The primary function of white adipose tissues is to store excess energy. The current study aimed to investigate the roles of Gremlin 2 (Grem2), a glycoprotein in adipogenesis. Using polymerase chain reaction-based microarrays, it was determined that Grem2 was markedly downregulated in adipose tissues from obese animals and humans. In addition, 3T3-L1 cells were used to investigate the details of the mechanisms underlying the anti-adipogenic effects of Grem2. Grem2 expression was markedly decreased upon the induction of adipocyte differentiation, as demonstrated by reverse transcription-quantitative polymerase chain reaction and western blot analysis. Notably, Grem2 overexpression inhibited adipogenesis, while knockdown of Grem2 led to an increase in adipogenesis. At the molecular level, Grem2 promotes nuclear translocation of β-catenin, an integral Wnt signaling component. Consistently, inhibition of Wnt/β-catenin signaling using a retrovirus targeting the β-catenin coding region attenuated the anti-adipogenic effects of Grem2. Therefore, to the best of our knowledge, the current study shows for the first time that Grem2 may be an important regulator of adipocyte differentiation.
When dietary nutrient intake chronically exceeds energy expenditure, obesity occurs due to an increase in the size and/or number of white adipocytes (
Decades of studies have identified a number of transcription factors involved in adipocyte differentiation, including peroxisome proliferator-activated receptor γ (PPARγ) and members of the C/EBP family (
In the present study, polymerase chain reaction (PCR)-based microarrays were performed in obese and lean mice to screen potential genes involved in adipogenesis.
Male C57BL/6,
Human subcutaneous fat tissues from obese (BMI>25; n=25) and lean (BMI<23; n=20) adults were collected at the Department of Endocrinology at Chongqing Ninth People's Hospital. All subjects had a stable weight, defined as the absence of fluctuations of >1% of body weight for at least 2 months prior to surgery. Adipose tissue was immediately frozen in liquid nitrogen following extraction. All samples were obtained with informed consent and the study was approved by the Chongqing Ninth People's Hospital Institutional Review Board.
3T3-L1 fibroblasts were obtained from The Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China), and cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco-BRL, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco-BRL), 100 IU/ml penicillin and 100
Total RNA was isolated from tissues or cells using the TRIzol reagent (Invitrogen Life Technologies, Shanghai, China), and reverse transcription was performed using the RT kit from Promega Corporation (Madison, WI, USA), according to the manufacturer's instructions. Affymetrix arrays were performed using Mouse Genome 2.0 chips from Gene Tech Company Limited (Shanghai, China). In order to quantify the transcripts of the interest genes, qPCR was performed using a SYBR Green Premix Ex Taq kit (Takara Bio Inc., Dalian, China) on a Light Cycler 480 (Roche, Basel, Switzerland) with 5 mg RNA. The following primer sequences form Bioyare Biotechnology Company (Shanghai, China) were used: Mouse Grem2, forward 5′-TGTGCTGGTAAAGGTAGCTGA-3′ and reverse 5′-CCACCTCTCTGAGTTGTTGCT-3′; human Grem2, forward 5′-ATCCCCTCGCCTTACAAGGA-3′ and reverse 5′-TCTTGCACCAGTCACTCTTGA-3′; C/EBPa, forward 5′-CAAGAACAGCAACGAGTACCG-3′ and reverse 5′-GTCACTGGTCAACTCCAGCAC-3′; PPARg, forward 5′-CTCCAAGAATACCAAAGTGCGA-3′ and reverse 5′-GCCTGATGCTTTATCCCCACA-3′; LPL, forward 5′-ATGGATGGACGGTAACGGGAA-3′ and reverse 5′-CCCGATACAACCAGTCTACTACA-3′; PEPCK, forward 5′-ATGAGCCGCAAACTGGGTC-3′ and reverse 5′-AGAGCCGAACTCCACAATCTC-3′; CD36, forward 5′-AGCATTCAACGCCAGGTTC-3′ and reverse 5′-CGAGTCTGTCAGTTCAATACCAA-3′; aP2, forward 5′-AAGGTGAAGAGCATCATAACCCT-3′ and reverse 5′-TCACGCCTTTCATAACACATTCC-3′; mouse HPRT1, forward 5′-TCAGTCAACGGGGGACATAAA-3′ and reverse 5′-GGGGCTGTACTGCTTAACCAG-3′; human HPRT1, forward 5′-CCTGGCGTCGTGATTAGTGAT-3′ and reverse 5′-AGACGTTCAGTCCTGTCCATAA-3′. The PCR cycling conditions were as follows: Initial period at 94°C for 3 min, followed by a two-step PCR program consisting of 95°C for 5 sec and 60°C for 30 sec for 55 cycles. Gene expression was calculated relative to the mRNA expression of HPRT1, and relative quantitation analysis of gene expression data was performed using the 2−ΔΔCt method.
Retroviruses containing empty vector or Grem2 cDNA were constructed by Invitrogen Life Technologies. siRNA oligos targeting Grem2 or β-catenin were purchased from Gene Pharma (Shanghai, China) and transfected into 3T3-L1 cells using Lipofectamine 2000 (Invitrogen Life Technologies), according to the manufacturer's instructions. Histological analysis was conducted using Oil Red O staining. Cells were fixed with 4% paraformaldehyde for 30 min, followed by incubation with Oil Red O (Sigma-Aldrich, St. Louis, MO, USA) for 45 min.
Cells or tissues were harvested and lysed with lysis buffer (150 mM NaCl, 50 mM Tris-HCl and 1% NP-40; pH 7.5). Proteins were quantified and separated by 10% SDS-PAGE and transferred to polyvinylidine difluoride membranes (Amersham Bioscience, Buckinghamshire, UK). After blocking with 10% non-fat milk in phosphate-buffered saline, membranes were immunoblotted with the following rabbit antibodies from Abcam (Cambridge, MA, USA): Polyclonal anti-Grem2 (ab102563; 1:1,000), monoclonal C/EBPα (ab40764; 1:1,000), polyclonal PPARγ (ab19481; 1:1,000), monoclonal P-P38 (ab178867; 1:2,000), polyclonal P-AKT (ab66138; 1:1,000), monoclonal P-CREB (ab32096; 1:2,000), monoclonal CREB (ab32515; 1:2,000), polyclonal β-catenin (ab6302; 1:1,000), polyclonal lamin B (ab16048; 1:2,000) and monoclonal GAPDH (ab181602; 1:1,000). The blots were then incubated with horseradish peroxidase-linked secondary antibodies (Cell Signaling Technology, Inc. Danvers, MA, USA). The signals were detected using the SuperSignal West Pico Chemiluminescent Substrate kit (Pierce, Rockford, IL, USA) according to manufacturer's instructions.
The results are expressed as the mean ± standard error of the mean and analyzed using Student's t-test or one way analysis of variance with GraphPad Prism software, version 5.0.1 (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.
To identify potential genes involved in adipogenesis and obesity, male C57BL/6 mice aged 10 weeks were placed on high-fat or normal diets for 12 weeks. The mice were sacrificed by cervical dislocation and gene expression arrays were performed using epididymal adipose tissues. The clustering analysis of the Affymetrix arrays revealed that 1,174 genes were upregulated and 1,572 genes were downregulated in the adipose tissues of mice fed a high-fat-diet compared with those fed a normal diet (P<0.05; data not shown). Among these genes, it was determined that Grem2 was significantly downregulated (P<0.001). Its downregulation was further confirmed by RT-qPCR and western blot analysis (
To determine whether the inhibition of Grem2 represents a common feature of obesity,
To explore the functions of Grem2 in adipogenesis, its gene expression was investigated at numerous time points during 3T3-L1 differentiation. The RT-qPCR results revealed that Grem2 mRNA gradually reduced from day 0 post-induction (
The role of Grem2 in adipogenesis was tested using retroviruses carrying Grem2 cDNA or empty vector, which resulted in forced overexpression of Grem2 in 3T3-L1 preadipocytes (
To further examine the roles of Grem2, its specific siRNA was transfected into 3T3-L1 cells (
Finally, the molecular basis for the observed phenotypic changes in 3T3-L1 cells with Grem2 overexpression was investigated. As shown in
In the present study, it was determined that the Grem2 gene was significantly downregulated in white adipose tissue from obese mice and humans. Using 3T3-L1 cells cultured
Members of the Gremlin family, including Grem2, have been shown to be bone morphogenetic protein (BMP) antagonists (
At the molecular level, it was revealed that Grem2 activates Wnt/β-catenin signaling, as evidenced by its nuclear accumulation. Initial studies have demonstrated that ectopic expression of Wnt1, an activator of Wnt/β-catenin signaling, may inhibit adipogenesis (
In conclusion, the results of the current study highlight a pivotal role for Grem2 in white fat-cell differentiation by controlling Wnt/β-catenin signaling. Further studies are required to investigate the functions and mechanisms of Grem2
Downregulation of Gremlin 2 (Grem2) in adipose tissues from obese mice and humans. (A–F) mRNA and protein levels of Grem2 were determined in epididymal adipose tissues from obese mice. C57BL/6 mice were fed a high-fat-diet (HFD) or normal chow diet (NCD) for 12 weeks. After fasting for 8 h, mice were sacrificed and tissues were collected. n=5–8. *P<0.05, **P<0.01, ***P<0.001. (G and H) mRNA and protein levels of Grem2 were determined in subcutaneous adipose tissues from obese and lean human subjects.
Gremlin 2 (Grem2) expression during 3T3-L1 adipocyte differentiation. Expression of Grem2 mRNA and protein during differentiation of 3T3-L1 cells was analyzed using (A) reverse transcription-quantitative polymerase chain reaction and (B) western blot analysis at the indicated times *P<0.05, **P<0.01, ***P<0.001 compared with day 0.
Gremlin 2 (Grem2) overexpression inhibits differentiation of 3T3-L1 preadipocytes into adipocytes. (A) Grem2 protein levels in 3T3-L1 cells after transfection with retrovirus containing empty vector (EV) or Grem2 cDNA. (B) 3T3-L1 cells were infected with retrovirus containing empty vector (EV) or Grem2 cDNA. On day 8 after induction of differentiation, cell morphology was determined with Oil Red O staining and a microscope at ×200. (C and D) mRNA and protein levels of C/EBPα and PPARγ in 3T3-L1 cells on day 8 after induction of differentiation. (E) mRNA levels of LPL, PEPCK, CD36 and aP2 in 3T3-L1 cells **P<0.01, ***P<0.001, compared with EV.
Gremlin 2 (Grem2) knockdown promotes 3T3-L1 cell differentiation. (A) Grem2 protein levels in 3T3-L1 cells after transfection with small interfering (si)RNA oligos targeting Grem2 or negative controls. Cells were harvested at day 2 and day 4 after differentiation induction for western blot analysis. (B) 3T3-L1 cells were infected with siRNA oligos targeting Grem2 or negative controls. On day 8 after induction of differentiation, cell morphology was determined with Oil Red O staining and a microscope at ×200. (C and D) Expression levels of C/EBPα, PPARγ, LPL, PEPCK, CD36 and aP2 in 3T3-L1 cells **P<0.01, ***P<0.001, compared with NC.
Gremlin 2 (Grem2) activates Wnt/β-catenin signaling to inhibit adipogenesis. (A) Protein levels of nuclear β-catenin was determined by western blot analysis in 3T3-L1 cells after transfection of Grem2 or empty vector (EV). (B) Protein levels of phosphorylated p38, AKT and CREB were examined by western blot analysis in 3T3-L1 cells after transfection of Grem2 or EV. (C) Grem2 protein levels in 3T3-L1 cells after transfection with siRNA oligos targeting β-catenin or negative controls. Cells were harvested at days 2 and 4 after differentiation induction for western blot analysis. (D) Expression levels of C/EBPα and PPARγ in 3T3-L1 cells. **P<0.01.