Journal of Cosmetics, Dermatological Sciences and Applications, 2013, 3, 99-106
http://dx.doi.org/10.4236/jcdsa.2013.31014 Published Online March 2013 (http://www.scirp.org/journal/jcdsa)
99
Suppression of Sebum Production and Accumulation by
β-Cryptoxanthin Due to the Inhibition of the Expression of
Diacylglycerol Acyltransferase-1 and Perilipin in Hamster
Sebocytes*
Takashi Sato1#, Yoshiyuki Shirakura2, Katsuyuki Mukai2, Akira Ito1
1Department of Biochemistry and Molecular Biology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Ha-
chioji, Tokyo, Japan; 2R & D Center, Unitika Ltd., Kyoto, Japan.
Received December 13th, 2012; revised January 15th, 2013; accepted January 24th, 2013
ABSTRACT
Background: Acne vulgaris is characterized by the enhancement of sebaceous lipogenesis and sebum secretion, and
apart from retinoids and some natural products there are few effective anti-acne agents that directly suppress sebum
production and accumulation in sebaceous glands. Objective: We examined the effects of β-cryptoxanthin (β-CRX),
which is a carotenoid pigment most abundant in Citrus unshiu Marcovich (Satsuma mandarin orange) and plays a role
as a Vitamin A precursor on sebum production and accumulation in hamster sebaceous gland cells (sebocytes). Materi-
als and methods: The regulation of sebum production was examined by the measurement of triacylglycerols (TGs), the
major sebum component, and oil red O staining in insulin-differentiated hamster sebocytes. The expression of diacyl-
glycerol acyltransferase-1 (DGAT-1), a rate-limiting enzyme of TG biosynthesis, and perilipin 1 (PLIN1), a lipid stor-
age droplet protein, was analyzed using real-time PCR and Western blotting. Results: Hamster sebocytes constitutively
produced TGs during cultivation and the production of TGs was enhanced by insulin treatment. Both constitutive and
insulin-enhanced TG productions were dose- and time-dependently inhibited by β-CRX as well as 13-cis retinoic acid.
In addition, the gene expression of DGAT-1 was suppressed by β-CRX in the sebocytes. Furthermore, the insulin-en-
hanced sebum accumulation as lipid droplets was reduced in the β-CRX-treated cells. Moreover, β-CRX was found to
suppress the gene expression and production of PLIN1 in insulin-differentiated hamster sebocytes. Conclusions: These
results provide novel evidence that β-CRX is an effective candidate for acne therapy by its ability to exert dual inhibi-
tory actions against DGAT-1-dependent TG production and PLIN1-mediated lipid-droplet formation in hamster sebo-
cytes.
Keywords: β-Cryptoxanthin; Sebocytes; Triacylglycerol Biosynthesis; Diacyglycerol Acyltransferase; Perilipin;
Lipid-Droplet Formation; Sebum
1. Introduction
The pathogenesis of acne, a common inflammatory skin
disease [1,2], is characterized by: 1) excess sebum pro-
duction in sebaceous glands; 2) the formation of micro-
comedones, which is closely associated with the hyperk-
eratinization of the follicular wall and infundibulum; 3)
the hyperproliferation of Propionibacterium acnes (P.
acnes); and 4) the induction of inflammatory reactions
such as the acceleration of cytokine production and the
biosynthesis of arachidonic acid metabolites in keratino-
cytes, sebocytes, and invaded inflammatory cells [3,4].
The aggravation and duration of the inflammation are
likely to result in acne scar that causes a psychological
and social impact in the patient’s quality of life [4].
Sebum production in acne lesions has been reported to
be increased by 5α-dihydrotestosterone, insulin, insu-
lin-like growth factor 1, and prostaglandins [5-9]. In ad-
dition, the biosynthesis of sebum components such as
triacylglycerols (TGs) and sapienic acids is regulated by
diacylglycerol acyltransferase (DGAT), a rate-limiting
enzyme of TG synthesis [6,10], and ∆6 desaturase
(FADS2) [11], respectively, in human and hamster se-
bocytes. Furthermore, we have previously reported that
the production of perilipin (PLIN), a lipid storage droplet
protein, is augmented in differentiated hamster sebocytes
and localized on the surface of intracellular lipid droplets,
*Conflict of interest: The authors have no conflict of interest to declare.
#Corresponding author.

Suppression of Sebum Production and Accumulation by β-Cryptoxanthin Due to the Inhibition of the Expression of
Diacylglycerol Acyltransferase-1 and Perilipin in Hamster Sebocytes
100
indicating that perilipin is a differentiation marker in ham-
ster sebocytes [12]. On the other hand, retinoic acids
such as tretinoin (all-trans retinoic acid; atRA) and isot-
retinoin (13-cis retinoic acid; 13-cisRA) have been topi-
cally and/or systemically used for acne therapy [13,14].
They have been reported to exhibit comedolytic, anti-
inflammatory, and anti-lipogenetic actions in sebaceous
glands and pilosebaceous units in humans, rats, and ham-
sters in vivo and in vitro [14-19]. However, the use of
retinoids in acne therapy has been limited in acceptance
because of adverse effects such as skin irritation, scaling,
and teratogenicity [13].
β-Cryptoxanthin (β-CRX), a carotenoid pigment most
abundant in Citrus unshiu Marcovich (Satsuma mandarin
orange), has been reported to exhibit multiple disease pre-
ventive actions; e.g. anti-tumorigenic, anti-obesity, anti-
atherogenic, and immunopotentiative ones [20-23]. In ad-
dition, carotenoids such as β-CRX and β-carotene have
been reported to be present in the blood of people from
different countries including Japan, which is associated
with some health benefits [22,24]. Indeed, epidemiologic
studies have shown that higher intakes or blood levels of
β-CRX result in a reduced risk of lung cancer and rheu-
matoid arthritis development [25-27]. On the other hand,
it has also been reported that low plasma level of Vita-
min A is associated with the development and aggrava-
tion of acne [28]. In addition, β-CRX is a vitamin A pre-
cursor, which is oxidatively cleaved to vitamin A by
β-carotene 15,15’-dioxygenase [29], and can be stably
stored in some tissue for several months [30]. Taken to-
gether with a recent report by Shirakura et al. [31] where
β-CRX inhibits the intracellular lipid-droplet formation
in mouse 3T3-L1 adipocytes, we hypothesize that, in-
stead of retinoids such as atRA and 13-cisRA, β-CRX is
an effective candidate for acne therapy by modulating
sebaceous lipogenesis. However, it is not fully under-
stood whether β-CRX directly suppresses sebum pro-
duction and accumulation in sebaceous gland cells (se-
bocytes) or not.
In the present study, we demonstrated that β-CRX dose-
and time-dependently inhibited the production and intra-
cellular accumulation of sebum in insulin-differentiated
hamster sebocytes. Furthermore, the β-CRX-mediated in-
hibition of sebum production and accumulation is closely
associated with the transcriptional suppression of DGAT-
1 and PLIN1, respectively, in differentiated hamster se-
bocytes.
2. Materials and Methods
2.1. Cell Culture and Treatment
Hamster sebocytes (2.4 × 104 cells/cm2) [32] were plated
onto 96-well multiplates, 35-mm or 100-mm diameter
culture dishes (Becton Dickinson, Tokyo, Japan) and
then cultured for 24 h in DMEM/F12 (Invitrogen, Carls-
bad, CA) supplemented with 6% heat-denatured fetal
bovine serum (Nichirei Biosciences Inc., Tokyo, Japan),
2% human serum (C-C Biotech Co., Valley Center, CA),
0.68 mM L-glutamine (Invitrogen), and recombinant
human epidermal growth factor (10 nM) (Progen Bio-
technik GmbH, Heidelberg, Germany) to achieve com-
plete cell adhesion as previously described [6,18]. The
hamster sebocytes were treated every two days for up to
8 days with or without β-CRX (purity ≥ 95%; Shikoku
Yashima Pure Chemicals, Tokushima, Japan) (Figure 1)
or 13-cisRA (Sigma Chemical, St. Louis, MO) in the
presence or absence of a sebocyte-differentiation inducer,
insulin (10 nM) (Sigma Chemical) [12] in DMEM/F12
supplemented with heat-denatured fetal bovine serum,
human serum, and L-glutamine. In this series of experi-
ments, hamster sebocytes were used as far as the 3rd pas-
sage level.
2.2. Analyses of Sebum Production and
Accumulation
After treating sebocytes with β-CRX, 13-cisRA, and/or
insulin, the cells were subjected to the quantification of
TGs, the major sebum component, using Liquitech TG-II
(Roche Diagnostics, Tokyo, Japan) as previously de-
scribed [12]. The amounts of intracellular TGs were cal-
culated using an authentic trioleinate-standard solution
(0.6 mg/ml). Intracellular DNA content was measured
using salmon sperm DNA (6.25 - 100 mg/ml) and 3,5-
diaminobenzoic acid dihydrochloride (Sigma Chemical).
For the analysis of intracellular sebum accumulation, oil
red O staining was performed. Briefly, the cells were
washed once with Ca2+- and Mg2+-free phosphate-buff-
ered saline [PBS(-)] and fixed with 4% paraformalde-
hyde (Wako Pure Chemicals, Osaka, Japan) diluted with
PBS(-) for 1 h at room temperature. The cells were
washed with distilled H2O and then stained with 0.3% oil
red O (Sigma Chemical) in isopropanol:distilled H2O
(3:2, vol:vol) at 37˚C for 15 min. The stained cells were
washed with distilled H2O, and then viewed with a light
microscope furnished with a digital camera (Olympus Op-
tical Co., Tokyo, Japan).
2.3. Real-Time PCR
For the quantification of DGAT-1 and PLIN1 mRNA,
total RNA was isolated from cells using ISOGEN (Nip-
pon Gene, Toyama, Japan) and then the aliquot of RNA
(500 ng) was subjected to reverse transcriptase reaction
Figure 1. Chemical structure of β-cryptoxanthin (β-CRX).

Suppression of Sebum Production and Accumulation by β-Cryptoxanthin Due to the Inhibition of the Expression of
Diacylglycerol Acyltransferase-1 and Perilipin in Hamster Sebocytes
101
for the synthesis of cDNA using a PrimeScript RT re-
agent Kit (Takara Bio, Shiga, Japan) according to the
manufacturer’s instructions. Aliquots (an equivalent of
2.5 ng of total RNA) of the transcript were subjected to
real-time PCR using SYBR Premix Ex Taq II (Takara
Bio) and the following specific primers: human DGAT-1
(NM_012079); 5’-TCTACAAGCCCATGCTTCGAC-3’
(sense) and 5’-GGACGCTCACCAGGTACT-3’ (an-
tisense), hamster PLIN1 (AB091681); 5’-ACCTTGCT
GGATGGAGACC-3’ (sense) and 5’-CCAGGACCTTG
TCTGAAGT-3’ (antisense), and hamster glyceralde-
hyde-3-phosphate dehydrogenase (GAPDH) (X52123);
5’-CAGAACATCATCCCTGCAT-3’ (sense) and 5’-TA
GGAACACGGAAGGCCAT-3’ (antisense) as previ-
ously described [33]. The amplification cycle was per-
formed at 94˚C for 5 s and 60˚C for 30 s using a Thermal
Cycler Dice Real Time System TP-800 (Takara Bio).
The obtained threshold cycle (CT) value for DGAT-1
and PLIN1 was normalized by that for GAPDH, and the
relative expression level was expressed as the mean
value of the control as 1.
2.4. Western Blot Analysis
The harvested cell lysate (50 μg protein) was subjected to
Western blot analysis using 12.5% acrylamide gel as
previously described [33]. The membrane was reacted
with rabbit anti-(human PLIN1) IgG, which was custom-
ized by Operon Biotechnologies (Tokyo, Japan). To
evaluate the level of β-actin as an internal control, the
harvested cell lysates (50 μg protein) were similarly sub-
jected to Western blot analysis using rabbit anti-(human
β-actin) IgG (Medical & Biological Laboratories, Nagoya,
Japan). Immunoreactive PLIN1 and β-actin were visual-
ized with Amersham enhanced chemiluminescence-Wes-
tern blotting detection reagents (GE Healthcare Bio-
Sciences, Tokyo, Japan) according to the manufacturer’s
instructions. Relative amounts of PLIN1 protein against
β-actin were quantified by densitometric scanning using
an Image Analyzer LAS-1000 Plus (GE Healthcare), and
the relative expression level was expressed as the mean
value of the control as 1.
2.5. Statistical Analysis
A one-way ANOVA was used for the statistical analysis,
and then the Fisher test was applied when multiple com-
parisons were performed.
3. Results
3.1. Inhibition of TG Production and DGAT-1
Gene Expression by β-CRX in Hamster
Sebocytes
Since hamster sebocytes constitutively produce TGs dur-
ing cultivation in vitro [32], we first examined the effect
of β-CRX on constitutive TG production in cultured
hamster sebocytes. As shown in Figure 2(A), β-CRX
was found to decrease the level of TGs in a dose-de-
pendent manner (70% inhibition at 10 μM). In addition,
the sebocyte differentiation inducer, insulin (10 nM), was
found to enhance the production of TGs in hamster se-
bocytes, and the enhanced level of TGs was dose-de-
pendently decreased by β-CRX (72% inhibition at 10
μM). Furthermore, a similar decrease in TG level was
time-dependently observed in hamster sebocytes treated
with β-CRX as well as 13-cis RA (Figure 3), as we pre-
viously reported [18,34]. On the other hand, the gene
expression of DGAT-1 was constitutively detectable, and
was found to be increased in response to insulin treat-
ment in hamster sebocytes (7.7 ± 3.9 fold, p < 0.05)
(Figure 2(B)). In addition, β-CRX was found to suppress
the gene expression of DGAT-1 in both the insulin-un-
treated and treated hamster sebocytes (52% and 88%
inhibition, respectively) (Figure 2(B)). Therefore, these
results suggest that β-CRX inhibits the production of TGs
due to the suppression of DGAT-1 expression in hamster
sebocytes.
3.2. Suppression of Sebum Accumulation by
Decreasing Gene Expression and Production
of Perilipin in Hamster Sebocytes
As the intracellular accumulation of sebum as lipid-dro-
plets has been reported to be due to the increase of TG
production in differentiated hamster sebocytes [32], oil
red O staining revealed that the lipid-droplet formation
was augmented in the insulin-differentiated hamster se-
bocytes (Figures 4(B) vs. (A)). In addition, the enhanced
sebum accumulation was found to be abolished by add-
ing β-CRX (10 μM) (Figures 4(C) vs . (B)). On the other
hand, perilipin, a lipid-droplet surface protein, has been
reported to play an important role in the formation of
intracellular lipid droplets in differentiated adipocytes,
steroidogenic cells, and sebocytes [12,35]. As β-CRX
inhibited sebum accumulation as lipid droplets (Figure
4), we examined whether β-CRX influenced the produc-
tion of PLIN1 in hamster sebocytes. As shown in Figure
5, the production of PLIN1 was barely detectable in insu-
lin-untreated sebocytes, but was augmented by insulin
treatment. In addition, β-CRX was found to dose-depen-
dently suppress the insulin-enhanced production of
PLIN1 in hamster sebocytes. Furthermore, both basal and
insulin-augmented levels of PLIN1 mRNA were found to
be decreased in the β-CRX-treated cells (Figure 6). Thus,
these results suggest that the suppression of PLIN1 pro-
duction by β-CRX is associated with the inhibition of
sebum accumulation in differentiated hamster sebocytes.

Suppression of Sebum Production and Accumulation by β-Cryptoxanthin Due to the Inhibition of the Expression of
Diacylglycerol Acyltransferase-1 and Perilipin in Hamster Sebocytes
102
(A) (B)
Figure 2. The decrease of intracellular TG level by β-CRX in hamster sebocytes. Hamster sebocytes at the 3rd passage were
treated every two days for 8 days with or without β-CRX (1.1 - 10 μM) in the presence or absence of insulin (Ins) (10 nM). A:
The harvested cell lysate was subjected to the measurement of intracellular TG level as described in the Materials and
Methods. B: Isolated RNA (an equivalent of 2.5 ng of total RNA) from the cells was subjected to the analysis of DGAT-1
mRNA expression as described in the Materials and Methods. Data are shown as mean ± SD of three dishes. * and **, signifi-
cantly different from untreated cells (Cont) (p < 0.05 and 0.01, respectively). # and ##, significantly different from insulin (Ins)
(10 nM)-treated cells (p < 0.05 and 0.01, respectively).
Figure 3. Time-dependent inhibition of TG production by β-CRX in hamster sebocytes. Hamster sebocytes at the 3rd passage
were treated every two days for up to 8 days with or without β-CRX (10 μM) or 13-cisRA (1 μM) in the presence or absence
of insulin (10 nM), and then the intracellular level of TGs was measured. Data are shown as mean ± SD of three dishes. Lane
1, untreated cells; Lane 2, β-CRX (10 μM)-treated cells; Lane 3, 13-cisRA (1 μM)-treated cells; Lane 4, insulin (10
nM)-treated cells; Lane 5, cells treated with insulin (10 nM) and β-CRX (10 μM); and Lane 6, cells treated with insulin (10
nM) and 13-cisRA (1 μM). *, **, and ***, significantly different from untreated cells (p < 0.05, 0.01, and 0.001, respectively). #
and ###, significantly different from insulin (10 nM)-treated cells (p < 0.05 and 0.001, respectively).
4. Discussion as the DGAT-1 transcript in differentiated hamster sebo-
cytes [18]. In addition, Harris et al. (2011) [36] reported
that DGAT is required for not only TG synthesis but also
lipid droplet formation in adipocytes from DGAT
knockout mice. Therefore, these results provide novel evi-
dence that β-CRX inhibits de novo synthesis of TGs
We demonstrated that β-CRX suppresses the production
of TGs and the gene expression of DGAT-1 in differenti-
ated hamster sebocytes. Our previous study showed that
the decrease of TG production by both 13-cisRA and
atRA is closely related to that of DGAT activity as well

Suppression of Sebum Production and Accumulation by β-Cryptoxanthin Due to the Inhibition of the Expression of
Diacylglycerol Acyltransferase-1 and Perilipin in Hamster Sebocytes
103
Figure 4. β-CRX decreases sebum accumulation in insulin-
differentiated hamster sebocytes. Hamster sebocytes treated
with insulin (10 nM) and/or β-CRX (10 μM) as shown in
Figure 2 were subjected to oil red O staining for the analy-
sis of intracellular sebum accumulation as described in the
Materials and Methods. A: untreated cells; B: insulin (10
nM)-treated cells; and C: cells treated with insulin (10 nM)
and β-CRX (10 μM). Bars: 50 μm.
Figure 5. β-CRX and 13-cisRA suppress the production of
PLIN1 in insulin-differentiated hamster sebocytes. Cell ly-
sate (50 μg protein) prepared from hamster sebocytes treat-
ed every two days for 8 days with insulin (10 nM), β-CRX
(1.1 - 10 μM), and/or 13-cisRA (1 μM) was subjected to
Western blot analysis for PLIN1 and β-actin as described in
the Materials and Methods. Data are shown as mean ± SD
of four independent experiments. ***, significantly different
from untreated cells (p < 0.001). # and ###, significantly dif-
ferent from insulin (10 nM)-treated cells (p < 0.05 and 0.001,
respectively).
by suppressing DGAT-1 expression, which may in turn
participate in the inhibition of lipid droplet formation in
differentiated sebocytes. Furthermore, taken together with
previous reports that TGs from sebaceous glands are a
source of nutrition of P. acnes [1,3], β-CRX-decreased TG
production is very likely to indirectly result in the preven-
tion of P. acnes proliferation in acne lesions.
The PAT-family of intracellular lipid storage droplet
proteins such as PLIN1-5 has been reported to play im-
portant roles in the regulation of TG storage and lipolysis
in adipocytes and steroidogenic cells [35]. Our previous
study showed that PLIN1 localizes to the surface of in-
tracellular sebum-droplets in differentiated hamster se-
bocytes [12]. In the present study, we found that not only
intracellular lipid-droplet formation but also the gene
expression and production of PLIN1 was suppressed by
β-CRX in differentiated hamster sebocytes. A similar
Figure 6. Suppression of PLIN1 mRNA expression by b-
CRX in insulin-differentiated hamster sebocytes. Isolated
RNA (an equivalent of 2.5 ng of total RNA) from the cells
treated with insulin (10 nM), β-CRX (1.1 - 10 mM), and/or
13-cisRA (1 mM) as shown in Figure 5 was subjected to the
analysis of PLIN1 mRNA expression as described in the
Materials and Methods. Data are shown as mean ± SD of
four different experiments. *, significantly different from
untreated cells (p < 0.05). # and ##, significantly different
from insulin (10 nM)-treated cells (p < 0.05 and 0.01, re-
spectively).
inhibition of lipid-droplet formation has been reported to
be observed in β-CRX-treated mouse 3T3-L1 adipocytes
[31]. Therefore, the β-CRX-inhibition of lipid-droplet for-
mation is likely to include the transcriptional suppression
of PLIN1 production in hamster sebocytes. Moreover,
intracellular lipid-droplet formation has been reported to
be associated with the protein kinase A (PKA)-dependent
phosphorylation of PLIN, which facilitates the hormone-
sensitive lipase-mediated lipolysis of neutral lipid drop-
lets in adipocytes [37,38]. We previously reported that
neither the level of cyclic AMP (cAMP) nor PKA activ-
ity was augmented by 13-cisRA [18], of which its sup-
pressive action against sebum accumulation is similar to
that of β-CRX in differentiated hamster sebocytes. How-
ever, β-carotene, which is structurally and functionally
similar to β-CRX [39], has been reported to increase the
level of cAMP and PKA activity in human pulmonary
adenocarcinoma cells and epithelial cells from small air-
ways and pancreatic ducts [40,41]. Thus, β-CRX may
also influence the phosphorylation of PLIN1, which is
coordinately associated with the abolishment of lipid-
droplets in differentiated hamster sebocytes. Further ex-
periments are needed to clarify these hypotheses.
The development of comedones has been related to the
enhancement of sebum production in sebaceous glands,
under which various lipogenetic signaling through an-
drogen and/or insulin-like growth factor 1/insulin path-
ways is activated [42,43]. In the inflamed acne lesions
resulting from comedogenesis, the expression and activa-
tion of PPARγ have been reported to be augmented and
closely associated with the aggravation of acne pathology
[44,45]. Shirakura et al. [31] reported that β-CRX does
not affect peroxisome proliferators activating receptor γ

Suppression of Sebum Production and Accumulation by β-Cryptoxanthin Due to the Inhibition of the Expression of
Diacylglycerol Acyltransferase-1 and Perilipin in Hamster Sebocytes
104
(PPARγ) activation. We have preliminarily demonstrated
that β-CRX did not alter the PPARγ ligand, troglita-
zone-augmented production of TGs and PLIN1 produc-
tion in hamster sebocytes (Sato T and Ito A, unpublished
data). Therefore, the inhibitory actions of β-CRX against
sebum production and storage in sebocytes may at least
partially account for the beneficial efficacy in the pre-
vention of comedogenesis in acne lesions under non-
inflammatory conditions.
Carotenoids split into two groups; xanthophylls and
carotenes [21]. Xanthophylls such as β-CRX exist as an
ester form with fatty acids in fruits or vegetables [46,47].
In addition, xanthophyll esters have been reported to be
present in human skin, where the xanthophylls are re-
esterified following absorption [48]. Since the blood
concentration of β-CRX has been reported to be higher
than that of β-carotene or lycopene [47], it is suggested
that β-CRX is easily absorbed and accumulated in the
skin, where it may behave as Vitamin A for the control
of cutaneous functions. Taken together with a report of
El-Akawi et al. [28] that low plasma levels of Vitamin A
are associated with acne development and aggravation,
therefore, the supplementation of β-CRX is likely to be
effective for the prevention of acne or acne maintenance.
In conclusion, our findings provide novel evidence
that β-CRX is an effective candidate for acne therapy by
inhibiting not only sebaceous lipogenesis but also sebum
accumulation against insulin-differentiated hamster se-
bocytes, in which acne pathology is at least partly mim-
icked in vitro. Furthermore, these findings may contrib-
ute to a novel understanding of the molecular mecha-
nisms of dietary carotenoids in the maintenance of skin
barrier functions.
5. Acknowledgements
This work was supported by a Grant-in-Aid for Scientific
Research (C) (#22590506). We wish to thank Dr. K. Ki-
tamura and Miss. M. Kuwata for their technical assis-
tance.
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