Cholesterol, a Cell Size-dependent Signal That Regulates Glucose Metabolism and Gene Expression in Adipocytes*,

Soazig Le Lay, Stéphane KriefDagger , Céline Farnier, Isabelle LefrèreDagger , Xavier Le Liepvre, Raymond Bazin, Pascal Ferré, and Isabelle Dugail§

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 Dagger  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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta -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 alpha , 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

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-alpha 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.

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.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-/- 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).

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-beta -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-beta -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-beta -cyclodextrins (data not shown). However, treatment with methyl-beta -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-beta -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-beta -cyclodextrins (CD, filled circles) or methyl-beta -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-beta -cyclodextrins (black bars) or methyl-beta -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.

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.


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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.

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-alpha , CAAT enhancer-binding protein alpha , 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 alpha  and peroxisomal proliferator-activated receptor gamma  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-alpha , 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.

                              
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Table I
Effect of sterol depletion on adipocyte gene expression
Total RNA was extracted from 3T3-L1 adipocytes differentiated in standard conditions or sterol-depleted medium as above. mRNA levels were assessed by real-time RT-PCR as described. The expression of each gene was quantified and normalized using simultaneous amplification of a ribosomal 18 S RNA fragment. The results are expressed as the ratio between depleted and standard conditions. Values are means ± S.D. obtained from at least three independent determinations on separated cultures. For genes indicated as positively or negatively regulated, differences in expression were statistically significant. Abbreviations used are as follows: UCP, uncoupling protein; G3PDH, glycerol-3-phosphate dehydrogenase; PPAR, peroxisome proliferator-activated receptor; PAI-1, plasminogen activator inhibitor-1; SCD-1, stearoyl CoA desaturase1; aP2, adipocyte protein 2; FAT, fatty acid transporter; Id, inhibitor of differentiation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; C/EBP, CAAT enhancer-binding protein; VLDL, very low density lipoprotein.


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Fig. 5.   Effect of sterol depletion on HMG-CoA reductase, TNF-alpha , FAS, glycerol-3-phosphate dehydrogenase, and CAAT enhancer-binding protein alpha  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.


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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

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-alpha , 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.

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.

    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.

    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

    ABBREVIATIONS

The abbreviations used are: TNF-alpha , tumor necrosis factor alpha ; 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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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[Abstract/Free Full Text]
11. Boizard, M., Le Liepvre, X., Lemarchand, P., Foufelle, F., Ferre, P., and Dugail, I. (1998) J. Biol. Chem. 273, 29164-29171[Abstract/Free Full Text]
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[Free Full Text]
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[Free Full Text]
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
28. Yamamoto, M., Toya, Y., Schwencke, C., Lisanti, M. P., Myers, M. G. J., and Ishikawa, Y. (1998) J. Biol. Chem. 273, 26962-26968[Abstract/Free Full Text]
29. Nystrom, F. H., Chen, H., Cong, L. N., Li, Y., and Quon, M. J. (1999) Mol. Endocrinol. 13, 2013-2024[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
33. Lopez, D., and McLean, M. P. (1999) Endocrinology 140, 5669-5681[Abstract/Free Full Text]
34. Costet, P., Luo, Y., Wang, N., and Tall, A. R. (2000) J. Biol. Chem. 275, 28240-28245[Abstract/Free Full Text]
35. Fried, S. K., Bunkin, D. A., and Greenberg, A. S. (1998) J. Clin. Endocrinol. Metab. 83, 847-850[Abstract/Free Full Text]
36. Van, H. V., Ariapart, P., Hoffstedt, J., Lundkvist, I., Bringman, S., and Arner, P. (2000) Obes. Res. 8, 337-341[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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[Free Full Text]
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]


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