Institut de biologie animale (J.R., L.M., P.E., B.D., W.W.), Université de Lausanne, CH-1015 Lausanne, Switzerland; and Ilex Oncology (F.T., E.N.), CH-1290 Versoix/Geneva, Switzerland
Address all correspondence and requests for reprints to: Pr. Walter Wahli, Institut de biologie animale, Université de Lausanne, Bâtiment de biologie, CH-1015 Lausanne, Switzerland. E-mail: Walter.Wahli{at}iba.unil.ch.
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ABSTRACT |
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INTRODUCTION |
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PPAR is expressed predominantly in adipose tissue, where it is known to play a critical role in adipocyte differentiation and fat deposition (3, 4). It can be activated by arachidonic acid-metabolites generated by the cyclooxygenase and lipooxygenase pathways (5, 6, 7) and by fatty acid-derived components released from oxidized low density lipoproteins (8). The antidiabetic thiazolidinediones (TZDs), currently used as insulin sensitizers, are the best synthetic PPAR
ligands in terms of specificity and affinity, although the mechanism by which activation of PPAR
leads to an improvement of insulin action is still debated (3, 4, 9). Moreover, deletion of one allele of PPAR
was recently shown to protect mice from high fat diet (HFD)-induced adipocyte hypertrophy and insulin resistance, underlying the complexity of the role of PPAR
in insulin sensitivity (10).
The most extensively studied therapeutic utility for PPAR has been in the treatment of type 2 diabetes. TZDs were shown to enhance the sensitivity of target tissues to insulin and to reduce plasma glucose, lipid, and insulin levels in animal models of type 2 diabetes, as well as in human (11, 12). However, due to their ability to induce gene expression in adipocytes and to enhance adipocyte differentiation (13), TZDs also have negative side-effects. Indeed, they induce adipocyte differentiation and weight gain in patients with already serious metabolic disorders (14, 15). For this reason, important efforts are made to identify new PPAR
modulators having antidiabetic action, without promoting weight increase. Recently, a novel PPAR
ligand (GW 0072), which is a partial agonist in transactivation assays, was shown to inhibit adipocyte differentiation in cell culture (16). In addition, new antagonists for PPAR
are being characterized: bisphenol A diglycidyl ether (BADGE) inhibits adipocyte differentiation (17), PD 068235 also blocks adipocyte differentiation but does not revert the phenotype of terminally differentiated adipocytes (18), LG 100641 blocks adipocyte differentiation as well, and stimulates glucose uptake in 3T3-L1 adipocytes (19). So far, none of these inhibitors has been tested in vivo to verify whether they may prevent obesity and reduce insulin resistance, which would delay the onset of type 2 diabetes.
Here, we report that SR-202 [dimethyl -(dimethoxyphosphinyl)-p-chlorobenzyl phosphate] is a novel PPAR
-specific antagonist that blocks adipocyte differentiation induced either by thiazolidinediones or by the combination of dexamethasone, insulin, and 3-isobutyl-1-methylxanthine (IBMX). We have investigated the activity of this antagonist in vivo and demonstrate that blocking PPAR
activity gives rise to a decrease in fat deposit and increase in insulin sensitivity. This new PPAR
antagonist may serve to develop compounds that will be more beneficial in the treatment of obesity and type 2 diabetes than thiazolidinediones because it yields both antiobesity and antidiabetic effects.
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RESULTS |
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SR-202 Inhibits PPAR-Dependent Differentiation of Adipocytes
A major role of PPAR is to stimulate adipogenesis. We thus designed experiments to see whether SR-202 could also antagonize PPAR
activity in a functional assay, more particularly in adipocyte differentiation. For that purpose, preadipocyte 3T3-L1 cells were pretreated with different concentrations of SR-202 or vehicle for 24 h and then induced to differentiate with medium containing either BRL 49653 (25 nM) and insulin (5 µg/ml) or a mixture containing dexamethasone (1 µM), insulin (10 µg/ml), and IBMX (0.5 mM). As shown in Fig. 3
, A and B, SR-202 was able to significantly inhibit BRL 49653- and hormone-induced adipocyte differentiation of 3T3-L1 cells in a dose-dependent manner, as shown by the decrease in lipid content revealed by Oil red O staining. The antiadipogenic effect of SR-202 in this experimental setting was also revealed by the reduced expression of an adipocyte differentiation marker, the adipocyte fatty acid binding protein (aP2) (Fig. 3C
). The inhibition of adipogenesis at high concentrations of SR-202 was not due to a toxic effect of the antagonist because it could be attenuated by cotreating the cells with a high concentration of BRL 49653 (1 µM instead of 25 nM, data not shown). Furthermore, the lack of toxicity of SR-202 was again confirmed by quantification of LDH activities released in the culture media (reaching only 5% of total cellular LDH activity after a 48-h treatment with 400 µM SR-202) and by the total number of cells that remained unchanged at the end of the differentiating protocols (data not shown).
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Thus, decreasing PPAR activity, either by treatment with a specific antagonist or by invalidation of one allele of the PPAR
gene, leads to decreased adiposity in mice. These data indicate that the phenotype of SR-202-treated mice is a bona fide consequence of the reduction of PPAR
activity in vivo.
SR-202 Decreases Adipocytokine Secretion by WAT
White adipocytes are not only forming a passive tissue mass devoted to the storage of excess energy but are also endocrine secretory cells (22). For example, adipsin, TNF, leptin, and plasminogen activator inhibitor-1 are all secreted by fat cells, under control of nutritional (feeding and fasting) and pathological (obesity) states (22). In particular, regulatory roles in energy homeostasis and/or systemic insulin sensitivity have been shown for leptin and TNF
(22). In addition, the amount of circulating leptin and TNF
correlates with body fat stores and/or hyperinsulinemia, suggesting a potential involvement of these cytokines in obesity-induced insulin resistance (23, 24). Because SR-202 treatment is associated with decreased adiposity, we tested the effects of SR-202 treatment on leptin and TNF
secretion by the adipose tissue. Interestingly, SR-202-treated wt mice, under SD, had normal plasma leptin levels but lower plasma TNF
levels (Fig. 7
, A and B, left panels). As expected, we found a strong increase of plasma leptin and TNF
levels in untreated wt mice under HFD (Fig. 7
, A and B, left panels; compare CTL SD mice with CTL HFD mice). Under SR-202 treatment, these mice were protected from HFD-induced hyperleptinemia and HFD-related increase in TNF
levels (Fig. 7
, A and B, left panels). These data provide further evidence that the secretion levels of leptin and TNF
are closely related to the size of adipocytes. In addition, it suggests that inhibiting PPAR
activity by treatment with an antagonist can prevent from obesity-induced secretion of these cytokines.
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In conclusion, decreasing PPAR activity, either by treatment with a specific antagonist or by invalidation of one of the two alleles of the PPAR
gene, leads to decreased leptin and TNF
plasma levels, which is consistent with the smaller size of adipocytes. Treating PPAR
+/- mice with SR-202, further decreased plasma levels of these signaling molecules, in agreement with a more pronounced decrease in the size of adipocytes (see Fig. 5
).
SR-202 Protects against HFD-Induced Insulin Resistance
Both reducing the adipocyte size and modulating the secretion of two major adipocytokines, leptin and TNF, are known to influence insulin sensitivity (3, 25). We thus investigated whether SR-202 treatment could increase insulin sensitivity in mice. For that purpose, we measured glucose and insulin levels, in addition to plasma free fatty acid (FFA) levels, which are known to be linked to insulin sensitivity. Treatment of wt mice on SD with SR-202 did not affect the plasma glucose levels but strongly decreased the plasma insulin levels (Fig. 8
, A and B, left panels). No change in FFA plasma levels was observed in SR-202-treated wt mice under SD (Fig. 8C
, left panel). As expected, HFD feeding in untreated wt mice was associated with an increase in FFA levels (Fig. 8C
, left panel), a high hyperinsulinemia (Fig. 8B
, left panel), and a slight, however statistically not significant, increase in glucose levels (Fig. 8A
, left panel), indicating a compensated state of insulin resistance. In contrast, SR-202 treated mice were protected from HFD-induced insulin resistance, as measured by the maintenance of relatively low insulin and FFA plasma levels (Fig. 8
, B and C, left panels). These data demonstrate that decreasing PPAR
activity by treatment with an antagonist potentiates insulin sensitivity and improves lipid parameters under HFD.
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In conclusion, a partial decrease in PPAR activity leads to increased insulin sensitivity and protects mice from HFD-induced obesity and insulin resistance. However, a combined pharmacological and genetic action to reduce PPAR
activity, such as in SR-202-treated PPAR
+/- mice, did not further improve insulin sensitivity compared with untreated PPAR
+/- mice.
SR-202 Increases Insulin Sensitivity in ob/ob Mice
After demonstrating the ability of SR-202 to prevent the development of an insulin resistance as seen upon a HFD challenge, we next tested whether SR-202 could improve insulin sensitivity in the context of an already developed obesity with hyperglycemia and hyperinsulinemia. For that purpose, we used ob/ob mice, a well characterized strain of obese and diabetic mice. Treatment of the ob/ob mice with SR-202 was started at 8 wk of age (experimental d 0), when mice are already overtly diabetic (see glucose and insulin levels, d 0, Fig. 9, A and B). The treatment as food admixture was maintained for 20 d, after which metabolic parameters were measured (experimental d 20).
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We then conducted glucose and insulin tolerance tests to determine whether SR-202 could improve glucose disposal and insulin sensitivity in ob/ob mice. As shown in Fig. 9C, SR-202-treated ob/ob mice showed an enhanced glucose disposal compared with untreated ob/ob mice despite a strikingly reduced insulin induction (Fig. 9D
), suggesting an increase of insulin sensitivity. In agreement with these data, ip administration of insulin resulted in a stronger glucose lowering effect in SR-202-treated ob/ob mice compared with untreated mice (Fig. 9E
).
Finally, SR-202 treatment directly affected the expression of PPAR target genes. 20 d of treatment of ob/ob mice with SR-202 induced a decrease of PPAR
activity as illustrated by the decrease of LPL, CD36, and aP2 mRNA levels in WAT (Fig. 9F
).
In conclusion, SR-202 functions as an insulin sensitizer, markedly decreasing hyperglycemia and hyperinsulinemia in ob/ob mice, a mouse model of obesity and insulin resistance.
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DISCUSSION |
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Two Animal Models of PPAR![]() |
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The PPAR Antagonist SR-202 Inhibits Adipocyte Differentiation
There is now strong evidence supporting a key role of PPAR in adipogenesis based on both gain or loss of function experiments. Indeed, it has been shown that the ectopic expression and activation of PPAR
in fibroblasts are sufficient to induce an adipogenic response (26). Moreover, genetic studies using chimeric mice have confirmed that PPAR
is required for adipogenesis in vivo (27). Finally, it was recently demonstrated that reducing PPAR
activity with a selective PPAR
antagonist, BADGE, inhibits adipocyte differentiation in vitro (17). Herein, we describe a new synthetic PPAR
antagonist belonging to the phosphonophosphate family with antiadipogenic activity. In vitro, SR-202 inhibits both TZD and hormonally induced adipocyte differentiation of 3T3-L1 cells, as shown by the decrease in lipid accumulation and aP2 mRNA levels. Hormone-induced aP2 expression was even more dramatically reduced indicating that SR-202 is more or less effective depending on the differentiation protocol applied. Recently, it was suggested that there may be at least two pathways for 3T3-L1 adipocyte differentiation (19). The first one would be regulated by PPAR
and its ligands and accordingly could be blocked by the PPAR
antagonist LG 100641. The second pathway would be independent of PPAR
action. Indeed, it was insensitive to LG 100641 (19). The fact that SR-202 antagonized both hormone- and TZD-induced adipocyte differentiation reveals that SR-202 has effects on both pathways. In addition to antagonizing PPAR
activity, SR-202 would inhibit another actor of adipocyte differentiation that responds to the IBMX/dexamethasone/insulin cocktail. A similar double antiadipogenic effect has also been described for two other PPAR
antagonists, BADGE (17) and PD 068235 (18). The in vitro observations are corroborated by in vivo experiments where SR-202 treated wt mice have decreased PPAR
activity, together with a decreased WAT and BAT mass, and are protected from HFD-induced adipocyte hypertrophy.
Treatment of Mice with SR-202 Improves Insulin Sensitivity
In addition to regulating adipocyte differentiation, PPAR plays a key role in insulin signaling and agonists of PPAR
are used in the treatment of type 2 diabetes (11). However, the mechanism for this role remains obscure.
We demonstrate herein that the reduction of PPAR activity by partial gene targeting improves insulin sensitivity in mice, under HFD, as previously reported by others (10). Importantly, we show that treatment of wt mice with SR-202 reproduces the insulin-sensitizing phenotype, confirming that reduction of PPAR
activity is associated with an increased insulin sensitivity. This observation suggests that antagonists of PPAR
could be useful to explore the signal transduction pathways involved in PPAR
-mediated insulin sensitization. Although our experiments do not provide a molecular mechanism to explain the increase of insulin sensitivity associated to the reduction of PPAR
activity, they suggest that this increased sensitivity is a consequence of the diminution of adipose tissue mass. Indeed, HFD feeding, which increases adipocyte size, also increases secretion of molecules causing insulin resistance such as FFA and TNF
. In contrast, SR-202-treated wt mice and untreated PPAR
heterozygous mice, which are protected from HFD-induced adipocyte hypertrophy, showed a significant decreased FFA and TNF
levels, and a concomitant improved insulin sensitivity. This is associated with a decreased expression in WAT of genes involved in fatty acid transport and lipogenesis, which participate in preventing adipocyte hypertrophy and therefore obesity under HFD. We thus can conclude that reduction of PPAR
activity increases insulin sensitivity, presumably by preventing adipocyte hypertrophy and consequently decreasing the secretion of FFA and TNF
. These results are in agreement with human genetic studies. One rare mutation renders PPAR
more active, leading to increased adipocyte differentiation and obesity (28). An other more common mutation impedes PPAR
activity and is associated with a lower body mass index, improved insulin sensitivity, and higher levels of plasma high density lipoprotein cholesterol (29).
The role of leptin secretion in participating to the modulation of insulin sensitivity remains unclear. Indeed, although lipoatrophic animals without leptin are insulin resistant, there is evidence that leptin can interfere with insulin signaling in some cell types in vitro (30, 31). Previous reports have shown that activation of PPAR also results in a decrease of leptin secretion (32, 33, 34) and that PPAR
+/- mice under HFD overexpress and hypersecrete leptin (10). Thus, at first sight, our results concerning the concomitant decrease in PPAR
activity and plasma levels of leptin may appear surprising. However, the fact that we observe a similar reduced leptin secretion in two models of decreased PPAR
activity (deletion of one allele of the PPAR
gene or treatment with an antagonist of PPAR
) and a further decrease in SR-202 treated PPAR
+/- mice are clearly consistent. In addition, SR-202 treatment also modulates two other major regulators of leptin secretion, the size of adipocytes and plasma insulin levels, in favor of a decrease in leptin plasma levels. As mentioned above, how to relate this effect on leptin secretion and insulin sensitivity remains unclear. In particular, the fact that SR-202 increased insulin sensitivity in ob/ob mice, a mouse model of obesity without leptin, indicates that the action of the drug is not mediated by leptin.
PPAR Antagonists Like SR-202 Could Be Clinically Useful in the Treatment of Obesity and Type 2 Diabetes
Our data and other recent reports (10, 35) underline the interesting paradox that both PPAR overactivity, due to TZD, and PPAR
underactivity, due to either haploinsufficiency or treatment with a PPAR
antagonist, protect against obesity-induced insulin resistance. One explanation of this paradox may reside in the number and size of adipocytes. Indeed, TZD-mediated activation of PPAR
in rodents results in an increase in the number of small adipocytes and a decrease in the number of large, hypertrophic fat cells (25). In parallel, expression of two cytokines that influence energy homeostasis and insulin sensitivity, namely leptin and TNF
, is repressed by PPAR
agonists in adipocytes (30, 32, 36). Remarkably, untreated PPAR
+/- mice or SR-202-treated wt mice have smaller adipocytes than untreated wt mice, and they secrete less leptin and TNF
and release less fatty acids, contributing to enhanced insulin sensitivity. Although the ways of modulating PPAR
activity are distinct, the outcome appears similar: generation of small adipocytes and attenuated secretion of cytokines that interfere with insulin sensitivity. Consequently, not only agonists but also antagonists of PPAR
could be clinically useful in the treatment of obesity and type 2 diabetes. As a proof of concept, the treatment of ob/ob mice clearly establishes the efficacy of SR-202 in improving glucose disposal and insulin sensitivity, confirming that SR-202 is an insulin sensitizer in a model of preexisting obesity and insulin resistance. In addition, if SR-202, like BADGE, has a low affinity for PPAR
, it presents the specific advantages to be soluble in water and nontoxic in vivo, which is a significant bonus from a therapeutic point of view. Furthermore, it is possible that higher affinity PPAR
antagonists can be derived based on the structure of SR-202.
This work on SR-202, which directly acts on PPAR, might be compared with studies on RXR, the obligate partner of PPARs. Very recently, mice lacking RXR in adipocytes (37) or mice treated with an antagonist of RXR, HX531 (38), have been reported to be resistant to HFD-induced obesity and insulin resistance. However, treatment of PPAR
+/- mice with HX531 severely depleted the WAT, leading to a reemergence of insulin resistance (38). This biphasic effect may raise concerns for the potential clinical use of molecules such as HX531, more particularly in patients with the variant PPAR
Ala12 allele. HX531 might worsen insulin resistance in patients that have lower PPAR
activity (29). The lack of effect of SR-202 in PPAR
+/- mice suggest that such an effect would not occur in this situation. The use of SR-202 type compounds could be considered for patients with normal PPAR
activity (Pro12 allele) or for patients with the more active variant Ala 12 PPAR
. Finally, PPAR
antagonists seem more adapted for clinical use than RXR antagonists that can also act on RXR homodimers or on other RXR containing heterodimers, triggering undesired side effects.
In conclusion, we have characterized a first PPAR antagonist compound, SR-202, which is effective both in vitro and in vivo. We propose that, by preventing adipocyte differentiation and lipid accumulation, SR-202 protects mice from HFD-induced adipocyte hypertrophy and insulin resistance. SR-202 also improves insulin sensitivity in a model of established obesity, confirming that SR-202 is an insulin sensitizer. Treatment with compounds having the characteristics of SR-202 could be extremely interesting if they behave in human like in mouse. They would prevent adipogenesis and obesity while increasing insulin sensitivity, in contrast to TZDs, which induce weight gain. Consequently, SR-202 offers a potential for the development of compounds that could improve the therapy of metabolic diseases.
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MATERIALS AND METHODS |
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CARLA
The CARLAs were performed as previously described with small modifications (7). Briefly, bacteria expressing GST-human (h) PPAR LBD were incubated at 37 C until the culture reached A600 of 0.7. At that point, isopropyl ß-D-1-thiogalactopyranoside was added at the final concentration of 0.5 mM and the cells were incubated for a further 3 h at 28 C. Then, bacteria were lysed by three freeze cycles in liquid nitrogen. Sepharose 4B beads equilibrated in NETN (20 mM Tris-HCl, pH 8; 100 mM NaCl; 1 mM EDTA; 0.5% Nonidet P-40; 1 mM dithiothreitol) were added to the cell extract (250 µl for PPAR
culture). GST-PPAR
fusion protein was allowed to bind to the beads overnight at 4 C. SRC-1 (7) was produced using the TNT quick coupled transcription/translation system of Promega Corp. (Madison, WI) and labeled with methionine 35S. Beads, SRC-1, and the compounds were mixed overnight at 4 C. After three centrifugation-wash cycles with NETN, the beads were loaded on a 10% SDS-PAGE gel. Autoradiography was used to localize SRC-1 and the amount of this protein was determined by image analysis on an image master Amersham Pharmacia Biotech (Buckinghamshire, UK).
Cell Culture
3T3-L1 cells were cultured in DMEM supplemented by 10% bovine calf serum. Two days after reaching confluency, differentiation was induced in DMEM supplemented with 10% fetal calf serum and dexamethasone (1 µM)/IBMX (0.5 mM)/insulin (10 µg/ml). After 48 h, the medium was changed with only the addition of insulin for an additional 2 d. Thereafter, the cells were grown in DMEM supplemented with 10% fetal calf serum. Alternatively, cells were also differentiated with BRL 49653 (25 nM) and insulin (5 µg/ml). SR-202 or vehicle (H2O) was added 24 h before induction of differentiation. Medium was replenished with ligands every 2 d. Adipogenesis was determined by the staining of lipids with Oil Red O and by measuring the expression of adipocyte markers. RNA was isolated using the Trizol reagent (Life Technologies, Inc.).
Transfections
Human HeLa cells were transiently transfected using the phosphate calcium method with expression vectors for murine (m) PPAR (39), mPPARß (40), or mPPAR
(41), the single PPRE-containing reporter plasmid AcoA.TK.CAT (chloramphenicol acetyl transferase) and a ß-galactosidase encoding plasmid as control. Cells were also transfected with a hFXR encoding plasmid and pCAT-promIBABP reporter plasmid. They were exposed to the ligands for 36 h, lysed, and assayed for CAT and ß-galactosidase activity. Transfections were performed in quadruplicates.
Measurement of LDH Activity
Viability of the cells was estimated by measuring the LDH activity released in the culture medium. Because LDH is a cytosolic enzyme, the activity of the enzyme found in the medium reflects cellular lysis. The LDH activity is measured by adding 15 µl of medium samples in 1 ml of the following reaction medium (200 mM phosphate buffer, pH 6.8; 20 mM NADH; and 1 mM pyruvate). LDH activity was determined with a spectrophotometer. Individual values are then expressed as percentage of the total quantity of LDH present in cells, determined after sonication of the cells.
Animals
Wild-type and PPAR heterozygous mice, on a mixed background (sv129/C56Bl6), were maintained at 20 C with a 12-h light, 12-h dark cycle. Generation of PPAR
heterozygous mice will be described elsewhere. Animal experiments were approved by the animal authorization committee of the canton of Vaud (Switzerland). At 3 wk of age, the animals were separated by sex, genotyped for the PPAR
gene, and started to be fed with SD or HFD (D12451, 45 kcal% fat, Research Diets, Inc.) supplemented or not with SR-202 (400 mg/kg). All mice were weighted on a weekly basis, starting at 3 wk of age. Ten weeks later, animals were killed by cervical dislocation and tissues were removed, weighed, and frozen. Eight-week-old male ob/ob mice were purchased from Janvier Breeding (Le Genest Saint Isle, France). These mice were fed with a SD supplemented or not with SR-202 (400 mg/kg) for 20 d. Blood was withdrawn at the fed state on the indicated days from the tip of the tail for the measurement of plasma glucose and insulin levels. Glucose and insulin tolerance tests were performed on 6-h-fasted mice. Animals were injected ip with 2 mg/g body weight of glucose or 0.75 mU/g body weight of insulin. Blood was taken by tail puncture immediately before and 15, 30, 60, and 120 min after injection for measurements of plasma glucose or insulin levels.
Plasma Metabolites Measurement
Whole blood was withdrawn at the fed state, at the end of the dark cycle, from the orbital sinus by using heparinized microcapillary tubes. After withdrawal, the blood samples were immediately centrifuged, the serum removed, placed in a fresh tube and immediately frozen. Blood glucose levels were measured with a glucometer (Roche Molecular Biochemicals, Mannheim, Germany) on whole blood. Serum insulin, leptin, and TNF levels were assayed using the murine ELISA kits (CrystalChem, Downers Grove, IL; or R&D Systems, Minneapolis, MN). Serum levels of nonesterified FFA were measured using a colorimetric assay (Roche Molecular Biochemicals).
Histological Analysis
Adipose tissue was removed from three animals per group, fixed in 10% paraformaldehyde/PBS and maintained at 4 C until used. Fixed specimens were embedded in tissue-freezing medium (Leica Instruments, Nussloch, Germany) and frozen in dry ice and isopentane. Twenty-micrometer cryostat tissue sections were mounted on slides and fixed. After washing in PBS, sections were used for histological staining (hematoxylin-eosin and Oil Red O). The number of adipocytes was manually quantified on at least three different microscopic fields from three different animals per group.
Measurement of mRNA
LPL, CD36/FAT, aP2, and SREBP-1c mRNA levels were determined by reverse transcription followed by real-time PCR using the Light-cycler-FastStart DNA Master Sybr Green I (Roche Molecular Biochemicals). Total RNA (1 µg) was reverse-transcribed in a 20-µl reaction containing 1x TR buffer (50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2), 10 mM dithiothreitol, 0.5 mM deoxy-NTPs, 0.25 µg of random and oligo(deoxythymidine) primers (Promega Corp.) and 100 U of Superscript II enzyme (Invitrogen, Basel, Switzerland), after the conditions recommended by the manufacturer. PCRs were carried out in duplicate by adding 5 µl of diluted first-strand reaction in 15 µl of Hot Start reaction mix containing FastStart Taq DNA polymerase, Sybr green as dye, 23 mM MgCl2 and 0,5 µM of each specific primers. The following primer combinations were used: 5'-CAGTGGGGCATGTTGACATT-3' and 5'-TGAGAGCGAGTCTTCAGGTA-3' for LPL; 5'-AAGATCCAAAACTGTCTGTA-3' and 5'-GTCCTGGCTGTGTTTGGAGG-3' for CD36/FAT; 5'-CTTGTCTCCAGTGAAAACTT-3' and 5'-GTGGAAGTCACGCCTTTCAT-3' for aP2; and 5'-ACGGAGCCATGGATTGCACA-3' and 5'-AAGGGTGCAGGTGTCACCTT-3' for SREBP-1c. After 10 min at 95 C, the tubes were subjected to 40 cycles of amplification including denaturation for 10 sec at 95 C, hybridization for 5 sec at 5860 C, and elongation for 10 sec at 72 C. For each target, a standard corresponding to a fragment of the cDNA, cloned in pGEM-T easy vector (Promega Corp.), was serially diluted and quantified in parallel to generate a eight-point serial standard curve for the PCR analysis. Results were expressed in fmol (10-15 mol) or amol (10-18 mol)/µg of total RNA.
Statistical Analysis
Values are reported as mean ± SEM. Statistical significance was determined by the unpaired Students t test.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 J.R. and F.T. contributed equally to this work.
2 Present address: Institut National de la Santé et de la Recherche Médicale Unité 449, Faculté de médecine R.T.H. Laennec, rue Guillaume Paradin, 69372 Lyon cedex 08, France.
3 Present address: Department of Physiology/Neurobiology, University of Basel, Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland.
Abbreviations: aP2, Adipocyte fatty acid binding protein; BADGE, bisphenol A diglycidyl ether; BAT, brown adipose tissue; CARLA, coactivator-dependent receptor ligand assay; CAT, chloramphenicol acetyl transferase; FAT/CD36, fatty acid translocase; FFA, free fatty acids; FXR, farnesoid X receptor; HFD, high fat diet; h, human; IBMX, 3-isobutyl-1-methylxanthine; LDH, lactate deshydrogenase; LPL, lipoprotein lipase; m, murine; PPAR, peroxisome proliferator-activated receptor
; PPREs, peroxisome proliferator response elements; RXR, receptor of 9-cis-retinoic acid; SD, standard diet; SR-202, dimethyl
-(dimethoxyphosphinyl)-p-chlorobenzyl phosphate; SRC-1, steroid receptor coactivator-1; SREBP-1c, sterol regulatory element-binding protein 1c; TZDs, thiazolidinediones; WAT, white adipose tissue; wt, wild-type.
Received for publication January 23, 2002. Accepted for publication July 23, 2002.
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REFERENCES |
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