Dexras1 mediates glucocorticoid-associated
adipogenesis and diet-induced obesity
Jiyoung Y. Chaa,1, Hyo Jung Kimb,1, Jung Hwan Yub,c, Jing Xua, Daham Kimb,c, Bindu D. Paula, Hyeonjin Choib,c,
Seyun Kima,2, Yoo Jeong Leeb, Gary P. Hoa, Feng Raoa, Solomon H. Snydera,d,e,3, and Jae-woo Kimb,c,f,3
aThe Solomon H. Snyder Department of Neuroscience and Departments ofdPsychiatry and Behavioral Sciences andePharmacology and Molecular Sciences,
The Johns Hopkins University School of Medicine, Baltimore, MD 21205;bDepartment of Biochemistry and Molecular Biology, Integrated Genomic Research
Center for Metabolic Regulation, Institute of Genetic Science, Yonsei University College of Medicine, Seoul 120-752, Korea;cBrain Korea 21 PLUS Project for
Medical Science, Yonsei University, Seoul 120-752, Korea; andfDepartment of Integrated OMICS for Biomedical Sciences, World Class University Program of
Graduate School, Yonsei University, Seoul 120-749, Korea
Contributed by Solomon H. Snyder, November 5, 2013 (sent for review October 2, 2013)
Adipogenesis, the conversion of precursor cells into adipocytes, is
associated with obesity and is mediated by glucocorticoids acting
via hitherto poorly characterized mechanisms. Dexras1 is a small G
protein of the Ras family discovered on the basis of its marked
induction by the synthetic glucocorticoid dexamethasone. We
show that Dexras1 mediates adipogenesis and diet-induced obe-
sity. Adipogenic differentiation of 3T3-L1 cells is abolished with
Dexras1 depletion, whereas overexpression of Dexras1 elicits adi-
pogenesis. Adipogenesis is markedly reduced in mouse embryonic
fibroblasts from Dexras1-deleted mice, whereas adiposity and diet-
induced weight gain are diminished in the mutant mice.
insulin|cyclic AMP|nitric oxide|Cushing disease
hypertension, and cardiovascular disability (1, 2). Obesity is as-
sociated with hypertrophy of adipocytes, as well as increases in
adipogenesis, which reflects the differentiation of precursor cells
into adipocytes (3–6). The adipogenic process involves multiple
factors, especially cAMP, insulin, and glucocorticoids (7, 8).
Thus, in the best-characterized model of adipogenesis, 3T3-L1
cells, a fibroblast line, are treated with the phosphodiesterase
inhibitor 3-isobutyl-1-methylxanthine (IBMX) to increase cAMP
levels, insulin, and the synthetic glucocorticoid dexamethasone.
When exposed to a hormonal mixture of IBMX, insulin, and
dexamethasone, 3T3-L1 cells accumulate lipid and develop the
characteristic morphology of mature adipocytes (7–9).
Adipocyte differentiation is controlled by a complex network of
transcription factors that temporally regulate adipocyte gene ex-
pression (4). An early response to hormonal stimuli of adipogenesis
is activation of two members of the CCAAT/enhancer binding
protein (C/EBP) family of transcription factors, C/EBPβ and C/
EBPδ, which induce the expression of C/EBPα and peroxisome
proliferator-activated receptor (PPAR)γ, the two principal adipo-
genic transcription factors. Although transcriptional regulation of
adipogenic differentiation has been well characterized, less is known
about how hormonal inducers promote this process. In particular,
how glucocorticoids induce adipogenesis is poorly understood.
Dexras1 (also known as Rasd1) is a small G protein of the Ras
family discovered on the basis of its marked induction by the syn-
thetic glucocorticoid dexamethasone (10). Dexras1 interacts with
neuronal nitric oxide synthase via the scaffolding protein CAPON,
with nitric oxide serving as a guanine nucleotide exchange factor for
Dexras1 (11). Dexras1 also participates in the glutamate–NMDA
neurotransmission cascade that leads to cellular iron entry and
neurotoxicity (12). Dexras1 also influences circadian rhythms (13).
Disruption of circadian rhythms leads to the development of met-
abolic disorders, including obesity and diabetes (14–17).
Here, we show that Dexras1 mediates adipogenesis and diet-
induced obesity. Dexras1, which is induced by glucocorticoids
during adipogenic differentiation, is essential for adipogenesis.
Overexpression of Dexras1 rescues impaired adipogenesis in
besity presents a major public health problem, with its
widespread occurrence leading to increases in diabetes,
mouse embryonic fibroblasts (MEFs) from Dexras1-deleted mice.
Dexras1 knockout mice display impaired adiposity and are resistant
to diet-induced weight gain. Accordingly, agents impacting Dexras1
may offer benefit in the treatment of obesity.
Dexras1 Is Induced by Glucocorticoids During Adipogenic Differentiation.
Preliminary microarray analysis sought genes altered in 3T3-L1
cells with adipogenesis initiated by treatment with IBMX,
dexamethasone, and insulin (designated as MDI), as well as
genes highly expressed in murine or human adipose tissue. These
experiments revealed high levels of Dexras1. Among diverse
organs, we observe highest levels of Dexras1 in fat-enriched
organs, especially white adipose tissue (WAT) (Fig. 1A). Dexa-
methasone treatment markedly augments Dexras1 levels in
mouse tissues (Fig. 1B). Adipogenic differentiation of 3T3-L1
cells is also associated with a striking induction of Dexras1, with
peak sevenfold enhancement at 4–8 h (Fig. 1C and Fig. S1A).
Omission of dexamethasone from the MDI mixture abolishes
Dexras1 mRNA expression (Fig. S1B), indicating that Dexras1
expression is transcriptionally regulated by interactions of
dexamethasone and the glucocorticoid receptor.
Dexras1 Is Required for Adipogenic Differentiation. To determine
the impact of Dexras1 upon adipogenesis, we depleted Dexras1
Glucocorticoids are well known to play a major role in obesity,
but underlying mechanisms have been obscure. We demon-
strate that the small G protein Dexras1, first identified based
on its dramatic induction by glucocorticoids, mediates adipo-
genic differentiation of preadipocytes, as well as diet-induced
obesity in intact rodents. Thus, the adipogenesis of preadipocytes
is abolished by Dexras1 deletion and selectively induced by
Dexras1 expression. Relevance to intact animals is evident from
our experiments wherein diet-induced obesity is prevented in
mice with knockout of Dexras1. Thus, pharmacotherapy in-
volving Dexras1 may afford a promising approach to the therapy
Author contributions: J.Y.C., H.J.K., S.H.S., and J.-w.K. designed research; J.Y.C., H.J.K.,
J.H.Y., J.X., D.K., B.D.P., H.C., S.K., Y.J.L., G.P.H., and F.R. performed research; J.Y.C.,
H.J.K., S.H.S., and J.-w.K. analyzed data; and J.Y.C., H.J.K., S.H.S., and J.-w.K. wrote
The authors declare no conflict of interest.
1J.Y.C. and H.J.K. contributed equally to this work.
2Present address: Department of Biological Sciences, Korea Advanced Institute of Science
and Technology, Daejeon 305-701, Korea.
3To whom correspondence may be addressed. E-mail: email@example.com or japol13@yuhs.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| December 17, 2013
| vol. 110
| no. 51
by cotransfecting 293FT cells with the lentivirus expression plasmid and
packaging plasmids using Lipofectamine 2000 (Invitrogen) according to the
manufacturer’s instructions. Infectious lentiviruses were harvested at 48 h
after transfection and filtered through 0.45-μm cellulose acetate filters; 3T3-
L1 cells were plated in 6-well plates at 50% confluence. After overnight
incubation, medium containing viral particles was removed and replaced
with fresh medium containing 2 μg/mL puromycin. Cells were grown to 90%
confluence and subcultured in medium containing puromycin. Dexras1
knockdown efficiency was confirmed by RT-PCR. The sequences for lentiviral
shRNAs were as follows: sh-Dexras1, #1, 5′-CCGGG CTGGT CATTT GCGGT
AACAA CTCGA GTTGT TACCG CAAAT GACCA GCTTT TTG-3′; #2, 5′-CCGGC
AAGCG CTCT GAACT GAGTA CTCGA GTACT CAGTT CAGAG TCGCT TGTTT
TTG-3′; and #3, 5′-CCGGG ACCTC ATGTA CATTC GTGAA CTCGA GTTCA
CGAAT GTACA TGAGG TCTTT TTG-3′.
siRNA. The 3T3-L1 cells were plated into 60-mm-diameter dishes 18–24 h
before transfection. Cells were transfected with control or gene-specific
siRNA at 50 nM (Dharmacon) in OPTI-MEM medium using Lipofectamine
RNAiMAX (Invitrogen), according to the manufacturer’s protocol. The next
day, the medium was replaced with fresh DMEM containing 10% calf serum,
and the cells were incubated for 24 h before the induction of differentia-
tion. Cellular total RNA and protein were assayed at the indicated time
points respectively by RT-PCR and immunoblot. Oil red O staining of Dexras1
knockdown was performed at day 8. The siRNA sequences were as follows:
si-Dexras1, #1, 5′-CAGGU UAUCA ACGAA ACUUU U-3′; #2, 5′-GGUCA UUUGC
GGUAA CAAAU U-3′; #3, 5′-UCAAA CAGCA GAUCC UAGAU U-3′; and si-
glucocorticoid receptor, 5′-GAUCC CCGAA AGCAU UGCAA CCTCA-3′.
Transient Transfection Assay. Dexras1-overexpressing vector (pcDNA3-Dex-
ras1-FLAG) was generated by inserting the whole ORF of mouse Dexras1 with
anN-terminal FLAG taginto pcDNA3.0(Invitrogen).To maximize transfection
efficiency, we used microliter volume electroporation of 3T3-L1 preadipocytes
with OneDrop MicroPorator MP-100 (Digital Bio). The cells were trypsinized,
washed with 1× PBS, and resuspended in 10 μL of resuspension buffer R with
0.5 μg of plasmid at a concentration of 200,000 cells per pipette. The cells
were then microporated at 1,300 V, with a 20-ms pulse width and two
pulses. Following microporation, the cells were seeded in 35-mm cell culture
dishes and placed at 37 °C in a 10% CO2-humidified atmosphere.
Animals. We generated Dexras1 knockout mice by deleting whole Dexras1
exons flanked by loxp sites with Cre recombinase (28). Then, Dexras1 mice
were backcrossed onto C57BL/6J background for eight generations. Mice
were housed in a 12-h light, 12-h dark cycle. At 4 wk of age, mice were either
fed normal chow diet or 60% HFD (Harlan Teklad). All mice used for studies
were littermate males unless otherwise noted. Animal protocols were
performed in accordance with National Institutes of Health guidelines and
approved by the Johns Hopkins University Committee on Animal Care.
MEF Adipogenesis Assays. Wild-type and knockout E14 embryos were isolated
from a single heterozygous female that had been paired with a heterozygous
male. The head and organs were removed, and the remaining carcasses were
minced and incubated in trypsin to obtain single cells. MEFs were seeded in
six-well plates and propagated to confluence. Forty-eight hours later, dif-
ferentiation was initiated using DMEM containing 10% FBS, 0.5 mM IBMX,
1 μM dexamethasone, 10 μg/mL insulin, and 10 μM rosiglitazone for 2 d.
Subsequently, cells were maintained in DMEM supplemented with 10% FBS,
10 μg/mL insulin, and 1 μM rosiglitazone. After 48 h, the medium was
replaced with maintenance medium containing DMEM supplemented with
10% FBS. Cells were stained with oil red O as described.
Histology and Adipocyte Size Analysis. Epididymal WAT specimens were fixed
in 10% neutral-buffered formalin for 24 h before trimming for histology
processing. Paraffin-embedded tissue sections were stained with hematox-
ylin/eosin according to standard protocols. Adipocyte size was determined
Body Composition Analysis. Whole-body lean mass and total fat of mice was
measured with an EchoMRI whole-body composition analyzer. One-month-
lean and fat mass per body weight.
Statistical Analysis. All results are expressed as means ± SD. Statistical com-
parisons of groups were made using an unpaired Student t test or two-
ACKNOWLEDGMENTS. We thank Prof. Su-Jae Lee (Hanyang University) for
providing the H-Ras and K-Ras expression vectors. We thank Drs. Tetsuya
Hosooka and Masato Kasuga (Kobe University) for providing a protocol for
mouse embryonic fibroblast adipogenesis assays. This work was supported
by US Public Health Service Grant MH18501 (to S.H.S.) and by National
Research Foundation of Korea Grants 2011-0030711 and 2011-0015665,
funded by the Korean government, Ministry of Science, ICT and Future
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