest that cholesterol depletion of
fat cell membranes can influence hormonal response of adipocytes and
induce a state of insulin resistance characteristic of the enlarged
adipocyte.
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Fig. 3.
Effect of short term cholesterol depletion by
cyclodextrin treatment on insulin-stimulated glucose metabolism in
isolated adipocytes. A, membrane cholesterol content of
adipocytes treated for 1 h with increasing concentrations of
methyl- -cyclodextrins (CD, filled circles) or
methyl-
-cyclodextrins loaded with cholesterol (CD-Chol,
open circles). Values are means ± S.E. obtained in
three independent experiments. Significant differences relative to
untreated cells (Student's t test) are indicated as
follows: *, p < 0.05; **, p < 0.01;
***, p < 0.001. B, glucose oxidation in
isolated adipocytes treated for 1 h with methyl-
-cyclodextrins
(black bars) or methyl-
-cyclodextrins loaded with
cholesterol (hatched bars). After cyclodextrin treatment,
cells were incubated with [U-14C] glucose for 2 h in
basal (no insulin) or insulin-stimulated (2.5 nM insulin,
maximal stimulation) conditions. Values are means ± S.E. of
maximal insulin effects (stimulated versus basal
incorporation) obtained in three independent experiments. No variation
of basal values was observed after cyclodextrin treatment. Significant
differences relative to untreated cells (Student's t test)
are indicated as follows: *, p < 0.05. C,
insulin dose-response curves of [U-14C]glucose oxidation
in untreated isolated adipocytes (filled circles) or in
adipocytes treated with 1 mM cyclodextrins loaded
(open circles) or not (asterisks) with
cholesterol. Values are means ± S.E. obtained from five
independent experiments.
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Fig. 4.
Effect of cholesterol depletion on the
expression of SREBP isoforms and SREBP target genes in 3T3-L1
adipocytes. 3T3-L1 cells were differentiated in either the
standard medium or the cholesterol-depleted medium and harvested at J7.
mRNA were extracted and used for real-time quantitative fluorescent
RT-PCR as described under "Materials and Methods." A,
relative expression of the three SREBP isoforms. Results were obtained
from six independent cultures and are represented as means ± S.E.
relative to the lowest expression values (SREBP-1a in standard medium)
arbitrarily set to 1. B, effect of cholesterol depletion on
the expression of SREBP target genes. mRNA encoding the low density
lipoprotein receptor (LDL-R), HMG-CoA reductase
(HMGCoA-red), or HMG-CoA synthase (HMGCoA-synth)
were amplified by real-time fluorescent RT-PCR. For each gene, results
were normalized to 18 S ribosomal RNA and are expressed
relative to the expression in cells differentiated in standard medium,
arbitrarily set to 1. Values are means ± S.E. from six
independent cultures. Significant differences are indicated as follows:
*, p < 0.05; ***, p < 0.005.
, CAAT enhancer-binding protein
,
and HMG-CoA reductase genes), Northern blot analysis was performed,
which always confirmed the variations measured in real-time RT-PCR
(Fig. 5). In Table I, among the genes
whose expression is unaffected by sterol depletion are many genes
important for the mature adipocyte phenotype and function. These
include the glycerol-3-phosphate dehydrogenase gene and key genes of
adipocyte differentiation such as CAAT enhancer-binding protein
and peroxisomal proliferator-activated receptor
genes. This
further establishes that sterol depletion in our experiments did not
alter an ongoing adipogenic program. In cholesterol-depleted cells, in
addition to the LDL receptor, HMG-CoA reductase, and HMG-CoA synthase
genes, the expression of seven genes was up-regulated, including genes
encoding adipocyte-secreted products like angiotensinogen, TNF-
, and
interleukin-6, but also genes encoding proteins involved in
intracellular cholesterol metabolism like SR-BI and caveolin 2 (Table
I). Two of the up-regulated genes, the gene encoding the glucose
transporter GLUT 1 and the gene encoding the lipogenic enzyme fatty
acid synthase, are involved in glucose utilization. Three of the
genes, including the gene encoding a cholesterol transporter, ABC1, the
gene encoding a glucose transporter, GLUT 4, and surprisingly the gene
encoding a protein involved in mitochondrial metabolism, UCP3, were
down-regulated. We ensured that some of the variations in gene
expression, observed here by Taqman analysis of mRNAs, were
followed by parallel changes at the protein level. Fig.
6 shows a Western blot analysis of GLUT 1 and GLUT 4 in total membranes of 3T3-L1 cells. It demonstrates that the
effects of cholesterol depletion on GLUT mRNAs (decreased GLUT 4 and increased GLUT 1 expression; see Table I) were also evident at the
level of the total number of glucose transporters in membranes.
This set of experiments establishes that cholesterol depletion
has effects on gene expression, which are not related to genes involved only in cholesterol metabolism.
Effect of sterol depletion on adipocyte gene expression
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Fig. 5.
Effect of sterol depletion on HMG-CoA
reductase, TNF- , FAS, glycerol-3-phosphate dehydrogenase, and
CAAT enhancer-binding protein
mRNA assessed by Northern
blot analysis. Pooled RNA samples used in real-time RT-PCR were
analyzed by Northern blot as described under "Materials and
Methods." The cDNA probes were described elsewhere.
View larger version (43K):
[in a new window]
Fig. 6.
Effect of long term sterol depletion on
glucose transporters in 3T3-L1 adipocytes. Western blot analysis
of GLUT 1 and GLUT 4 in 3T3-L1 adipocytes cultured in cholesterol
deprived (+) or standard medium ( ). Cells were
harvested at J7. Total membranes (20 or 100 µg of protein) were
separated by SDS-polyacrylamide gel electrophoresis, blotted, and
probed with GLUT 1 and GLUT 4 antibodies. Bands were revealed using the
ECL system (Amersham Pharmacia Biotech). Two membrane preparations from
independent experiments are shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, angiotensinogen, and interleukin-6
are overexpressed in obesity (5, 35-37). It has been suggested that
these products can participate in the morbid complications of obesity
such as insulin resistance (5), hypertension (38), or atherosclerosis
and coronary heart disease (39). We show here that cholesterol
depletion up-regulates the expression of these genes. We also observed
changes in the expression of genes involved in energy metabolism, such
as FAS, GLUT 4, and UCP3, in cholesterol-depleted fat cells. FAS
expression was increased 2-fold, in accordance with previous
observations showing that FAS expression is activated by insulin
through SREBP-1c (31, 32) but also by cholesterol (40) through the
SREBP-2 pathway. The induction of FAS gene expression through the
induction of SREBP-2 is also reminiscent of our previous work on the
mechanism of FAS gene overexpression in obese Zucker rat adipocytes
(11). GLUT 4 and UCP3 expression were decreased 3- and 5-fold,
respectively. UCP3 might potentially play an important role in energy
expenditure (41). However, adipocytes express low levels of UCP3
mRNA relative to UCP2 (approximately one order of magnitude), and
thus, the functional significance of decreased UCP3 expression in
cholesterol-depleted fat cells can be questioned. Nevertheless,
decreased UCP3 is generally observed in muscle (42) during obesity,
which is consistent with our observation. Given the importance of GLUT
4 in the regulation of glucose homeostasis (43), our observation of
decreased GLUT 4 expression in cholesterol-depleted adipocytes might be
important in the context of obesity. Accordingly, down-regulated GLUT 4 is a major contributor to impaired glucose homeostasis in all forms of
obesity and diabetes (44), and it was found recently that
adipose-selective depletion of GLUT 4 in transgenic mice led to
impaired glucose tolerance and insulin resistance (45) at the whole
body level.
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ACKNOWLEDGEMENTS |
---|
We thank Francine Diot-Dupuy for excellent
technical assistance. We thank Dr. G. J. Murphy for preparation of
ob mice adipocytes and M. Guerre-Millo for the gift of
fatcpe/
mice.
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FOOTNOTES |
---|
* This work was supported by contract FAIR 97-3011 from the European Community.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.
The on-line version of this article (available at
http://www.jbc.org) contains supplemental data.
§ To whom correspondence should be addressed. Tel.: 33 1 42 34 69 22/23/24; Fax: 33 1 40 51 85 86; E-mail: idugail@bhdc.jussieu.fr.
Published, JBC Papers in Press, February 27, 2001, DOI 10.1074/jbc.M010955200
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ABBREVIATIONS |
---|
The abbreviations used are:
TNF-, tumor necrosis factor
;
SREBP, sterol regulatory element-binding
protein;
HMG, hydroxy-methyl-glutaryl;
LDL, low density lipoprotein;
PCR, polymerase chain reaction;
RT-PCR, reverse transcription-PCR;
FAS, fatty acid synthase;
SR-BI, scavenger receptor BI;
ABC1, ATP binding
cassette 1.
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REFERENCES |
---|
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---|
1. | Friedman, J. M. (2000) Nature 404, 632-634[Medline] [Order article via Infotrieve] |
2. | Chlouverakis, C., and Hojnicki, D. (1974) Steroids Lipids Res. 5, 351-358[Medline] [Order article via Infotrieve] |
3. | Olefsky, J. M. (1977) Endocrinology 100, 1169-1177[Abstract] |
4. | Molina, J. M., Ciaraldi, T. P., Brady, D., and Olefsky, J. M. (1989) Diabetes 38, 991-995[Abstract] |
5. | Hotamisligil, G. S., Shargill, N. S., and Spiegelman, B. M. (1993) Science 259, 87-91[Medline] [Order article via Infotrieve] |
6. | Krause, B. R., and Hartman, A. D. (1984) J. Lipid Res. 25, 97-110[Abstract] |
7. | Kovanen, P. T., Nikkila, E. A., and Miettinen, T. A. (1975) J. Lipid Res. 16, 211-223[Abstract] |
8. | Schreibman, P. H., and Dell, R. B. (1975) J. Clin. Invest. 55, 986-993[Medline] [Order article via Infotrieve] |
9. | Guerre-Millo, M., Guesnet, P., Guichard, C., Durand, G., and Lavau, M. (1994) Lipids 29, 205-209[Medline] [Order article via Infotrieve] |
10. |
Storch, J.,
Shulman, S. L.,
and Kleinfeld, A. M.
(1989)
J. Biol. Chem.
264,
10527-10533 |
11. |
Boizard, M.,
Le Liepvre, X.,
Lemarchand, P.,
Foufelle, F.,
Ferre, P.,
and Dugail, I.
(1998)
J. Biol. Chem.
273,
29164-29171 |
12. | Brown, M. S., and Goldstein, J. L. (1997) Cell 89, 331-340[Medline] [Order article via Infotrieve] |
13. | Sheng, Z., Otani, H., Brown, M. S., and Goldstein, J. L. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 935-938[Abstract] |
14. | Simons, K., and Toomre, D. (2000) Nat. Rev. Mol. Cell Biol. 1, 31-39[CrossRef][Medline] [Order article via Infotrieve] |
15. | Peet, D. J., Janowski, B. A., and Mangelsdorf, D. J. (1998) Curr. Opin. Genet. Dev. 8, 571-575[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Rodbell, M.
(1964)
J. Biol. Chem.
239,
375-380 |
17. | Goldstein, J. L., Basu, S. K., and Brown, M. S. (1983) Methods Enzymol. 98, 241-260[Medline] [Order article via Infotrieve] |
18. | Hainault, I., Guerre-Millo, M., Guichard, C., and Lavau, M. (1991) J. Clin. Invest. 87, 1127-1131[Medline] [Order article via Infotrieve] |
19. |
Folch, J.,
Lees, M.,
and Sloane-Stanley, G. H.
(1956)
J. Biol. Chem.
226,
497-509 |
20. | Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve] |
21. | Cathala, G., Savouret, J. F., Mendez, B., West, B. L., Karin, M., Martial, J. A., and Baxter, J. D. (1983) DNA (N. Y.) 2, 329-335[Medline] [Order article via Infotrieve] |
22. | Guichard, C., Dugail, I., Le Liepvre, X., and Lavau, M. (1992) J. Lipid Res. 33, 679-687[Abstract] |
23. | PerkinElmer Applied Biosystems. (1999) Bulletin 2 |
24. |
Rothblat, G. H.,
de la Llera-Moya, M.,
Atger, V.,
Kellner-Weibel, G.,
Williams, D. L.,
and Phillips, M. C.
(1999)
J. Lipid Res.
40,
781-796 |
25. | Guerre-Millo, M., Leturque, A., Girard, J., and Lavau, M. (1985) J. Clin. Invest. 76, 109-116[Medline] [Order article via Infotrieve] |
26. |
Parpal, S.,
Karlsson, M.,
Thorn, H.,
and Strålfors, P.
(2001)
J. Biol. Chem.
276,
9670-9678 |
27. |
Gustavsson, J.,
Parpal, S.,
Karlsson, M.,
Ramsing, C.,
Thorn, H.,
Borg, M.,
Lindroth, M.,
Peterson, K. H.,
Magnusson, K. E.,
and Stralfors, P.
(1999)
FASEB J.
13,
1961-1971 |
28. |
Yamamoto, M.,
Toya, Y.,
Schwencke, C.,
Lisanti, M. P.,
Myers, M. G. J.,
and Ishikawa, Y.
(1998)
J. Biol. Chem.
273,
26962-26968 |
29. |
Nystrom, F. H.,
Chen, H.,
Cong, L. N.,
Li, Y.,
and Quon, M. J.
(1999)
Mol. Endocrinol.
13,
2013-2024 |
30. | Smart, E. J., Ying, Y. S., Conrad, P. A., and Anderson, R. G. (1994) J. Cell Biol. 127, 1185-1197[Abstract] |
31. |
Kim, J. B.,
Sarraf, P.,
Wright, M.,
Yao, K. M.,
Mueller, E.,
Solanes, G.,
Lowell, B. B.,
and Spiegelman, B. M.
(1998)
J. Clin. Invest.
101,
1-9 |
32. |
Foretz, M.,
Pacot, C.,
Dugail, I.,
Lemarchand, P.,
Guichard, C.,
Le Liepvre, X.,
Berthelier-Lubrano, C.,
Spiegelman, B.,
Kim, J. B.,
Ferre, P.,
et al..
(1999)
Mol. Cell. Biol.
19,
3760-3768 |
33. |
Lopez, D.,
and McLean, M. P.
(1999)
Endocrinology
140,
5669-5681 |
34. |
Costet, P.,
Luo, Y.,
Wang, N.,
and Tall, A. R.
(2000)
J. Biol. Chem.
275,
28240-28245 |
35. |
Fried, S. K.,
Bunkin, D. A.,
and Greenberg, A. S.
(1998)
J. Clin. Endocrinol. Metab.
83,
847-850 |
36. |
Van, H. V.,
Ariapart, P.,
Hoffstedt, J.,
Lundkvist, I.,
Bringman, S.,
and Arner, P.
(2000)
Obes. Res.
8,
337-341 |
37. | Frederich, R. C., Jr., Kahn, B. B., Peach, M. J., and Flier, J. S. (1992) Hypertension 19, 339-344[Abstract] |
38. |
Engeli, S.,
Negrel, R.,
and Sharma, A. M.
(2000)
Hypertension
35,
1270-1277 |
39. | Yudkin, J. S., Kumari, M., Humphries, S. E., and Mohamed-Ali, V. (2000) Atherosclerosis 148, 209-214[CrossRef][Medline] [Order article via Infotrieve] |
40. |
Bennett, M. K.,
Lopez, J. M.,
Sanchez, H. B.,
and Osborne, T. F.
(1995)
J. Biol. Chem.
270,
25578-25583 |
41. | Clapham, J. C., Arch, J. R., Chapman, H., Haynes, A., Lister, C., Moore, G. B., Piercy, V., Carter, S. A., Lehner, I., Smith, S. A., et al.. (2000) Nature 406, 415-418[CrossRef][Medline] [Order article via Infotrieve] |
42. | Schrauwen, P., Walder, K., and Ravussin, E. (1999) Obes. Res. 7, 97-105[Abstract] |
43. | Zisman, A., Peroni, O. D., Abel, E. D., Michael, M. D., Mauvais-Jarvis, F., Lowell, B. B., Wojtaszewski, J. F., Hirshman, M. F., Virkamaki, A., Goodyear, L. J., et al.. (2000) Nat. Med. 6, 924-928[CrossRef][Medline] [Order article via Infotrieve] |
44. |
Kahn, B. B.,
and Flier, J. S.
(2000)
J. Clin. Invest.
106,
473-481 |
45. | Abel, E. D., Peroni, O. D., Kim, J. K., Kim, Y. B., Boss, O., Hadro, E., Minnemann, T., Shulman, G. I., and Kahn, B. B. (2001) Nature 409, 729-733[CrossRef][Medline] [Order article via Infotrieve] |
46. |
Horton, J. D.,
Shimomura, I.,
Brown, M. S.,
Hammer, R. E.,
Goldstein, J. L.,
and Shimano, H.
(1998)
J. Clin. Invest.
101,
2331-2339 |
47. |
Mandrup, S.,
Loftus, T. M.,
MacDougald, O. A.,
Kuhajda, F. P.,
and Lane, M. D.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
4300-4305 |