Retinoic Acid Upregulates Preadipocyte Genes to Block Adipogenesis and Suppress Diet-Induced Obesity

All-trans-retinoic acid (RA) was from Calbiochem. Antibodies against Pref-1, Sox9, KLF2, and RARγ were from Santa Cruz Biotechnology. FABP4 antibodies were from R&D Systems. Pref-1 antibodies used in immunoblots were from Cell Signaling Technology. Lentiviruses harboring short hairpin RNAs (shRNAs) were from Open Biosystems. The triglyceride assay kit was from Zen-Bio.

Preadipocytes were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% calf serum. Two days postconfluence, cells were induced to differentiate in medium supplemented with 10% fetal bovine serum, 10 μg/mL insulin, 0.5 mmol/L 3-isobutyl-1-methylxanthine, and 0.25 mmol/L dexamethasone. NIH3T3-L1 cells were used unless otherwise specified. Experiments using preadipocytes were carried out at 70–80% confluency.

RNA was extracted and cDNA generated as described (13). TaqMan chemistry and assays on demand probes (Applied Biosystems) were: Pref-1, Mm00494477_m1, Hs00171584_m1; Sox9, Mm00448840, Hs00165814_m1; KLF2, Mm01244979_g1, Hs00360439_g1; C/EBPα, Mm00514283_s1; C/EBPβ, Mm00843434_s1; Adiponectin, Mm01343606_m1; FABP4, Mm00445880_m1; PPARγ, Mm01184322_m1; RARα, Mm043626_m; RARγ, Mm00441083_m1; CRABP-II, Mm00801691_m1; PPARβ/δ, Mm00803184; FABP5, Mm00783731_s1; and GAPDH, Mm99999915_g1.

Chromatin immunoprecipitation (ChIP) assays were carried out as described (13). PCR was performed using the following primer sets: RAR response element (RARE) Pref-1 sense, 5′-cttttcgtggtggttttcgt-3′ and antisense, 5′-gcaagtctcaggaaccaagc-3′; RARE Sox9 sense, 5′-agagcttcgccctcttagg-3′ and antisense, 5′-gttctgcttcttgcgtcctg-3′; RARE KLF2 sense, 5′-cctcttgctttctccagagc-3′ and antisense, 5′-gtgcagggaagagctgttgt-3′; KLF2 site RARγ sense, 5′-ccttattggtggccatggta-3′ and antisense, 5′-gacttgggttgcttggtgat-3′; and KLF2 site CRABP-II sense, 5′-tcaccgcactttcagctcta-3′ and antisense, 5′-gaggctagaagggcagaggt-3′.

Human adipose tissues were collected from patients undergoing abdominoplasty. Mouse preadipocytes were isolated from epididymal and inguinal fat depots. Preadipocytes were isolated as described (35) and passaged three times prior to experimentation.

Preadipocytes were grown in DMEM supplemented with 10% calf serum. Differentiation media (DMEM supplemented with 10% fetal bovine serum, 10 μg/mL insulin [Sigma-Aldrich], 0.5 mmol/L 3-isobutyl-1-methylxanthine [Sigma-Aldrich], and 0.25 mmol/L dexamethasone [ThermoFisher]) was added 2 days past confluence. Three days later, medium was replaced with DMEM supplemented with 10% FBS for an additional 4 days.

Stromal-vascular (preadipocyte) and mature adipocyte fractions were isolated as described (35) and pooled. Pooled cells were stained with Nile red (50 μg/mL) and Hoechst dye 33342 (10 μmol/L) for 1 h in the dark. Cells were analyzed by flow cytometry (at 530 and 430 nm) to assess the fraction of Nile red-positive (lipid-containing) cells within total the Hoechst-positive cells.

Mouse studies were performed by the Mouse Metabolic Phenotyping Center of the Case Western Reserve University. All experiments used male mice. Lean mice were fed chow diet (LabDiet 5P76 Irradiated Isopro RMH 3000; Prolab, St. Louis, MO). Eight-week-old C57BL/6Ntac mice (Taconic Farms) mice were subcutaneously implanted with an RA pellet or mock pelleted by using a 10-gauge precision trochar (Innovative Research of America). Eight-week-old mice were placed on a high-fat/high-sucrose diet (HFHS; research diet D12331) as indicated. At the end of the experimental duration, mice were killed and tissues harvested following an overnight fast.

Twenty-four–hour food consumption was measured once a week. During each 24-h period, mice were housed individually with a thin layer of bedding, and a known quantity of diet was provided. Food remaining after 24 h was weighed. Body weights were measured before and after the 24-h feeding period.

Following an overnight fast, mice were killed and blood collected by a heart puncture. Plasma was isolated by using Microtainer plasma separator tubes (BD Biosciences). Leptin levels were measured using a leptin (mouse/rat) ELISA kit (ALPCO). Other parameters were measured by Veterinary Diagnostic Services (Marshfield Laboratories, Marshfield, WI).

Tissues were preserved in 10% formalin and then processed and sectioned by the Case Western Reserve University Histology Core. Sections were analyzed by using a Zeiss camera (Carl Zeiss).

Data were analyzed by Student paired t test.

Eight-week-old C57BL/6Ntac male mice were used to examine effects of RA on dietary-induced obesity. Mice were fed an HFHS diet (research diet D12331). Animals in the experimental group were systemically treated by subcutaneous implantation of slow-release pellets (7.5 mg, 60-day release; Innovative Research of America). Preliminary results showed that this supraphysiological dose was effective but not toxic to the mice. Mice treated with RA were resistant to diet-induced weight gain despite a higher food intake as compared with control animals (Fig. 1A and B). The weight of white adipose tissue (WAT; combined epididymal and inguinal fat) of RA-treated mice was markedly lower as compared with WAT of nontreated animals (Fig. 1C), and histological examination showed that adipocyes in RA-treated mice were markedly smaller than in control animals (Fig. 1D and Supplementary Fig. 1A). RA treatment blunted diet-induced elevation in levels of plasma cholesterol (Fig. 1E) and plasma triglycerides (Fig. 1F) but did not affect levels of plasma free fatty acids (Fig. 1G).

RA treatment upregulated adipose PPARβ/δ target genes (e.g., 3-phosphoinositide-dependent protein kinase 1), which is involved in insulin signaling, and adipose differentiation-related protein, which is associated with lipid droplets. In addition, RA induced the expression of genes involved in regulating energy homeostasis that are under control of both RAR and PPARβ/δ, including uncoupling protein (UCP) 3, which mediates energy dissipation, and hormone-sensitive lipase (HSL), which catalyzes lipid hydrolysis (Fig. 1H). The data indicate that the ability of RA to protect animals from diet-induced obesity and insulin resistance is mediated, at least in part, by increasing the expression of adipose proteins that enhance lipid oxidation and energy dissipation and that promote insulin signaling.

To examine whether RA inhibits adipogenesis in vivo, adipose cells were isolated from WAT of control and RA-treated mice. Cells were stained with Hoechst stain and with the fluorescent lipid dye Nile red and analyzed by flow cytometry to assess the number of mature, lipid-accumulating cells in the tissues. The data (Fig. 1I) indicate that RA treatment decreased the content of mature adipocytes in WAT. Concomitantly, RA upregulated the expression of the preadipocyte marker Pref-1 in total adipose tissue (Fig. 1J and Supplementary Fig. 1B) and in the adipose stromal vascular fraction (Fig. 1K). Hence, the data indicate that RA treatment both upregulated the expression level of Pref-1 in cells that express this protein and increased the number of Pref-1–expressing cells. RA thus seem to contribute to maintenance of preadipocytes and inhibits adipocyte differentiation in vivo.

To investigate the mechanism by which RA inhibits adipocyte differentiation, the well-established cultured cell model of preadipocytes, NIH-3T3-L1 cells, and the pluripotent mesenchymal stem cell line C3H10T1/2 were used. Differentiation was induced by a standard protocol (see research design and methods) and monitored by examining the appearance of the adipocyte marker FABP4. Including RA in differentiation mixtures markedly inhibited adipogenesis both in NIH3T3-L1 and in C3H10T1/2 cells (Fig. 2A and Supplementary Fig. 5A). In agreement with the reports that RA functions in preadipocytes primarily through the CRABP-II/RAR pathway (13,14), the hormone did not inhibit differentiation in the presence of the pan-RAR antagonist LE540 (Fig. 2A). All of the components of the CRABP-II/RAR pathway in preadipocytes (i.e., CRABP-II, RARα, and RARγ) are downregulated following induction of differentiation, but loss of RARγ is more pronounced than that of RARα (Fig. 2B). Overexpression of RARγ suppressed differentiation and enhanced the ability of RA to inhibit the process (Fig. 2C and E). Conversely, decreasing the level of RARγ enhanced differentiation and abrogated inhibition by RA (Fig. 2D and E). The observations thus demonstrate that downregulation of this receptor is critical for allowing differentiation to proceed.

Several proteins have been reported to effectively inhibit adipocyte differentiation. Among these is Pref-1, a signaling protein for which the downstream effector, transcription factor Sox9, inhibits adipogenesis by suppressing the expression of the adipogenic factors C/EBPβ and C/EBPδ (30,31,36,37). Another protein that was reported to inhibit adipogenesis is the transcription factor KLF-2, which suppresses PPARγ, C/EBPα, and SREBP1c expression (33,34).

RA and the pan-RAR agonist (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthylenyl)-1-propenyl] benzoic acid (TTNPB) induced expression of Pref-1, Sox9, and KLF2 in NIH-3T3-L1 cells (Fig. 2F–I and Supplementary Fig. 1C), C3H10T1/2 cells (Supplementary Fig. 5B), and primary human preadipocytes (Fig. 2J). The three genes were also upregulated in adipose tissue of mice treated with TTNPB or RA in vivo (Fig. 2K). RA and TTNPB induced the expression of these genes both in the absence and presence of the protein synthesis inhibitor cycloheximide (Fig. 2F–H), and the effect was abolished upon pretreatment with the pan-RAR antagonist LE540 (Fig. 2L and Supplementary Fig. 1D and E). Hence, Pref-1, SOX9, and KLF2 are direct targets for RAR. Examination of their respective promoter regions using Nubiscan (http://www.nubiscan.unibas.ch/) revealed putative RAR response elements in all three genes. Specifically, Pref-1, SOX9, and KLF2 harbor the putative RAREs GGGTCAgaAGCCCA, AGTCCAagtcaAGGGCC, and AGTCCAagtcaAGGGCC at 563, 1370, and 1300 bp upstream from the respective transcription start sites. ChIP analyses were carried out to identify the RAR isotype that regulates the expression of the genes. The data (Fig. 3A) indicated that RARγ, but not RARα, is associated with the RAREs in the Pref-1 and Sox9 promoters. It is worth noting that SOX9 was previously found to also comprise a direct target for RAR in breast cancer cells (38). Both RAR isotypes were found to be associated with the RARE of the KLF2 promoter, but binding of RARγ was stronger than that of RARα. The elements were also occupied by RXR, demonstrating the presence of a functional RAR-RXR heterodimer. It is worth noting that recruitment of the RAR-RXR heterodimers to the response element was independent of the presence of RA. To further examine the relative contributions of RARγ and RARα, their expression levels were modulated. Ectopic overexpression of RARγ, but not RARα, upregulated Pref-1 (Fig. 3B) and Sox9 (Supplementary Fig. 2A). KLF2 was induced by both RARs (Supplementary Fig. 2B). Critical involvement of RARγ was further demonstrated by the observation that decreasing the expression of this isotype abolished the RA response of all three genes (Fig. 3C). As expected from bona fide RAR targets, upregulation of Pref-1, SOX9, and KLF2 by RA was enhanced by ectopic expression of CRABP-II (Fig. 3D and Supplementary Fig. 2D and E).

RA potently induced the expression of Pref-1, SOX9, and KLF2 at early stages of adipocyte differentiation but was much less effective at later stages (Fig. 3E–G). These observations, likely reflecting the loss of CRABP-II and RARγ following differentiation induction (Fig. 2B), provide a rationale for understanding why RA is effective in inhibiting adipocyte differentiation only early in the process (9).

The expression of Pref-1 and Sox9 in preadipocytes was decreased using respective shRNAs (Supplementary Fig. 3A), and cells were then induced to differentiate in the presence or absence of RA. Differentiation was assessed by Oil Red O staining, measurements of the triglyceride content of cells, and monitoring the expression of various adipocyte markers. Downregulation of Sox9 or of both Sox9 and Pref-1 enhanced differentiation and abrogated the inhibitory activity of RA both in NIH3T3-L1 cells (Fig. 3H–J and Supplementary Fig. 3B–D) and C3H10T1/2 cells (Supplementary Fig. 5C and D). In contrast, reducing the expression of Pref-1 alone was not as effective (Fig. 3H and I). The latter observation can be readily understood considering that SOX9, the downstream effector of Pref-1, is itself a direct target for RAR. As SOX9 is induced by RA independently of Pref-1 (Supplementary Fig. 4A and B), RA can inhibit differentiation even in the absence of the latter.

To examine the involvement of KLF2 in RA-induced inhibition of adipogenesis, the factor was ectopically overexpressed in preadipocytes. Cells were then induced to differentiate in the presence of varying concentrations of RA. Ectopic expression of KLF2 effectively inhibited adipogenesis and markedly potentiated the inhibitory activity of RA (Fig. 4A and B). Conversely, decreasing the expression of KLF2 enhanced adipogenesis and abrogated the ability of RA to inhibit the differentiation of NIH3T3-L1 (Fig. 4C–E) and C3H10T1/2 cells (Supplementary Fig. 5E and F).

Interestingly, ectopic expression of KLF2 led to upregulation of CRABP-II and RARγ (Fig. 4F and G). Examination of the promoters of these genes revealed the presence of KLF2 response elements in both. Specifically, the RARγ gene harbors a 5′-CACCC-3′ sequence at +105 bp, and CRABP-II harbors three tandem KLF2 elements,, 5′-CACCCCCACCCCCACCACCC-3′, at −763 bp. ChIP assays demonstrated that KLF2 occupies these elements in preadipocytes (Fig. 4H). Hence, KLF2 is placed within a positive-feedback loop that promotes the transcriptional activities of RA through the CRABP-II/RARγ path. In accordance with this notion, overexpressing KLF2 enhanced the ability of RA to upregulate the expression of the hallmark RAR target gene Cyp26a1 as well as the newly identified RAR targets Pref-1 and Sox9 (Fig. 4I).

Inhibition of adipocyte differentiation by RA, exerted by activating the CRABP-II/RAR path, may contribute to the ability of the hormone to suppress diet-induced obesity. To examine this notion, 8-week-old CRABP-II heterozygous (C57/Bl6/CRABP-II^+/−) mice and wild-type (WT) littermates were used. The reduced expression of CRABP-II in these mice is expected to decrease the transcriptional activity of RAR. Expression levels of the adipocyte markers FABP4 and PPARγ in WT and CRABP-II^+/− mice were similar, demonstrating that mature adipocytes in CRABP-II^+/− mice retain normal phenotype (Supplementary Fig. 4C). In agreement with the identification of Pref-1, SOX9, and KLF2 as CRABP-II/RAR targets, the levels of these genes were markedly lower in adipose tissue of CRABP-II^+/− versus WT mice (Fig. 5A). The reduced expression of these adipocyte inhibitors suggests that CRABP-II^+/− mice are prone to excess adipogenesis. If so, it may be predicted that high-fat feeding of these mice will result in enhanced adiposity. In accordance with this notion, CRABP-II^+/− mice fed an HFHS diet gained more weight than WT animals (Fig. 5B), although they displayed lower food intake (Fig. 5C). The increased weight stemmed from a higher weight of WAT and liver (Fig. 5D). The observed hepatic steatosis (Fig. 5E) is in keeping with the report that RA enhances lipid oxidation and inhibits lipid biosynthesis in the liver of mice (39). Remarkably, the size of adipocytes in WT and CRABP-II^+/− mice was similar (Fig. 5F and G). Hence, the increase in the weight of WAT in these animals did not result from enhanced hypertrophy but, instead, reflected accelerated generation of mature adipocytes. The levels of Pref-1, SOX9, and KLF2 in CRABP-II^+/− mice remained lower than in WT animals following 13 weeks on a high-fat diet (Fig. 5H).

In mature adipocytes, RAR upregulates HSL and UCP1 (13,40) and represses the expression of leptin (41). Reflecting a reduced RAR activity in CRABP-II^+/− mice, expression of adipose HSL and UCP1 was lower, and expression of leptin was higher in these animals (Fig. 6A). The resulting increased serum level of leptin (Fig. 6A, inset) provides a rationale for understanding the reduced food intake in these animals (Fig. 5C). In contrast with RAR target genes, the expression of adipose genes that are regulated by RA through activation of PPARβ/δ, including 3-phosphoinositide-dependent protein kinase 1, adipose differentiation-related protein, aldehyde dehydrogenase 9, and UCP3 (13), was similar in CRABP-II^+/− and WT mice (Fig. 6A). Hence, the reduced expression of CRABP-II did not hamper the ability of RA to activate PPARβ/δ in mature adipocytes. CRABP-II^+/− mice also displayed normal plasma levels of cholesterol, triglycerides, and free fatty acids, parameters that were previously shown to be controlled by RA through the FABP5/PPARβ/δ pathway (Fig. 6B–D).

Multiple reports demonstrated that the vitamin A metabolite RA induces weight lost and enhances insulin sensitivity in obese mice (13,23). In further support of the conclusion that RA plays an important role in regulating body weight, it was also reported that mice lacking retinol dehydrogenase 1, an enzyme that catalyzes the first step in the conversion of retinol to RA, display increased size and obesity (42). One mechanism by which RA induces weight loss involves activation of RAR and PPARβ/δ in adipocytes, liver, and skeletal muscle, resulting in upregulation of genes that promote lipolysis, lipid oxidation, energy dissipation, and insulin sensitivity (13,14) (Fig. 1H). RA thus prevents diet-induced weight gain and adipose hypertrophy (Fig. 1A–D) and protects mice from high-fat diet–induced elevation in serum levels of cholesterol and triglycerides (Fig. 1E and F). It is noteworthy that, whereas RA treatment lowered serum triglyceride levels in mice, it has been reported that administration of the agent to human patients leads to hypertriglyceridemia (43–46). The differential response may reflect species specificities. However, as biochemical abnormalities in human patients appear only at high RA doses (45,46), it is possible that hypertriglyceridemia may be avoided if RA is used at subtoxic doses. The efficacy of a low-dose RA treatment in overcoming obesity and insulin resistance in humans remains to be examined.

In addition to modulating lipid metabolism and energy utilization, RA suppresses the generation of new adipocytes in cultured cells and, as we show in this study, in vivo (Fig. 1I–K). The data demonstrate that this activity is mediated by CRABP-II cooperating mainly with the RAR isotype RARγ, albeit with some contribution of RARα (Fig. 3A–D). These RA-activated RARs inhibit adipogenesis through induction of target genes that encode the adipogenesis inhibitors Pref-1, Sox9, and KLF2 (Fig. 6E).

It was previously reported that inhibition of adipogenesis by RA originates from interference of RAR with the activity of the early adipogenic factor C/EBP-β and that the effect may be mediated by Smad3 (9–11). Our observations that RA induces the expression of Pref-1 suggest an alternative mechanism for inhibition of C/EBPs by RA. Pref-1 inhibits adipogenesis by triggering mitogen-activated protein kinase/extracellular signal-regulated kinase signaling, leading to upregulation of Sox9, which, in turn, suppresses the transcription of both C/EBP-β and C/EBP-δ (30,36). The data described in this study demonstrate that RARγ induces the expression of Pref-1 as well as SOX9, providing a direct rationale for understanding the inhibition of C/EBPs by RA. The involvement of Smad3 in the activity remains to be clarified. Transgenic mice that overexpress Pref-1 in adipose tissue display partial lipodystrophy and, accordingly, hypertriglyceridemia (47). Notably, induction of Pref-1 by RA was not accompanied by biochemical abnormalities and did not lead to lipodystrophy. The data thus indicate that RA treatment of mice fed a high-fat diet moderated adipogenesis but did not completely block the process. Likely, induction of Pref-1 by RA increased the expression level of the protein to a lesser extent than that achieved by transgenic overexpression (47). Taken together with the report that a low level of Pref-1 is associated with obesity in humans (32), these observations support the notions that RA may be efficacious in treatment of obesity and that it does so in part by increasing the expression of Pref-1.

KLF2 inhibits adipocyte differentiation by suppressing the expression of PPARγ, C/EBPα, and SREBP1c (33,34). We show in this study that KLF2 is a direct RAR target in preadipocytes (Fig. 2H) and that, in turn, the factor induces the expression of RARγ and CRABP-II (Fig. 4H). KLF2 is thus placed within a positive-feedback loop that promotes RA signaling through CRABP-II and RARγ, thereby further enhancing inhibition of adipogenesis by the hormone (Fig. 6E). Interestingly, a similar cross-talk between KLF2 and RA signaling was recently found to occur in MCF-7 mammary carcinoma cells in which KLF2 functions as a tumor suppressor (48). In MCF-7 cells, RA promotes proliferation and survival when directed toward PPARβ/δ by FABP5 but inhibits growth when targeted to RAR by CRABP-II (15,49,50). The tumor-suppressive activities of KLF2 may thus be exerted through its ability to modulate RA signaling (48). The cross-talk between KLF2 and the RA machinery in cancer development remains to be investigated.

CRABP-II^+/− mice fed an HFHS diet displayed increased adipose tissue mass (Fig. 5D) but a similar adipocyte size as compared with WT counterparts (Fig. 5F). These observations must reflect that the number of adipocytes is higher in the CRABP-II^+/− mice. Hence, enhanced weight gain in CRABP-II^+/− mice did not stem from adipocyte hypertrophy but originated directly from enhanced adipogenesis. These observations demonstrate that decreasing RA signaling through the CRABP-II/RAR pathway resulted in resistance to inhibition of adipogenesis by RA. Consequently, HFHS intake led to massive adipogenesis and enhanced adiposity in CRABP-II^+/− mice. Taken together, these and previous observations demonstrate that RA regulates adipose tissue biology by two distinct activities: in preadipocytes, RA activates CRABP-II and RAR to inhibit adipocyte differentiation, and in mature adipocytes, RA activates both the CRABP-II/RAR and FABP5/PPARβ/δ paths to promote lipid oxidation and energy utilization. RA thus suppresses dietary-induced obesity by counteracting both adipogenesis and adipocyte hypertrophy.

This work was supported by National Institutes of Health (NIH) Grant R01-DK-060684 to N.N. D.C.B. was supported in part by NIH Grant T3-DK-073195. The Mouse Metabolic Phenotyping Center of the Case Western Reserve University is supported by NIH Grant DK-59630.

No potential conflicts of interest relevant to this article were reported.

D.C.B. designed experiments, collected data, and wrote the article. D.D. collected data. H.S. provided human adipose tissue. C.M.C. provided advice and assisted in animal studies. N.N. formulated experiments, analyzed data, and cowrote the manuscript and is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

The authors thank Vincent Giguere (McGill University, Montreal, Quebec, Canada) for sharing CRABP-II^+/− mice, Hiroyuki Kagechika (Tokyo Medical and Dental University, Tokyo, Japan) for providing the RAR antagonist LE-540, Cecile Rochette-Egly (Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg, France) for CRABP-II antibodies, and Mukesh Jain (Case Western Reserve University, Cleveland, OH) for the KLF2 retrovirus.