Accepted Manuscript

A Co-drug of Butyric Acid Derived from Fermentation Metabolites of the Human Skin
Microbiome Stimulates Adipogenic Differentiation of Adipose-Derived Stem Cells:
Implications in Tissue Augmentation

Yanhan Wang, Lingjuan Zhang, Jinghua Yu, Stephen Huang, Zhenping Wang,
Kimberly Ann Chun, Tammy Ling Lee, Ying-Tung Chen, Richard L. Gallo, Chun-Ming
Huang
PII: S0022-202X(16)32239-4
DOI: 10.1016/j.jid.2016.07.030
Reference: JID 479

To appear in: The Journal of Investigative Dermatology

Received Date: 27 May 2015

Revised Date: 28 June 2016
Accepted Date: 12 July 2016

Please cite this article as: Wang Y, Zhang L, Yu J, Huang S, Wang Z, Chun KA, Lee TL, Chen Y-T,
Gallo RL, Huang C-M, A Co-drug of Butyric Acid Derived from Fermentation Metabolites of the Human
Skin Microbiome Stimulates Adipogenic Differentiation of Adipose-Derived Stem Cells: Implications in
Tissue Augmentation, The Journal of Investigative Dermatology (2016), doi: 10.1016/j.jid.2016.07.030.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.
 ACCEPTED MANUSCRIPT
Butyric Acid Co-drug Stimulates Adipogenic Differentiation of ADSCs Huang CM 03252015

A Co-drug of Butyric Acid Derived from Fermentation Metabolites of the

Human Skin Microbiome Stimulates Adipogenic Differentiation of Adipose-
Derived Stem Cells: Implications in Tissue Augmentation

PT
RI
Yanhan Wang1, Lingjuan Zhang1, Jinghua Yu2, Stephen Huang3, Zhenping
Wang1, Kimberly Ann Chun1, Tammy Ling Lee1, Ying-Tung Chen4,

SC
Richard L. Gallo1 and Chun-Ming Huang1,5*

U
1
Department of Dermatology, School of Medicine, University of California, San
Diego, CA, USA AN
2
Sanford-Burnham Institute for Medical Research, La Jolla, CA, USA
M
3
Surface Bioadvances Inc., San Diego, CA, USA
4
Nerd SkinCare Inc., San Francisco, USA
D

4
Moores Cancer Center; University of California, San Diego, CA, USA
TE

Running title: Butyric Acid Co-drug Stimulates Adipogenic Differentiation of

EP

ADSCs
C

*
Reprint requests to: Professor Chun-Ming Huang, Department of Dermatology,
University of California, San Diego. 3525 John Hopkins Court, Rm276, San
AC

Diego, CA, 92121, USA

E-mail: chunming@ucsd.edu
Tel: 858-822-4627
Fax: 858-642-1435

Keywords:

Adipogenic, Butyric acid, Differentiation, Microbiome, Skin

1
 ACCEPTED MANUSCRIPT
Butyric Acid Co-drug Stimulates Adipogenic Differentiation of ADSCs Huang CM 03252015

Abbreviations:
A, alanine; Ac, acetic acid; ADD1/SREBP1c, adipocyte determination and
differentiation-dependent factor 1/sterol response element-binding protein 1c;
ADSCs, adipose-derived stem cells; aP2, adipocyte protein 2; ATCC, American
Type Culture Collection; BA, butyric acid; BA-DEG-BA, butyric acid 2-(2-
butyryloxyethoxy) ethyl ester; BLASTn, basic local alignment search tool; BrdU,
5-bromo-2'-deoxyuridine; BSA, bovine serum albumin; CCK-8, Cell Counting Kit-

PT
8; C/EBPs, CCAAT/enhancer-binding proteins; CFSE, carboxyfluorescein
succinimidyl ester; CFU, colony-forming unit; 1-D, one-dimensional; 2-D, two-
dimensional; DAPI, 4',6-diamidino-2-phenylindole; DEG, diethylene glycol; DM,

RI
differentiation medium; DMEM, Dulbecco’s Modified Eagle’s Medium; DMSO,
dimethyl sulfoxide; D-PBS, Dulbecco’s Phosphate Buffered Saline; FBS, fetal
bovine serum; FDA, Food and Drug Administration; Ffar1, free fatty acid receptor

SC
1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GPR41, G-protein
coupled receptor 41; H&E, hematoxylin and eosin; HDAC, histone deacetylase;
HRP, horseradish peroxidase; HSQC, heteronuclear single quantum coherence;
ICR, Institute of Cancer Research; NF-S. epidermidis, non-fermenting

U
Staphylococcus epidermidis; NIH, National Institutes of Health; NMR, Nuclear
magnetic resonance; NS, non-significant; PA, propionic acid; P. acnes,
AN
Propionibacterium acnes; PBS, phosphate buffered saline; PKA, protein kinase
A; PPAR-γ, peroxisome proliferator-activated receptor gamma; Rplp0, ribosomal
protein, large, P0; Rref-1, preadipocyte factor 1; RNAi, RNA interference; RT-
qPCR, Reverse transcription-quantitative polymerase chain reaction; S. aureus,
M
Staphylococcus aureus; SCFAs, Short-chain fatty acids; SD, standard deviation;
S. epidermidis, Staphylococcus epidermidis; siRNA, small interfering RNA; 16S
rRNA, 16S ribosomal RNA; TCI, triple resonance inverse.
D
TE
C EP
AC

2
 ACCEPTED MANUSCRIPT
Butyric Acid Co-drug Stimulates Adipogenic Differentiation of ADSCs Huang CM 03252015

ABSTRACT

Here we demonstrate that Staphylococcus epidermidis (S. epidermidis), a

commensal bacterium in the human skin microbiome, produces short-chain fatty

acids (SCFAs) by glycerol fermentation that can induce adipogenesis. Although

PT
the antimicrobial and anti-inflammatory activities of SCFAs have been previously

RI
well characterized, little is known about the contribution of SCFAs to the

adipogenic differentiation of adipose-derived stem cells (ADSCs). We show that

SC
ADSCs differentiated into adipocytes and accumulated lipids in the cytoplasm

when cultured with butyric acid, a principal SCFA in the fermentation metabolites

U
of S. epidermidis. Additionally, a co-drug, butyric acid 2-(2-butyryloxyethoxy)
AN
ethyl ester (BA-DEG-BA), released active butyric acid when it was intradermally

injected into mouse ears and induced ADSC differentiation, characterized by an

M
increased expression of cytoplasmic lipids and perilipin A. The BA-DEG-BA-
D

induced adipogenic differentiation was mediated via peroxisome proliferator-

TE

activated receptor gamma (PPAR-γ). Furthermore, intradermal injection of

ADSCs along with BA-DEG-BA into mouse ears markedly enhanced the
EP

adipogenic differentiation of ADSCs, leading to dermal augmentation. Our study

introduces BA-DEG-BA as a enhancer of ADSC adipogenesis and suggests an

C

integral interaction between the human skin microbiome and ADSCs.

AC

3
 ACCEPTED MANUSCRIPT
Butyric Acid Co-drug Stimulates Adipogenic Differentiation of ADSCs Huang CM 03252015

INTRODUCTION

Human skin is a large, heterogeneous organ that defends the body

against invasion by pathogens while sustaining microbes that influence human

health and disease. The skin is colonized by a diverse milieu of microbes, most

PT
of which are commensal microbes that are harmless or sometimes even

RI
beneficial to their host (Grice et al., 2009, Lai et al., 2009, Naik et al., 2012)

although the link between commensal microbes and disease pathology has been

SC
reported (Achermann et al., 2014). Commensal microbes on the skin surface can

regulate the behavior of cells beneath the epidermis. Propionibacterium and

U
Staphylococcus species, two predominant groups of skin commensal bacteria,
AN
were primarily detected on the surface of sebaceous sites when collected by

superficial sampling methods (Grice et al., 2009). However, recent reports

M
indicate that the microbiome is distributed uniformly throughout the skin,
D

extending to the dermal stroma and superficial adipose tissue of normal human
TE

skin (Nakatsuji et al., 2013, Zeeuwen et al., 2012). The distribution of the

microbiome in the deep skin allows a physical interaction between commensal

EP

microbes and adipocytes in normal skin.

Previously, we have demonstrated that P. acnes can exploit glycerol

C

fermentation to produce various short-chain fatty acids (SCFAs) which play a role
AC

in skin immunity against Staphylococcus aureus (S. aureus) (Shu et al., 2013). In

addition to exhibiting bactericidal activity, SCFAs also act on host cells through at

least two mechanisms: the inhibition of histone deacetylase (HDAC) and the

activation of free fatty acid receptors, such as Ffar1, also known as G-protein

4
 ACCEPTED MANUSCRIPT
Butyric Acid Co-drug Stimulates Adipogenic Differentiation of ADSCs Huang CM 03252015

coupled receptor 41 (GPR41), and Ffar2 (GPR43) (Vinolo et al., 2011). Butyric

acid has been shown to enhance preadipocyte differentiation to adipocytes via its

HDAC inhibitory activity (Chen et al., 2007, Toscani et al., 1990).

Adipogenesis is a multi-step process involving a cascade of transcription

PT
factors, which regulate gene expression and lead to adipocyte development.

RI
Several positive and negative regulators of this network have been elucidated in

past years (Lefterova and Lazar, 2009). In vitro studies using murine 3T3-L1 and

SC
3T3-F442A adipose tissue-derived stem cells (ADSCs) have revealed complex

transcriptional cascades that are essential for adipocyte differentiation (Morrison

U
and Farmer, 2000, Rosen et al., 2000). Peroxisome proliferator-activated
AN
receptor gamma (PPAR-γ), CCAAT/enhancer-binding proteins (C/EBPs) and

adipocyte determination and differentiation-dependent factor 1/sterol response

M
element-binding protein 1c (ADD1/SREBP1c) have been identified as key
D

transcription factors involved in adipogenesis (Hamm et al., 2001, Wu et al.,

TE

1996). When ADSCs are exposed to differentiation conditions, a complex

signaling cascade induces the expression of previously silent adipogenic genes.

EP

Although it is not clear how these genes are maintained in a quiescent state,

downregulation of HDACs at an early stage of adipogenesis is a rate-limiting step

C

(Yoo et al., 2006)

AC

Soft tissue augmentation is a minimally invasive procedure used to correct

facial wrinkles with durable soft tissue fillers. Various natural fillers are available

commercially, such as bovine collagen, hyaluronic acid, calcium hydroxylapatite,

poly-L-lactic acid (Sculptra®), polymethylmethacrylate and liquid silicone, which

5
 ACCEPTED MANUSCRIPT
Butyric Acid Co-drug Stimulates Adipogenic Differentiation of ADSCs Huang CM 03252015

have been widely used for soft tissue augmentation (Alster and West, 2000,

Duranti et al., 1998, Garfein et al., 2003, Knapp et al., 1977). Although these

materials have been shown to be safe to administer, their resorption requires

repeated injections for maintenance. Alternatively, ADSCs are currently used as

PT
a cell source for soft tissue augmentation (Jeong et al., 2011). ADSCs, which

RI
include stem cells and various precursor cells, are capable of replication and

differentiation into adipocytes that are fully mature and are histologically similar to

SC
naturally formed adipocytes (Jeong et al., 2011). However, the viability of ADSCs

alone in the tissue is low, thereby leading to a high rate of failure in soft tissue

U
augmentation. Thus, repeated injections of ADSCs may be also required for soft

tissue augmentation.
AN
In the current study, we sought to investigate the effect of fermentation by-
M
products made by skin commensal bacteria on preadipocyte differentiation. We
D

demonstrate that the fermented metabolites of the skin microbiome can

TE

significantly enhance the differentiation of ADSCs in vitro. Furthermore, co-

injection of ADSCs with a co-drug of butyric acid into a mouse ear enhanced
EP

ADSC differentiation, resulting in dermal thickening. Our findings may therefore

introduce a area of research into the human skin microbiome in promoting

C

preadipocyte differentiation, and lead to a revolutionary approach to soft tissue

AC

augmentation.

6
 ACCEPTED MANUSCRIPT
Butyric Acid Co-drug Stimulates Adipogenic Differentiation of ADSCs Huang CM 03252015

RESULTS

Induction of adipogenic differentiation of ADSCs by fermented media of

Staphylococcus epidermidis (S. epidermidis)

S. epidermidis (ATCC 12228) bacteria were incubated in rich media in the

PT
presence of glycerol to detect their fermentation activity (Supplementary Figure

RI
1a). Rich media plus either glycerol or S. epidermidis were used as controls. To

monitor the fermentation process, cultures were tested with phenol red, a

SC
fermentation indicator, to assess SCFA production as a result of glycerol

fermentation. After six days of incubation, only the media of S. epidermidis with

U
glycerol became more acidic and turned yellow, indicating fermentation activity of
AN
S. epidermidis. For comparison, a S. epidermidis strain was isolated from human

skin in our laboratory. By 16S ribosomal RNA gene (16S rRNA) gene sequencing
M
using 16S rRNA 27 F and 534R primers (Lindh et al., 2005), we found that the
D

16S rRNA gene of this strain shared 99 % identity with that in S. epidermidis
TE

ATCC 12228 (Supplementary Figure 1b). However, media in the culture of this

strain with glycerol remained red after six days of incubation (Supplementary
EP

Figure 1a). We thus define this strain as a non-fermenting S. epidermidis (NF-S.

epidermidis).
C

To examine whether the fermentation product of S. epidermidis affects

AC

ADSC adipogenesis, the culture media of S. epidermidis (ATCC 12228) and non-

fermenting S. epidermidis (NF-S. epidermidis) with or without glycerol were

added into ADSCs for three days. A cell counting kit-8 (CCK-8) assay was used

to count ADSC cells by measuring a change in absorbance at 450 nm. As shown

7
 ACCEPTED MANUSCRIPT
Butyric Acid Co-drug Stimulates Adipogenic Differentiation of ADSCs Huang CM 03252015

in Figure 1a, b, j), the number of ADSCs incubated with the culture media of S.

epidermidis (ATCC 12228) in the presence of glycerol was lower than the ADSCs

incubated with the culture media of S. epidermidis in the absence of glycerol. To

investigate whether glycerol fermentation of S. epidermidis stimulates adipogenic

PT
differentiation, cytoplasmic lipids were stained with Oil Red O (Figure 1a-d) after

RI
incubation of culture media for six days. ADSCs were also immunostained for

perilipin A, a lipid droplet surface protein associated with the formation of

SC
intracellular lipid droplets during adipogenesis (Blanchette-Mackie et al. , 1995,

Greenberg et al. , 1991), with 4',6-diamidino-2-phenylindole (DAPI) counterstain

U
(Figure 1f-i). The percentages of Oil Red O and perilipin A positive cells in
AN
ADSCs incubated with the culture media of S. epidermidis with glycerol were

considerably higher than those in ADSCs incubated with the culture media of S.
M
epidermidis without glycerol (Figure 1k). These results demonstrate that the
D

fermented media of S. epidermidis decreased the proliferation and enhanced the

TE

adipogenic differentiation of ADSCs. Conversely, in ADSCs incubated with the

culture media of non-fermenting S. epidermidis with and without glycerol (Figure

EP

1j, k), no significant change was noted in the number of ADSCs or Oil Red

O/perilipin A positive cells, indicating that glycerol fermentation is essential for S.

C

epidermidis to promote the adipogenic differentiation of ADSCs.

AC

SCFAs in fermented media of S. epidermidis

To identify the SCFAs in fermented media, S. epidermidis (ATCC 12228)

13
was incubated in rich medium in the presence of C3-glycerol (20 g/l) for six

8
 ACCEPTED MANUSCRIPT
Butyric Acid Co-drug Stimulates Adipogenic Differentiation of ADSCs Huang CM 03252015

days. The fermented media in 10 % deuterium oxide (D2O) were subjected to

one-dimensional (1-D) (Figure 2a, b) and two-dimensional (2-D) (Figure 2c) 1H

13
and C nuclear magnetic resonance (NMR) analysis. Three SCFAs (acetic,

butyric and succinic acids) were detected in the fermented media of S.

PT
epidermidis, with butyric acid displaying the strongest signal in the NMR spectra.

RI
These results illustrate that S. epidermidis is able to utilize fermentation to

metabolize glycerol into SCFAs.

SC
Induction of adipogenic differentiation of ADSCs by SCFAs

U
When preadipocyte growth medium in ADSC cultures was replaced with
AN
adipocyte differentiation medium, ADSCs ceased proliferation detected by a

CCK-8) assay (Figure 2p) and underwent adipogenic differentiation, resulting in

M
the formation of lipid droplets (Figure 2e) and an increased in expression of
D

perilipin A (Figure 2k). High percentages of Oil Red O - (~60%) and perilipin A-
TE

(~45%) positive cells were observed after a seven-day culture of ADSCs in

adipocyte differentiation medium (Figure 2q). To determine whether SCFAs can

EP

induce adipogenic differentiation, ADSCs were exposed to 4 mM butyric,

propionic, acetic and succinic acids, major fermentation products of P. acnes

C

(Shu et al., 2013). As shown in Figure 2p, butyric and propionic acids, but not
AC

acetic and succinic acids, ceased proliferation of ADSCs. A significant increase

in the percentages of Oil Red O- and perilipin A-positive cells were detected after

incubation of ADSCs with butyric or propionic acids, but not acetic or succinic

acid, for seven days. Since butyric acid induced higher percentages of Oil Red

9
 ACCEPTED MANUSCRIPT
Butyric Acid Co-drug Stimulates Adipogenic Differentiation of ADSCs Huang CM 03252015

O- and perilipin A-positive cells than propionic acid, we suggest that butyric acid

is a potent inducer of adipogenic differentiation.

Release of butyric acid (BA) from BA-DEG-BA in vivo and effect of BA-

PT
DEG-BA on adipogenesis

RI
SCFAs have been deemed safe for consumption by the Food and Drug

Administration (FDA) (Collins, 1971, Pradhan et al., 2009), however they have

SC
not yet been utilized as treatment options for human diseases. The major

problem has been to achieve and maintain their concentrations in vivo because

U
SCFAs can be metabolized as soon as they enter the cells via an active transport
AN
system (Schroder et al., 2000, Stein et al., 1995, Stein et al., 2000). To overcome

the challenges of instituting therapeutic SCFAs, which include an offensive odor,

M
rapid metabolization, and inability to achieve pharmacologic concentrations in
D

vivo, we synthesized a co-drug of butyric acid containing two butyric acid

TE

moieties esterified to a diethlyene glycol (DEG) linker.

To readily allow the release of BA in the skin, two BA molecules were

EP

linked with DEG by an ester bond to form butyric acid 2-(2-butyryloxyethoxy)

ethyl ester (BA-DEG-BA). To investigate whether BA-DEG-BA releases butyric

C

acid in skin, BA-DEG-BA and a control, butyric acid, were intradermally injected
AC

into the ears of Institute of Cancer Research (ICR) mice for two days. Mouse

ears were then homogenized and the released BA from BA-DEG-BA was
13
detected by 1-D C NMR analysis. Before injection into mice, the BA and BA-

DEG-BA in solution were subjected to carbon NMR analysis to generate the

10
 ACCEPTED MANUSCRIPT
Butyric Acid Co-drug Stimulates Adipogenic Differentiation of ADSCs Huang CM 03252015

reference spectra. Four signals at 13.1, 18.1, 35.5, and 179.4 ppm corresponded

to the chemical groups (-CH3, -CH2, -CH2, and -COOH) of BA (Figure 3a, blue).

Two additional signals at 60.9 and 68.7 ppm derived from four symmetric carbon

atoms of DEG were detected in a spectrum of BA-DEG-BA (Figure 3a, red). The

PT
-COOH signal of free BA at 179.4 ppm was distinguishable from the -COOH

RI
signal of DEG-linked BA at 172.5 ppm. Two days after injection of BA-DEG-BA or

BA into mouse ears, the supernatants of ear homogenates in 10% D2O were

SC
subjected to 1-D carbon NMR analysis. The spectrum of BA with four signals in

the ear homogenates (Figure 3b, blue) is identical to that in solution (Figure 3a,

U
red). However, in the spectrum of homogenates of BA-DEG-BA-injected ears, the
AN
-COOH signal of DEG-linked BA at 172.5 ppm was shifted to the position of the

free BA signal at 179.4 ppm (Figure 3b, red), indicating the release of BA from
M
BA-DEG-BA in skin.
D

To determine whether BA-DEG-BA can induce adipogenesis, ADSCs

TE

were exposed to either BA-DEG-BA or DEG for three to six days. Results in

Figure 3 demonstrate that treatment of ADSCs with BA-DEG-BA, not DEG,

EP

inhibits cell proliferation measured by a CCK-8 assay (Figure 3g) and

significantly enhances the production of cytoplasmic lipids (Figure 3c) and the
C

expression of perilipin A (Figure 3e). The percentages of Oil Red O- and perilipin
AC

A-positive cells increased to approximately 20% after treatment of BA-DEG-BA

for seven days (Figure 3h), suggesting BA-DEG-BA, as BA, can induce

adipogenic differentiation of ADSCs.

11
 ACCEPTED MANUSCRIPT
Butyric Acid Co-drug Stimulates Adipogenic Differentiation of ADSCs Huang CM 03252015

Involvement of PPAR-γ in the BA-DEG-BA-mediated adipogenic

differentiation

To determine the expression of genes known to regulate and/or serve as

markers for adipogenic differentiation, reverse transcription-quantitative

PT
polymerase chain reaction (RT-qPCR) analysis was performed on RNA isolated

RI
from ADSCs treated with BA-DEG-BA for five days. A gene (Pref-1) of

preadipocyte factor 1 (Pref-1), a negative regulator of adipogenesis, was

SC
downregulated (Supplementary Figure 2) and two genes (Pparg and Cebpb) of

PPAR-γ and C/EBP beta, markers of adipocyte differentiation, were significantly

U
upregulated (Figure 4a), confirming the adipogenic differentiation of ADSCs
AN
mediated by BA-DEG-BA. Differentiated ADSCs at day 5 exhibited very high

expression level of Pparg (Figure 4a). PPAR-γ is a member of the nuclear

M
receptor superfamily of ligand-activated transcription factors and is a prerequisite
D

for the differentiation of both brown and white adipocytes (Kajimura et al., 2008).
TE

Previous PPAR-γ gain and loss of function models validated the essential role of

PPAR-γ in lipid formation (Farmer, 2006) and regulation of adipogenesis (Rosen

EP

et al., 2002). Therefore, we examined whether the downregulation of PPAR-γ

influenced the BA-DEG-BA-mediated adipogenic differentiation in ADSCs. The

C

small interfering RNA (siRNA) specific for PPAR-γ mRNA was added into ADSC
AC

cultures for 6 h. RT-qPCR showed 87% PPAR-γ mRNA knockdown in ADSCs

treated with siRNA (Figure 4b). As shown in Figure 4c-fg, treatment of ADSCs

with BA-DEG-BA (4 mM) for seven days resulted in an increased Oil Red O stain

(Figure 4d) and expression of perilipin A (Figure 4f). Remarkably, knockdown of

12
 ACCEPTED MANUSCRIPT
Butyric Acid Co-drug Stimulates Adipogenic Differentiation of ADSCs Huang CM 03252015

PPAR-γ led to a dramatic attenuation of adipogenesis with a marked decrease in

Oil Red O stain and perilipin A expression (Figure 4c, e, g), indicating that PPAR-

γ mediates the BA-DEG-BA-induced adipogenic differentiation.

PT
Dermal augmentation and adipogenic differentiation with ADSCs plus BA-

RI
DEG-BA

Dermal augmentation and adipogenic differentiation of ADSCs were

SC
analyzed by immunostaining and flow cytometry, respectively. Although ADSCs

are valuable stem cells for dermal augmentation, its poor survival rate has limited

U
their therapeutic efficacy. Furthermore, injection of BA-DEG-BA alone may not be
AN
effective (Supplementary Figure 3) due to low ADSC abundance in tissue. Thus,

we co-injected ADSCs with BA-DEG-BA to observe for dermal augmentation.

M
ADSCs were labeled with carboxyfluorescein succinimidyl ester (CFSE) to
D

visualize their presence in tissue. Images using CFSE fluorescence and Oil Red
TE

O staining illustrated that intradermal injection of ADSCs, and not PBS, into the

ears of ICR mice significantly increased the dermal thickness (Supplementary

EP

Figure 4). To examine if BA-DEG-BA can enhance the ADSC-mediated dermal

thickness and adipogenic differentiation, CFSE-labeled-ADSCs along with 4 mM

C

BA, BA-DEG-BA, succinic acid or DEG were intradermally injected into the ears
AC

of ICR mice. The dermal thickness and expression of perilipin A were measured

seven days after injection by immunostaining and flow cytometry. As shown in

Figure 5a-f, the dermal layer in the ear injected with ADSCs plus BA or BA-DEG-

BA was thicker than in the ear injected with ADSCs alone or plus either succinic

13
 ACCEPTED MANUSCRIPT
Butyric Acid Co-drug Stimulates Adipogenic Differentiation of ADSCs Huang CM 03252015

acid or DEG. The expression of perilipin A in the ear injected with ADSCs plus

BA or BA-DEG-BA was indicated in Figure 5g, h. Results obtained from flow

cytometry analysis demonstrated that there was an increased in the total number

of CFSE-labeled ADSCs after injection of ADSCs plus BA or BA-DEG-BA,

PT
indicating that BA and BA-DEG-BA enhance the survival and proliferation of

RI
ADSCs (Figure 5i, j). Importantly, the percentage of perilipin A-stained cells

(0.74%) in the CFSE-labeled ADSCs was predominantly increased to 1.08% and

SC
1.28% when ear was injected with ADSCs plus BA or BA-DEG-BA, respectively

(Figure 5i, k). These results support the function of BA and BA-DEG-BA, but not

U
succinic acid, as enhancers for adipogenic differentiation of ADSCs.
AN
DISCUSSION
M
P. acnes and S. epidermidis, two major commensal bacteria in the human skin
D

microbiome (Grice and Segre, 2011), naturally reside on the surface of human
TE

skin, with P. acnes additionally living within hair follicles. Following skin

wounding, both P. acnes and S. epidermidis may irregularly migrate from the skin
EP

surface or hair follicle into the reticular dermis, in which a thick layer of

adipocytes exists. The migration of two bacteria to the deep dermis, which has a
C

relatively low oxygen microenvironment, may trigger bacteria to undergo

AC

fermentation using carbon sources, such as glycerol, that are naturally produced

in the skin (Fluhr et al., 2008) to form SCFAs. Although glycerol can serve as a

carbon source for production of SCFAs via the fermentation of skin probiotic

bacteria in vitro (Wang et al., 2014) it is enigmatic if SCFAs are produced by skin

14
 ACCEPTED MANUSCRIPT
Butyric Acid Co-drug Stimulates Adipogenic Differentiation of ADSCs Huang CM 03252015

probiotic bacteria on the skin under physiological conditions. It has been reported

that germ-free mice devoid of bacteria expressed little or no SCFAs (Maslowski

et al., 2009). Thus, it is worth determining the correlations of the number of skin

probiotic bacteria and the amounts of SCFAs in human skin. As shown in Figure

PT
2, butyric, acetic and succinic acids are major SCFAs produced by the glycerol

RI
fermentation of S. epidermidis (ATCC 12228). Although glycerol fermentation

was demonstrated using an ATCC 12228 strain of S. epidermidis in this study,

SC
results in our publication have shown that eight S. epidermidis strains isolated

from human skin can exert glycerol fermentation (Wang et al., 2014) and produce

U
SCFAs (data not shown). The ADSCs in adipose tissue are stimulated by
AN
SCFAs, which may induce their differentiation and promote skin regeneration

during the healing of skin wounds. Results in Nature Communications (Nakatsuji

M
et al., 2013) demonstrated that bacteria were consistently detectable within the
D

dermis and dermal adipose layer of normal human skin, thus permitting physical
TE

interaction between microbes and dermal cells, indicating that under normal

conditions, skin commensal bacteria and their metabolites, including SCFAs,

EP

may exist in the dermis where they can directly interact with adipocytes.

Perilipin serves as a marker of fully differentiated adipocytes. In humans,

C

perilipin is expressed in three different isoforms: A, B, and C (Greenberg et al.,

AC

1991). Perilipin A, a main substrate for protein kinase A (PKA) (Zhang et al.,

2003), is the most abundant protein on the periphery of lipid droplets in

adipocytes. The phosphorylated perilipin A may play a key role in catecholamine-

stimulated lipolysis (Miyoshi et al., 2006). Activation of PPAR-γ increased the

15
 ACCEPTED MANUSCRIPT
Butyric Acid Co-drug Stimulates Adipogenic Differentiation of ADSCs Huang CM 03252015

expression of perilipin A, indicating that PPAR-γ plays a role in lipid droplet

formation (Arimura et al., 2004, Dalen et al., 2004). Here, we show that ADSCs

treated with propionic or butyric acid enhanced the intracellular accumulation of

Oil Red O stained lipids (Figure 2) and that knockdown of PPAR-γ decreased the

PT
expression of BA-DEG-BA-induced perilipin A (Figure 4). These results suggest

RI
that butyric acid increases the accumulation of lipid droplets and induces

adipogenic differentiation of ADSCs via PPAR-γ.

SC
The precise mechanism in which SCFAs induce adipogenic differentiation

is currently unclear. Previous evidence has supported that butyric acid enhanced

U
preadipocyte differentiation by stimulating PPAR-γ, subsequently dislodging
AN
HDAC1 from the promoter to be degraded in the proteasome (Zhao et al., 2013,

Zuo et al., 2006). Furthermore, knockdown of HDAC1, potentiated preadipocyte

M
differentiation by increasing PPAR-γ expression at the onset of differentiation
D

(Kuzmochka et al., 2014). In contrast, other studies have observed that Ffar2 is
TE

activated by propionic acid and promotes adipocyte differentiation in 3T3-L1

ADSCs, inhibiting lipolysis in vitro and in vivo (Ge et al., 2008, Hong et al., 2005).
EP

After 3T3-L1 cells were transfected with Ffar2 siRNA, the expression of PPAR-γ

was downregulated and subsequently, fat synthesis diminished (Ge et al., 2008,
C

Hong et al., 2005). Although we confirmed that SCFAs and its derivatives
AC

mediated ADSCs differentiation via PPAR-γ, we cannot definitively say that

adipogenic differentiation of ADSCs was due to the inhibition of HDAC1 or the

activation of Ffar. Thus, further investigation is required to determine the

mechanism of how SCFAs influence preadipocyte differentiation.

16
 ACCEPTED MANUSCRIPT
Butyric Acid Co-drug Stimulates Adipogenic Differentiation of ADSCs Huang CM 03252015

A number of SCFAs, although in relatively low concentrations, are

detectable in the skin and in sweat. Human sweat has been noted to contain

0.1% lactic, 0.04% citric, 0.0096% acetic and 0.0062% propionic acid

(Burtenshaw, 1942). The amount of SCFAs produced by bacterial fermentation in

PT
skin has not yet been quantified. The total concentration of SCFAs in human

RI
peripheral blood is approximately 50-100 µM (Cummings et al., 1987). The

SCFAs produced by intestinal microbes in the human colon can locally reach a

SC
high level (20-140 mM) (Garland, 2011) that may elicit an innate immune

response from preadipocytes. We demonstrated that 4 mM butyric acid was

U
effective at stimulating adipogenic differentiation of ADSCs in vitro (Figure 2) and
AN
in vivo (Figure 5). Injection of 4 mM BA-DEG-BA along with ADSCs into mouse

ears induced a considerable augmentation of the dermal layer. The mouse back
M
skin has been widely used as a model for study of adipogenesis principally due
D

to the abundance of adipogenic progenitors and the changes in thickness of the

TE

adipose layer in harmony with the hair growth cycle (Wojciechowicz et al., 2013).

Future study includes injecting ADSCs into mouse back skin, quantifying the
EP

endogenous level of butyric acid in the dermal layer and measuring the amount

of butyric acid released from BA-DEG-BA. This will provide valuable insight into
C

the formulation and dosing of BA-DEG-BA as a enhancer of ADSC differentiation

AC

for soft tissue augmentation. Although an increase in dermal thickness (0.24 ±

0.01 to 0.67 ± 0.04 mm from day 0 to 7) and expression of perilipin A was

demonstrated on the seventh day (Figure 5), the increase in ear thickness (0.35

± 0.01 mm) is still detectable twenty-one days after injection of ADSCs and BA-

17
 ACCEPTED MANUSCRIPT
Butyric Acid Co-drug Stimulates Adipogenic Differentiation of ADSCs Huang CM 03252015

DEG-BA, indicating an enduring effect of ADSCs/BA-DEG-BA on tissue

augmentation. Both tissue immunostaining and flow cytometry analysis were

conducted to detect the perilipin A positive adipocytes (Figure 5) since flow

cytometry may be limited to analyzing the large size of adipocytes. DEG can be a

PT
nephrologic and neurologic toxin (Schep et al., 2009). Although no cell death was

RI
detected in ADSCs treated with BA-DEG-BA (data not shown), future work will

include using other linkers, such as 1,3-propanediol (Yu et al., 2011) or glycerol

SC
(Calderon et al., 2011), to conjugate SCFAs. Butyric acid is a four carbons acid

with a very unpleasant odor. Thus, although both BA and BA-DEG-BA can

U
induce adipogenic differentiation (Figures 2 and 5), BA-DEG-BA will be used as
AN
an alternative to butyric acid for tissue augmentation. As shown in Figure 5, BA-

DEG-BA can enhance the survival and adipogenic differentiation of ADSCs in

M
mouse skin. The 5-bromo-2'-deoxyuridine (BrdU) labelling was performed to
D

determine whether an increase in the population of perilipin A positive

TE

differentiated adipocytes is due to lipid filling in differentiated adipocytes or de

novo generation of new adipocytes. As shown in Supplementary Figure 5, almost

EP

all of the perilipin A positive differentiated adipocytes were BrdU negative,

suggesting no DNA synthesis occurs during adipogenic differentiation from

C

ADSCs. BA-DEG-BA may stimulate ADSCs to differentiate into lipid filled

AC

adipocytes.

Numerous soft tissue fillers are commercially available for correcting facial

wrinkles, although all require multiple injections due to tissue resorption. Injective

transfer of autologous aspirated fat (Matsumoto et al., 2006) is a popular option

18
 ACCEPTED MANUSCRIPT
Butyric Acid Co-drug Stimulates Adipogenic Differentiation of ADSCs Huang CM 03252015

for tissue augmentation, but several issues limit its clinical application, including

unpredictability and a low survival rate due to partial necrosis. An injectable

adipocyte filler has been developed from cultured ADSCs (Sajjadian et al., 2007).

However, multiple questions remain regarding the viability of ADSCs, the

PT
requirement of biological scaffolds, and the repeated injection schedule in the

RI
formation of future fat graft replacements. Our results demonstrate that propionic

acid, butyric acid, and BA-DEG-BA are potent inducers of adipocyte

SC
differentiation (Figures 2 and 3). Moreover, a noticeable expansion of the dermal

layer was observed upon co-injection of ADSCs with BA-DEG-BA into mouse

U
ears (Figure 5). These results demonstrate the potential for SCFAs to enhance
AN
the adipogenic differentiation and the viability of ADSCs, reducing the need for

repeated injections, and thereby achieving a more permanent solution for soft
M
tissue augmentation.
D
TE

MATERIALS AND METHODS

Ethics statement
EP

This study was carried out in strict accordance with the recommendations in the

Guide for the Care and Use of Laboratory Animals of the National Institutes of
C

Health (NIH) and an approved Institutional Animal Care and Use Committee
AC

(IACUC) protocol (no. S10058) at University of California, San Diego (UCSD).

ADSC isolation and treatment with fermented media or SCFAs

19
 ACCEPTED MANUSCRIPT
Butyric Acid Co-drug Stimulates Adipogenic Differentiation of ADSCs Huang CM 03252015

ADSCs were isolated from ICR mice (2-3 month-old females; River Lab,

Placentia, CA) according to previous published protocols (Huang et al. , 2012,

Huang et al. , 2013). ADSCs were cultured in preadipocyte growth medium

(CELL applications, CA) and treated with adipocyte differentiation medium (DM),

PT
4 mM SCFAs, BA-DEG-BA, DEG or fermented media of S. epidermidis (Shu,

RI
Wang, 2013). The 16S rRNA gene sequences using 16S rRNA 27 F and 534R

primers (Lindh et al., 2005) were analyzed using the basic local alignment search

SC
tool (BLASTn). SCFAs in fermented media were identified by NMR analysis

(Chitarra et al., 2000). The viability of ADSCs was determined using a CCK-8

U
assay (Dojindo Laboratory, Kumamoto, Japan) (Yu, 2011). ADSC isolation, S.
AN
epidermidis fermentation, NMR analysis and CCK-8 assay were described in

detail in Supplementary Information online.

M
D

Synthesis of BA-DEG-BA
TE

50 mmol butyric acid and 20 mmol DEG in 100 ml dichloromethane were added

to 60 mmol N,N’-dicyclohexyl carbodimide portionwise. The cloudy white

EP

suspensions were stirred at room temperature overnight, then filtered and

washed with hexane. The filtrate was concentrated under reduced pressure to
C

yield pure and colorless BA-DEG-BA (>97%, 2.3 g), which was purified by
AC

chromatography (silica gel) eluted with 10% ethyl ethanoate/ hexane.

The expression of adipogenic regulatory genes and PPAR-γ knockdown

20
 ACCEPTED MANUSCRIPT
Butyric Acid Co-drug Stimulates Adipogenic Differentiation of ADSCs Huang CM 03252015

The expression of adipogenic regulatory genes (Pref-1, Cebpb, and Pparg) was

determined by RT-qPCR as described in Supplementary Information online. For

PPAR-γ knockdown (Luu-The et al., 2005), ADSCs reached 100% confluence

and transfected with 300 nM mouse PPAR-γ siRNA (Thermo Fisher Scientific) for

PT
6 h with an Amaxa Cell Line Nucleofector Kit V for undifferentiated 3T3-L1

RI
(Lonza).

SC
Immunostaining and flow cytometry

Cells reaching 100% confluence were labeled by CFSE (Sigma Chemical Co.).

U
The labeled cells (2 × 106) plus 4 mM BA, BA-DEG-BA, succinic acid or DEG in
AN
10 µl 5% dimethyl sulfoxide (DMSO) were injected into ear of ICR mice for seven

days. Tissue sections of mouse ears were subjected to Oil Red O, H&E, or
M
immunohistochemical staining. Single-cell suspensions were prepared by
D

mincing ear tissues with scissors for flow cytometry analysis. After
TE

permeabilization with 0.1% saponin (Molecular Probes, Eugene, Oregon) in

phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA) for
EP

15 min and incubation with goat antibodies to perilipin A (Abcam), the

populations of CFSE-labeled and perilipin A-stained in single-cell suspensions

C

were quantified by the Guava EasyCyte 8HT two-laser, six-color microcapillary-

AC

based benchtop flow cytometer (Millipore, Billerica, MA). More detailed protocol

can be found in Supplementary Information online.

21
 ACCEPTED MANUSCRIPT
Butyric Acid Co-drug Stimulates Adipogenic Differentiation of ADSCs Huang CM 03252015

Statistical analysis

Comparisons were made using the two-tailed t-test. The P-values of <0.05 (*),

<0.01 (**), and <0.001 (***) were accepted for statistical significance.

PT
RI
U SC
AN
M
D
TE
C EP
AC

22
 ACCEPTED MANUSCRIPT
Butyric Acid Co-drug Stimulates Adipogenic Differentiation of ADSCs Huang CM 03252015

FIGURE LEGENDS

Figure 1. Induction of adipogenic differentiation of ADSCs by fermented

media of S. epidermidis. S. epidermidis (ATCC 12228) (S. epi) (a, b, f, g) and a

glycerol non-fermenting S. epidermidis (NF-S. epi) (c, d, h, i) 105 CFU/ml were

PT
incubated in rich medium in the absence (-G) and presence of 20 g/l glycerol

RI
(+G) for seven days. After removing bacteria, fermented media were added to

ADSCs (103 cell/slide) on 24-well microplates for seven days. ADSCs were

SC
stained with Oil Red O (a-d) or immunostained with perilipin A (red). A high

magnitude photo of one selected area of Oil Red O stained ADSCs was

U
displayed in (e). ADSCs cultured with fermented media of S. epi differentiated
AN
into adipocytes and accumulated lipid droplets in the cytoplasm. Cell nuclei were

counterstained with DAPI (blue) and merged views of immunostaining with DAPI
M
and perilipin A were shown (f-i). Bars (a-i) = 100 µm. After treatment of ADSCs
D

with fermented media for three days, the total number of ADSCs in a 96-well
TE

microplate was counted on the absorbance at 450 nm using a CCK-8 assay (j).

The percentages (%) of ADSCs positively stained with Oil Red O or perilipin A
EP

were shown (k). *P<0.05 or **P<0.01 was evaluated using t-tests. Data are the

mean ± standard deviation (SD) of three separate experiments. NS = non-

C

significant.
AC

23
 ACCEPTED MANUSCRIPT
Butyric Acid Co-drug Stimulates Adipogenic Differentiation of ADSCs Huang CM 03252015

Figure 2. SCFA identification and SCFA-induced ADSC adipogenic

differentiation. Fermented media of S. epidermidis (ATCC 12228) (105 CFU/ml)

13
in the presence of C3-glycerol were passed through a 0.22 µm filter and

subjected to 1-D (400 MHz) 1H- (a), 13

C- (b), and 2-D (600 MHz) 1H-13C HSQC

PT
(c) NMR spectrometers. Besides glycerol (G), three SCFAs [acetic acid (Ac),

RI
butyric acid (BA), and succinic acid (S)] were detected. Murine ADSCs (103

cell/well) were cultured in adipocyte differentiation medium (DM) for adipogenic

SC
differentiation. Cells were incubated in preadipocyte growth medium (M) alone or

with 4 mM butyric acid (M+BA), propionic acid (M+PA), acetic acid (M+Ac) or

U
succinic acid (M+S). After seven days of incubation, the ADSCs stained with Oil
AN
Red O (d-i) or perilipin A/DAPI (j-o) were demonstrated. The cell number of

ADSCs was determined by a CCK-8 assay, showing a change in absorbance at

M
450 nm (p). The percentages (%) of ADSCs positively stained with Oil Red O or
D

perilipin A (q) were quantified. Bars = 50 µm. UD = undetectable. **P<0.01 or

TE

***P<0.001 was evaluated using t-tests. Results are the mean ± SD of three

independent experiments.
C EP
AC

24
 ACCEPTED MANUSCRIPT
Butyric Acid Co-drug Stimulates Adipogenic Differentiation of ADSCs Huang CM 03252015

Figure 3. Butyric acid released from BA-DEG-BA in mouse skin and

adipogenic differentiation induced by BA-DEG-BA. A co-drug (BA-DEG-BA)

of two butyric acid (BA) moieties esterified to DEG was synthesized. The solution

(4 mM) of BA (blue) or BA-DEG-BA (red) was mixed with 10% D2O and analyzed

PT
by a NMR (400 MHz JEOL JNM-ECS) spectrometer. Open arrows denote the -

RI
COOH signal of free BA at 179.4 ppm. A solid arrow indicates the -COOH signal

of DEG-linked BA at 172.5 ppm. Data from 1,024 scans were accumulated (a).

SC
The ears of ICR mice were injected intradermally with 4 mM (20 µl) BA (blue) or

BA-DEG-BA (red) for two days (b). ADSCs (103 cell/well) in preadipocyte growth

U
medium were incubated with 4 mM BA-DEG-BA (M+BA-DEG-BA) or DEG
AN
(M+DEG). After seven days of incubation with BA-DEG-BA or DEG, ADSCs

stained with Oil Red O (c, d) or perilipin A/DAPI (e, f) were exhibited. Effect of
M
ADSC number on absorbance at 450 nm was measured using a CCK-8 assay
D

(g). The percentages (%) of ADSCs positively stained with Oil Red O or perilipin
TE

A were enumerated after cell incubation with human preadipocyte growth

medium plus BA-DEG-BA (M+BA-DEG-BA) or DEG (M+DEG) (h). UD =

EP

undetectable. Bars = 50 µm. **P<0.01 or ***P<0.001 was evaluated using t-tests.

Error bars represent the mean ± SD of three independent experiments performed

C

in triplicate.
AC

25
 ACCEPTED MANUSCRIPT
Butyric Acid Co-drug Stimulates Adipogenic Differentiation of ADSCs Huang CM 03252015

Figure 4. Effect of PPAR-γ knockdown on BA-DEG-BA-mediated adipogenic

differentiation. (a) The mRNA levels of Cebpb and Pparg were quantitatively

analyzed by RT-qPCR five days after treatment of ADSCs (105 cell/well) with and

without (control) 4 mM BS-DEG-BA. Expression level of each gene was

PT
normalized to the level of Rplp0 (ribosomal protein, large, P0) gene. (b) ADSCs

RI
were transfected with (knockdown) or without (control) mouse PPAR-γ siRNA for

6 h before isolation of RNA. PPAR-γ mRNA expression was determined by RT-

SC
qPCR and normalized to GAPDH mRNA. Control and PPAR-γ-knockdown cells

were treated with 4 mM BA-DEG-BA for seven days. The percentages (%) of

U
control and PPAR-γ-knockdown cells positively stained with Oil Red O or perilipin
AN
A were presented (c). Cells were stained with Oil Red O (d, e) and

immunostaining with perilipin A (green). Cell nuclear was counterstained with

M
DAPI (blue) and merged views of immunostaining with DAPI and perilipin A were
D

shown (f, g). Bars = 100 µm. *P<0.05, **P<0.01, or ***P<0.001 was evaluated
TE

using t-tests. Error bars represent the mean ± SD of three individual experiments

performed in triplicate.
C EP
AC

26
 ACCEPTED MANUSCRIPT
Butyric Acid Co-drug Stimulates Adipogenic Differentiation of ADSCs Huang CM 03252015

Figure 5. Dermal augmentation and adipogenic differentiation induced by

intradermal injection of ADSCs with BA-DEG-BA. (a-f) After labeled with

CFSE (green and white thin arrows), ADSCs (2 × 106) plus 4 mM BA, BA-DEG-

BA or succinic acid (S), DEG was injected into ear of ICR mice for seven days.

PT
Mouse ear without ADSC injection served as a control. The tissue sections of

RI
mouse ears were immunostained with DAPI (blue) and perilipin A (red and solid

arrowheads). Merge contains the combined image of green CFSE and both DAPI

SC
and perilipin A staining. The ear sections contain green autofluorescent hair

follicles and cartilages. Bar = 200 µm. The high magnitude photos of selected

U
areas in (e) and (f) were displayed in (g) and (h), respectively. Open arrows
AN
indicate colocalization of green CFSE with and red perilipin A staining. Bar = 50

µm. (i) Flow cytometric analysis of CFSE-labeled ADSCs. Single-cell

M
suspensions were isolated form mouse ears injected without (control) or with
D

CFSE-labeled ADSCs plus BA, BA-DEG-BA, succinic acid (S) or DEG for seven
TE

days. Subsequently, cells were incubated with antibodies to perilipin A (Alexa

Fluor® 647). Cells without ADSC injection were incubated with fluorescent
EP

isotype-matched IgG as a negative control (Isotype control). A representative plot

denoting the percentages of cell population in each quadrant was presented. The
C

percentages (%) of CFSE-labeled cells (upper left and right quadrants) (j) and
AC

CFSE-labeled and perilipin A-stained cells (upper right quadrant) (k) were

quantified. *P<0.05 or **P<0.01 was evaluated using t-tests. Error bars represent

the mean ± SD of three individual experiments performed in triplicate.

27
 ACCEPTED MANUSCRIPT
Butyric Acid Co-drug Stimulates Adipogenic Differentiation of ADSCs Huang CM 03252015

CONFLICT OF INTEREST

The authors state no conflict of interest

ACKNOWLEDGMENTS

PT
This work was supported by a NIH STTR grant (1R41AR064046-01) awarded to

RI
Surface Bioadvances Inc., in San Diego. We thank James A Stanford and

Andrew Borkowski for assistance for the siRNA-mediated PPAR-γ knockdown.

U SC
AN
M
D
TE
C EP
AC

28
 ACCEPTED MANUSCRIPT
Butyric Acid Co-drug Stimulates Adipogenic Differentiation of ADSCs Huang CM 03252015

REFERENCES

PT
Achermann Y, Goldstein EJ, Coenye T, Shirtliff ME. Propionibacterium acnes:
from commensal to opportunistic biofilm-associated implant pathogen. Clinical

RI
microbiology reviews. 2014;27:419-40.
Alster TS, West TB. Human-derived and new synthetic injectable materials for
soft-tissue augmentation: current status and role in cosmetic surgery. Plastic and

SC
reconstructive surgery. 2000;105:2515-25; discussion 26-8.
Arimura N, Horiba T, Imagawa M, Shimizu M, Sato R. The peroxisome
proliferator-activated receptor gamma regulates expression of the perilipin gene
in adipocytes. The Journal of biological chemistry. 2004;279:10070-6.

U
Blanchette-Mackie EJ, Dwyer NK, Barber T, Coxey RA, Takeda T, Rondinone
CM, et al. Perilipin is located on the surface layer of intracellular lipid droplets in
AN
adipocytes. Journal of lipid research. 1995;36:1211-26.
Burtenshaw JM. The mechanism of self-disinfection of the human skin and its
appendages. The Journal of hygiene. 1942;42:184-210.
Calderon M, Welker P, Licha K, Fichtner I, Graeser R, Haag R, et al.
M
Development of efficient acid cleavable multifunctional prodrugs derived from
dendritic polyglycerol with a poly(ethylene glycol) shell. Journal of controlled
release : official journal of the Controlled Release Society. 2011;151:295-301.
D

Chen TH, Chen WM, Hsu KH, Kuo CD, Hung SC. Sodium butyrate activates
ERK to regulate differentiation of mesenchymal stem cells. Biochemical and
TE

biophysical research communications. 2007;355:913-8.

Chitarra LG, Breeuwer P, Van Den Bulk RW, Abee T. Rapid fluorescence
assessment of intracellular pH as a viability indicator of Clavibacter
michiganensis subsp. michiganensis. Journal of applied microbiology.
EP

2000;88:809-16.
Collins EB. Preservatives in dairy foods. Journal of dairy science. 1971;54:148-
52.
C

Cummings JH, Pomare EW, Branch WJ, Naylor CP, Macfarlane GT. Short chain
fatty acids in human large intestine, portal, hepatic and venous blood. Gut.
AC

1987;28:1221-7.
Dalen KT, Schoonjans K, Ulven SM, Weedon-Fekjaer MS, Bentzen TG,
Koutnikova H, et al. Adipose tissue expression of the lipid droplet-associating
proteins S3-12 and perilipin is controlled by peroxisome proliferator-activated
receptor-gamma. Diabetes. 2004;53:1243-52.
Duranti F, Salti G, Bovani B, Calandra M, Rosati ML. Injectable hyaluronic acid
gel for soft tissue augmentation. A clinical and histological study. Dermatologic
surgery : official publication for American Society for Dermatologic Surgery [et al].
1998;24:1317-25.

29
 ACCEPTED MANUSCRIPT
Butyric Acid Co-drug Stimulates Adipogenic Differentiation of ADSCs Huang CM 03252015

Farmer SR. Transcriptional control of adipocyte formation. Cell metabolism.

2006;4:263-73.
Fluhr JW, Darlenski R, Surber C. Glycerol and the skin: holistic approach to its
origin and functions. The British journal of dermatology. 2008;159:23-34.
Garfein ES, Orgill DP, Pribaz JJ. Clinical applications of tissue engineered
constructs. Clinics in plastic surgery. 2003;30:485-98.
Garland SH. Short chain fatty acids may elicit an innate immune response from

PT
preadipocytes: a potential link between bacterial infection and inflammatory
diseases. Medical hypotheses. 2011;76:881-3.
Ge H, Li X, Weiszmann J, Wang P, Baribault H, Chen JL, et al. Activation of G

RI
protein-coupled receptor 43 in adipocytes leads to inhibition of lipolysis and
suppression of plasma free fatty acids. Endocrinology. 2008;149:4519-26.
Greenberg AS, Egan JJ, Wek SA, Garty NB, Blanchette-Mackie EJ, Londos C.

SC
Perilipin, a major hormonally regulated adipocyte-specific phosphoprotein
associated with the periphery of lipid storage droplets. The Journal of biological
chemistry. 1991;266:11341-6.
Grice EA, Kong HH, Conlan S, Deming CB, Davis J, Young AC, et al.

U
Topographical and temporal diversity of the human skin microbiome. Science.
2009;324:1190-2.
AN
Grice EA, Segre JA. The skin microbiome. Nature reviews Microbiology.
2011;9:244-53.
Hamm JK, Park BH, Farmer SR. A role for C/EBPbeta in regulating peroxisome
proliferator-activated receptor gamma activity during adipogenesis in 3T3-L1
M
preadipocytes. The Journal of biological chemistry. 2001;276:18464-71.
Hong YH, Nishimura Y, Hishikawa D, Tsuzuki H, Miyahara H, Gotoh C, et al.
Acetate and propionate short chain fatty acids stimulate adipogenesis via
D

GPCR43. Endocrinology. 2005;146:5092-9.

Huang SP, Hsu CC, Chang SC, Wang CH, Deng SC, Dai NT, et al. Adipose-
TE

derived stem cells seeded on acellular dermal matrix grafts enhance wound
healing in a murine model of a full-thickness defect. Annals of plastic surgery.
2012;69:656-62.
EP

Huang SP, Huang CH, Shyu JF, Lee HS, Chen SG, Chan JY, et al. Promotion of
wound healing using adipose-derived stem cells in radiation ulcer of a rat model.
Journal of biomedical science. 2013;20:51.
Jeong SH, Han SK, Kim WK. Soft tissue augmentation using in vitro
C

differentiated adipocytes: a clinical pilot study. Dermatologic surgery : official

publication for American Society for Dermatologic Surgery [et al]. 2011;37:760-7.
AC

Kajimura S, Seale P, Tomaru T, Erdjument-Bromage H, Cooper MP, Ruas JL, et

al. Regulation of the brown and white fat gene programs through a
PRDM16/CtBP transcriptional complex. Genes & development. 2008;22:1397-
409.
Knapp TR, Kaplan EN, Daniels JR. Injectable collagen for soft tissue
augmentation. Plastic and reconstructive surgery. 1977;60:398-405.
Kuzmochka C, Abdou HS, Hache RJ, Atlas E. Inactivation of histone deacetylase
1 (HDAC1) but not HDAC2 is required for the glucocorticoid-dependent

30
 ACCEPTED MANUSCRIPT
Butyric Acid Co-drug Stimulates Adipogenic Differentiation of ADSCs Huang CM 03252015

CCAAT/enhancer-binding protein alpha (C/EBPalpha) expression and

preadipocyte differentiation. Endocrinology. 2014;155:4762-73.
Lai Y, Di Nardo A, Nakatsuji T, Leichtle A, Yang Y, Cogen AL, et al. Commensal
bacteria regulate Toll-like receptor 3-dependent inflammation after skin injury.
Nature medicine. 2009;15:1377-82.
Lefterova MI, Lazar MA. New developments in adipogenesis. Trends in
endocrinology and metabolism: TEM. 2009;20:107-14.

PT
Lindh JM, Terenius O, Faye I. 16S rRNA gene-based identification of midgut
bacteria from field-caught Anopheles gambiae sensu lato and A. funestus
mosquitoes reveals new species related to known insect symbionts. Applied and

RI
environmental microbiology. 2005;71:7217-23.
Luu-The V, Paquet N, Calvo E, Cumps J. Improved real-time RT-PCR method for
high-throughput measurements using second derivative calculation and double

SC
correction. BioTechniques. 2005;38:287-93.
Maslowski KM, Vieira AT, Ng A, Kranich J, Sierro F, Yu D, et al. Regulation of
inflammatory responses by gut microbiota and chemoattractant receptor GPR43.
Nature. 2009;461:1282-6.

U
Matsumoto D, Sato K, Gonda K, Takaki Y, Shigeura T, Sato T, et al. Cell-
assisted lipotransfer: supportive use of human adipose-derived cells for soft
AN
tissue augmentation with lipoinjection. Tissue engineering. 2006;12:3375-82.
Miyoshi H, Souza SC, Zhang HH, Strissel KJ, Christoffolete MA, Kovsan J, et al.
Perilipin promotes hormone-sensitive lipase-mediated adipocyte lipolysis via
phosphorylation-dependent and -independent mechanisms. The Journal of
M
biological chemistry. 2006;281:15837-44.
Morrison RF, Farmer SR. Hormonal signaling and transcriptional control of
adipocyte differentiation. The Journal of nutrition. 2000;130:3116S-21S.
D

Naik S, Bouladoux N, Wilhelm C, Molloy MJ, Salcedo R, Kastenmuller W, et al.

Compartmentalized control of skin immunity by resident commensals. Science.
TE

2012;337:1115-9.
Nakatsuji T, Chiang HI, Jiang SB, Nagarajan H, Zengler K, Gallo RL. The
microbiome extends to subepidermal compartments of normal skin. Nature
EP

communications. 2013;4:1431.
Pradhan AK, Ivanek R, Grohn YT, Geornaras I, Sofos JN, Wiedmann M.
Quantitative risk assessment for Listeria monocytogenes in selected categories
of deli meats: impact of lactate and diacetate on listeriosis cases and deaths.
C

Journal of food protection. 2009;72:978-89.

Rosen ED, Walkey CJ, Puigserver P, Spiegelman BM. Transcriptional regulation
AC

of adipogenesis. Genes & development. 2000;14:1293-307.

Sajjadian A, Tandav Magge K. Treating facial soft tissue deficiency: fat grafting
and adipose-derived stem cell tissue engineering. Aesthetic surgery journal / the
American Society for Aesthetic Plastic surgery. 2007;27:100-4.
Schep LJ, Slaughter RJ, Temple WA, Beasley DM. Diethylene glycol poisoning.
Clinical toxicology. 2009;47:525-35.
Schroder O, Opritz J, Stein J. Substrate and inhibitor specificity of butyrate
uptake in apical membrane vesicles of the rat distal colon. Digestion.
2000;62:152-8.

31
 ACCEPTED MANUSCRIPT
Butyric Acid Co-drug Stimulates Adipogenic Differentiation of ADSCs Huang CM 03252015

Shu M, Wang Y, Yu J, Kuo S, Coda A, Jiang Y, et al. Fermentation of

Propionibacterium acnes, a commensal bacterium in the human skin
microbiome, as skin probiotics against methicillin-resistant Staphylococcus
aureus. PloS one. 2013;8:e55380.
Stein J, Schroder O, Milovic V, Caspary WF. Mercaptopropionate inhibits
butyrate uptake in isolated apical membrane vesicles of the rat distal colon.
Gastroenterology. 1995;108:673-9.

PT
Stein J, Zores M, Schroder O. Short-chain fatty acid (SCFA) uptake into Caco-2
cells by a pH-dependent and carrier mediated transport mechanism. European
journal of nutrition. 2000;39:121-5.

RI
Toscani A, Soprano DR, Soprano KJ. Sodium butyrate in combination with
insulin or dexamethasone can terminally differentiate actively proliferating Swiss
3T3 cells into adipocytes. The Journal of biological chemistry. 1990;265:5722-30.

SC
Vinolo MA, Rodrigues HG, Nachbar RT, Curi R. Regulation of inflammation by
short chain fatty acids. Nutrients. 2011;3:858-76.
Wang Y, Kuo S, Shu M, Yu J, Huang S, Dai A, et al. Staphylococcus epidermidis
in the human skin microbiome mediates fermentation to inhibit the growth of

U
Propionibacterium acnes: implications of probiotics in acne vulgaris. Applied
microbiology and biotechnology. 2014;98:411-24.
AN
Wojciechowicz K, Gledhill K, Ambler CA, Manning CB, Jahoda CA. Development
of the mouse dermal adipose layer occurs independently of subcutaneous
adipose tissue and is marked by restricted early expression of FABP4. PloS one.
2013;8:e59811.
M
Wu Z, Bucher NL, Farmer SR. Induction of peroxisome proliferator-activated
receptor gamma during the conversion of 3T3 fibroblasts into adipocytes is
mediated by C/EBPbeta, C/EBPdelta, and glucocorticoids. Molecular and cellular
D

biology. 1996;16:4128-36.
Yoo EJ, Chung JJ, Choe SS, Kim KH, Kim JB. Down-regulation of histone
TE

deacetylases stimulates adipocyte differentiation. The Journal of biological

chemistry. 2006;281:6608-15.
Yu L, Zhou L, Ding M, Li J, Tan H, Fu Q, et al. Synthesis and characterization of
EP

novel biodegradable folate conjugated polyurethanes. Journal of colloid and

interface science. 2011;358:376-83.
Yu ZH, K. H.; Zhong, W.; Yang, L. Q.; Chen, Q. K.; Zhu, Z. H. In vitro lethal
effect of photodynamic therapy on human pancreatic cancer cells and its major
C

influencing factors. Clinical Oncology and Cancer Research. 2011;8:8.

Zeeuwen PL, Boekhorst J, van den Bogaard EH, de Koning HD, van de Kerkhof
AC

PM, Saulnier DM, et al. Microbiome dynamics of human epidermis following skin
barrier disruption. Genome biology. 2012;13:R101.
Zhang HH, Souza SC, Muliro KV, Kraemer FB, Obin MS, Greenberg AS. Lipase-
selective functional domains of perilipin A differentially regulate constitutive and
protein kinase A-stimulated lipolysis. The Journal of biological chemistry.
2003;278:51535-42.
Zhao QH, Wang SG, Liu SX, Li JP, Zhang YX, Sun ZY, et al. PPARgamma forms
a bridge between DNA methylation and histone acetylation at the C/EBPalpha

32
 ACCEPTED MANUSCRIPT
Butyric Acid Co-drug Stimulates Adipogenic Differentiation of ADSCs Huang CM 03252015

gene promoter to regulate the balance between osteogenesis and adipogenesis

of bone marrow stromal cells. The FEBS journal. 2013;280:5801-14.
Zuo Y, Qiang L, Farmer SR. Activation of CCAAT/enhancer-binding protein
(C/EBP) alpha expression by C/EBP beta during adipogenesis requires a
peroxisome proliferator-activated receptor-gamma-associated repression of
HDAC1 at the C/ebp alpha gene promoter. The Journal of biological chemistry.
2006;281:7960-7.

PT
RI
U SC
AN
M
D
TE
C EP
AC

33
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
M
D
TE
EP
C
AC
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
M
D
TE
EP
C
AC
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
M
D
TE
EP
C
AC
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
M
D
TE
EP
C
AC
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
M
D
TE
EP
C
AC