Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Mar 30;12(3):e0174761.
doi: 10.1371/journal.pone.0174761. eCollection 2017.

Regulation of White and Brown Adipocyte Differentiation by RhoGAP DLC1

Affiliations
Free PMC article

Regulation of White and Brown Adipocyte Differentiation by RhoGAP DLC1

Choon Kiat Sim et al. PLoS One. .
Free PMC article

Abstract

Adipose tissues constitute an important component of metabolism, the dysfunction of which can cause obesity and type II diabetes. Here we show that differentiation of white and brown adipocytes requires Deleted in Liver Cancer 1 (DLC1), a Rho GTPase Activating Protein (RhoGAP) previously studied for its function in liver cancer. We identified Dlc1 as a super-enhancer associated gene in both white and brown adipocytes through analyzing the genome-wide binding profiles of PPARγ, the master regulator of adipogenesis. We further observed that Dlc1 expression increases during differentiation, and knockdown of Dlc1 by siRNA in white adipocytes reduces the formation of lipid droplets and the expression of fat marker genes. Moreover, knockdown of Dlc1 in brown adipocytes reduces expression of brown fat-specific genes and diminishes mitochondrial respiration. Dlc1-/- knockout mouse embryonic fibroblasts show a complete inability to differentiate into adipocytes, but this phenotype can be rescued by inhibitors of Rho-associated kinase (ROCK) and filamentous actin (F-actin), suggesting the involvement of Rho pathway in DLC1-regulated adipocyte differentiation. Furthermore, PPARγ binds to the promoter of Dlc1 gene to regulate its expression during both white and brown adipocyte differentiation. These results identify DLC1 as an activator of white and brown adipocyte differentiation, and provide a molecular link between PPARγ and Rho pathways.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Dlc1 is Associated with PPARγ Defined Super-enhancers in both White and Brown Adipocytes.
(A) Venn diagram showing the overlap of the Top-100 super-enhancer associated genes in both white and brown adipocytes. Genome-wide binding profiles of PPARγ were used to define SEs. Representative SE associated genes with their ranks in the corresponding lineages were shown. (B) PPARγ binding peaks and their defined SEs at the Dlc1 gene locus in brown adipocytes, white adipocytes, brown adipose tissue and epididymal white adipose tissue. (C) ENCODE RNA-seq data showing the expression of Dlc1 gene in various mouse tissues.
Fig 2
Fig 2. DLC1 is Required for White Adipogenic Differentiation.
(A) mRNA expression of Dlc1 gene during differentiation of 3T3-L1 white adipocytes. (B) qRT-PCR result showing the knockdown of Dlc1 gene expression in 3T3-L1 white adipocytes. Tbp was used as normalizing control for gene expression. (C) Oil-Red-O staining showing reduced lipid droplet formation in Dlc1 knockdown cells. (D) mRNA expression of adipogenic marker genes Adiponectin (AdipoQ) and Fabp4 in si-Dlc1 white adipocytes. (E) qRT-PCR result showing the knockdown of Dlc1 gene expression in primary iWAT SVF cells derived white adipocytes. (F) Oil-Red-O staining showing reduced lipid droplet formation in primary iWAT SVF cell derived white adipocytes upon Dlc1 knockdown. (G) mRNA expression of adipogenic marker genes Adiponectin and Fabp4 in si-Dlc1 white adipocytes derived from primary iWAT SVF cells. (H) Western blotting analysis of mature adipocyte marker perilipin in primary iWAT SVF cell derived white adipocytes upon Dlc1 knockdown. Data are presented as mean ± s.e.m. n = 3–4 biological replicates. Two-tailed Student’s t-test was used: ** P < 0.01.
Fig 3
Fig 3. DLC1 Regulates Brown Adipogenesis and Brown Cell Function.
(A) mRNA expression of Dlc1 gene during differentiation of BAT-WT1 brown adipocytes and C3H10T1/2 derived brown adipocytes. (B) mRNA expression of Dlc1, general adipogenic, BAT-specific and mitochondrial genes in BAT-WT1 brown adipocytes upon Dlc1 knockdown. (C) Oil-Red-O staining showing reduced lipid droplet formation in Dlc1 knockdown BAT-WT1 brown adipocytes. (D) Oxygen consumption rates (OCR) in BAT-WT1 brown adipocytes with or without Dlc1 knockdown. Vertical green lines indicate the time points of Oligomycin, FCCP and Rotenone/Antimycin A (ROT/AA) injection. n = 3. (E) qRT-PCR measurements of the fold change of Ucp1 expression after isoproterenol treatment compared with basal levels without treatment. Dlc1 knockdown significantly impaired the upregulation of Ucp1 expression after isoproterenol treatment. (F) mRNA expression of Dlc1, general adipogenic and BAT-specific genes in Dlc1 knockdown brown adipocytes derived from C3H10T1/2 cells. (G) Oil-Red-O staining showing reduced lipid droplet formation in Dlc1 knockdown brown adipocytes derived from C3H10T1/2 cells. Data are presented as mean ± s.e.m. n = 3–6 biological replicates. Two-tailed Student’s t-test was used: * P < 0.05, ** P < 0.01.
Fig 4
Fig 4. Adipogenic Role of DLC1 Involves the Rho Pathway.
(A) Western blots showing the absence of DLC1 protein in Dlc1-KO MEFs. Calnexin was included as a loading control. (B) Oil-Red-O staining of differentiated wild-type and Dlc1-KO MEFs. (C) Relative mRNA expression of adipogenic and BAT-specific genes in differentiated MEFs. (D) Western blots showing active RhoA levels in Dlc1-KO MEFs. Total RhoA was shown as a loading control. Band intensity was quantified using ImageJ and the ratio between Active / Total RhoA was presented as bar graph in the right panel. (E) Phalloidin staining of F-actin in MEFs (left panels) and quantification of F-actin stress fibers (right panel). n = 15. (F) Proposed model of DLC1-Rho pathway and the site of action for ROCK inhibitor Y-27632 and F-actin inhibitor Latrunculin-B. (G) Oil-Red-O staining to show the rescue of lipid droplet formation by the ROCK and F-actin inhibitors in Dlc1-KO MEFs. (H) qRT-PCR showing the restoration of adipogenic gene expression. Data are presented as mean ± s.e.m. n = 2–3 biological replicates. Two-tailed Student’s t-test was used: * P < 0.05, ** P < 0.01, *** P < 0.001.
Fig 5
Fig 5. Dlc1 is a Direct Target of PPARγ.
(A) PPARγ ChIP-seq data revealed direct binding of this master regulator of adipogenesis at the promoter of Dlc1 gene in mouse BAT, eWAT, 3T3-L1 white adipocytes and C3H10T1/2 derived brown adipocytes. The positions (Relative to the transcription start site of Dlc1 gene) of PPARγ binding peaks (P1-P3) are indicated under the graph. (B) PPARγ ChIP confirmed its binding to Peak 1–3 at the promoter/ upstream region of Dlc1 gene in C3H10T1/2 cells (Brown adipocytes). Fabp4 gene promoter and a chromosome 15 (Chr.15) region were used here as positive/negative controls for PPARγ ChIP. (C-F) qRT-PCR results showing the knockdown of Pparg and the reduced expression of Dlc1 gene in (C) 3T3-L1 white adipocytes, (D) primary iWAT SVF cell derived white adipocytes, (E) BAT-WT1 brown adipocytes and (F) C3H10T1/2 derived brown adipocytes. Data are presented as mean ± s.e.m. n = 3–6 biological replicates. Two-tailed Student’s t-test was used: * P < 0.05, ** P < 0.01.

Similar articles

See all similar articles

Cited by 7 articles

See all "Cited by" articles

References

    1. Singla P, Bardoloi A, Parkash AA. Metabolic effects of obesity: A review. World J Diabetes. 2010;1(3):76–88. Epub 2011/05/04. 10.4239/wjd.v1.i3.76 - DOI - PMC - PubMed
    1. Tang QQ, Lane MD. Adipogenesis: from stem cell to adipocyte. Annu Rev Biochem. 2012;81:715–36. Epub 2012/04/03. 10.1146/annurev-biochem-052110-115718 - DOI - PubMed
    1. Kajimura S, Saito M. A new era in brown adipose tissue biology: molecular control of brown fat development and energy homeostasis. Annu Rev Physiol. 2014;76:225–49. Epub 2013/11/06. 10.1146/annurev-physiol-021113-170252 - DOI - PMC - PubMed
    1. Ahmadian M, Suh JM, Hah N, Liddle C, Atkins AR, Downes M, et al. PPARgamma signaling and metabolism: the good, the bad and the future. Nat Med. 2013;19(5):557–66. Epub 2013/05/09. 10.1038/nm.3159 - DOI - PMC - PubMed
    1. Spiegelman BM, Frank M, Green H. Molecular cloning of mRNA from 3T3 adipocytes. Regulation of mRNA content for glycerophosphate dehydrogenase and other differentiation-dependent proteins during adipocyte development. J Biol Chem. 1983;258(16):10083–9. - PubMed

MeSH terms

Grant support

This work was supported by the intramural funding from the Agency for Science, Technology and Research (A*STAR) of Singapore to FX. Funding for the open-access charge was provided by A*STAR of Singapore. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Feedback