THE

JOURNAL • RESEARCH • www.fasebj.org

Niacin fine-tunes energy homeostasis through

canonical GPR109A signaling
Lingyan Ye,1 Zheng Cao,1 Xiangru Lai, Weiwei Wang, Zhiqiang Guo, Lili Yan, Yuyan Wang, Ying Shi,2
and Naiming Zhou3
Institute of Biochemistry, College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou, China

ABSTRACT: The incidence of overweight and obesity has become a global public health problem, constituting a major
risk factor for numerous comorbidities. Despite tremendous efforts, effective pharmacological agents for the
treatment of obesity are still limited. Here, we showed that in contrast to lactate receptor GPR81, niacin receptor
GPR109A-deficient mice had progressive weight gain and hepatic fat accumulation. Using high-fat diet–induced
mouse model of obesity, we demonstrated that niacin treatment apparently protected against obesity without
affecting food intake in wild-type mice but not in GPR109A-deficient mice. Further investigation showed that
niacin treatment led to a remarkable inhibition of hepatic de novo lipogenesis. Additionally, we demonstrated that
niacin treatment triggered brown adipose tissue and/or white adipose tissue thermogenic activity via activation of
GPR109A. Moreover, we observed that mice exposed to niacin exhibited a dramatic decrease in intestinal ab-
sorption of sterols and fatty acids. Taken together, our findings demonstrate that acting on GPR109A, niacin shows
the potential to maintain energy homeostasis through multipathways, representing a potential approach to the
treatment of obesity, diabetes and cardiovascular disease.—Ye, L., Cao, Z., Lai, X., Wang, W., Guo, Z., Yan, L., Wang,
Y., Shi, Y., Zhou, N. Niacin fine-tunes energy homeostasis through canonical GPR109A signaling. FASEB J.
33, 4765–4779 (2019). www.fasebj.org
KEY WORDS: obesity • de novo lipogenesis • thermogenesis • intestinal fat absorption

The incidence of overweight and obesity has become
a global epidemic, with over 1.9 billion overweight and
600 million obese individuals worldwide (1, 2). Obesity is
caused by an abnormal excess storage of energy as lipids in
adipose tissue due to a net imbalance between energy
ABBREVIATIONS: ACC, acetyl CoA carboxylase; ACC1, acetyl CoA car-
boxylase 1; AUC, area under the curve; BAT, brown adipose tissue;
DGAT2, diacylglycerol acyltransferase 2; DIO, diet-induced obesity;
eWAT, epididymal WAT; FAS, fatty acid synthase; FATP4, fatty acid
transport protein 4; FFA, free fatty acid; GTT, glucose tolerance test; H&E,
hematoxylin and eosin; HFD, high-fat diet; HOMA-IR, homeostasis model
assessment of insulin resistance; I-FABP, intestinal-type fatty acid–binding
protein; ITT, insulin tolerance test; NCD, normal chow diet; NPC1L1,
Niemann-Pick C1-Like 1; PDH, pyruvate dehydrogenase; PGC-1a, per-
oxisome proliferator-activated receptor g coactivator 1-a; PRDM16, PR
domain containing 16; pWAT, perirenal WAT; qRT-PCR, quantitative
RT-PCR; SCD-1, stearoyl-CoA desaturase-1; SREBP-1c, sterol regulatory
element–binding protein-1c; sWAT, subcutaneous WAT; TC, total cho-
lesterol; Tg, triglyceride; UCP1, uncoupling protein 1; WAT, white adi-
pose tissue; WT, wild-type
1
These authors contributed equally to this work.
2
Correspondence: Institute of Biochemistry, College of Life Sciences,
Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang 310058,
3
China. E-mail: shi_y@zju.edu.cn
Correspondence: Institute of Biochemistry, College of Life Sciences,
Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang 310058,
China. E-mail: zhounaiming@zju.edu.cn
doi: 10.1096/fj.201801951R
This article includes supplemental data. Please visit http://www.fasebj.org to
obtain this information.
intake and energy expenditure (3, 4). Obesity is considered
a major risk factor for numerous comorbidities including
major diseases such as cardiovascular disease, diabetes,
and cancer (5, 6). However, despite tremendous efforts,
effective antiobesity pharmacotherapies are still lim-
ited. Additionally, to date, current antiobesity agents are
designed to reduce food intake and appetite through
the hypothalamus-based molecular pathways, but un-
fortunately, these agents exhibit severe psychiatric or
cardiovascular side effects (7–9). Therefore, the magnitude
of this current health epidemic has heightened the need
for alternative therapeutic strategies aiming to control
body weight through potential targets in peripheral non-
neuronal tissues.
Since the discovery of decreasing plasma cholesterol
levels in 1955, nicotinic acid (niacin) has been used to treat
dyslipidemia as an approved drug for more than half a
century (10, 11). A number of randomized clinical trials
demonstrated that niacin monotherapy has been shown to
substantially increase levels of HDL-C and decrease levels
of triglycerides (Tgs) (12, 13). However, its exact mecha-
nism was not well understood until the orphan receptor
GPR109A (recently renamed as hydroxyl-carboxylic acid
receptor 2 or HCA2) was identified as a receptor with
high affinity for niacin in 2003, independently by 3 research
groups (14–17). Subsequently, the ketone body b-hydroxy-
butyrate was identified as an endogenous ligand for

0892-6638/19/0033-4765 © FASEB 4765

GPR109A (18). In adipocytes, upon stimulation by niacin,
GPR109A functions via Gi/o proteins to lower intra-
cellular cAMP production, leading to decreased PKA-
mediated activation of hormone-sensitive lipase, which in
turn reduces Tg hydrolysis to free fatty acids (FFAs) (19).
This FFA hypothesis is supported by evidence that niacin
failed to lower FFA and Tgs in GPR109A-knockout mice
(15).
The established clinical benefits of niacin treatment
on cardiovascular events have been challenged by re-
cent studies showing that niacin exerts its beneficial effects
on GPR109A-mediated antiatherosclerotic activity inde-
pendently from its antilipolytic properties (20, 21). How-
ever, these data did not exclude the possibility that niacin
provides cardiovascular benefits in an antilipolysis-
independent manner through GPR109A. A niacin-
mediated beneficial cardiovascular effect has been shown
to be transferable with GPR109A-competent bone mar-
row cells (21). In addition, accumulating evidence has
demonstrated that activation of GPR109A was able to
induce anti-inflammatory effects not only on atheroscle-
rosis but also on acute ischemic stroke, arthritis, chronic
renal failure, and sepsis through inhibition of immune cell
chemotaxis and proinflammatory cytokine production
(20–29). Therefore, more studies are needed to thoroughly
evaluate the GPR109A-mediated pleiotropic effects on
atherogenesis and other metabolic diseases.
GPR109A-induced inhibition in adipocyte lipolysis and
fatty acid mobilization in response to long-term treat-
ment with niacin would result in excessive synthesis of Tgs
and obesity (30). However, during breeding process of
GPR109A-knockout mice we obtained from Dr. Stefan
Offermanns (Max Planck Institute for Heart and Lung Re-
search, Bad Nauheim, Germany), we found that the
GPR109A-knockout mice are more prone to being obese
compared to the wild-type (WT) mice. Therefore, we initi-
ated this study to investigate roles of niacin-GPR109A in the
regulation of lipid metabolism using GPR109A-knockout
mice combined with high-fat diet (HFD)-induced obe-
sity mouse model. We show that upon activation by
niacin, GPR109A fine tunes lipid metabolism through
multipathways including inhibition of hepatocyte lipo-
genesis and fatty acid absorption and promotion of brown
adipose tissue (BAT) thermogenesis.
MATERIALS AND METHODS
Animal studies
All mice were housed in groups of 4–6 per cage under constant
temperature (23–25°C) with a 12-h light/dark cycle. Animals
were given ad libitum access to water and food. GPR109A het-
erozygous mice on C57BL/6 background were a generous gift
from Dr. Stefan Offermanns. Homozygous GPR109A knockout
and their littermate WT mice were generated by matting het-
erozygous mice and genotyped using PCR. Male mice were used
for all the experiments described in this study. 4-wk-old male
C57BL/6 mice were purchased from the Shanghai Laboratory
Animal Center (Shanghai, China), and after a week of adaptation,
mice were fed a HFD (60% kcal from fat) (D12492; Research Diets,
New Brunswick, NJ, USA) for generating a diet-induced obesity
(DIO) model and a normal chow diet (NCD) (10% kcal; Shanghai
4766 Vol. 33 April 2019 The FASEB Journal x www.fasebj.org
Laboratory Animal Center) in the control group. After 6 wk on an
HFD, mice were divided randomly into 2 groups. One group
received niacin (50 mM) (MilliporeSigma, Burlington, MA, USA)
dissolved in drinking water, and the other received vehicle
(water) for 8–9 wk as a control. Groups of age- and weight-
matched animals were used in all experiments. Food consump-
tion and body weight were recorded weekly. After the modeling
period, mice were briefly starved overnight (12 h) and anes-
thetized with 3% isoflurane in a dedicated chamber, and the
weights of major organs including fat pads and liver were
recorded. Whole blood was collected and processed to isolate
serum, and tissues were flash frozen in liquid nitrogen and stored
at 280°C for biochemical and histologic analyses. All animal
procedures were conducted in compliance with protocols ap-
proved by the Institutional Animal Care and Use Committee at
Zhejiang University (ZJU2010-1-01-020).
Mice primary hepatocyte extraction and culture
Mice (6 wk old) were anesthetized with isoflurane. The liver was
perfused with prewarmed calcium- and magnesium-free HBSS
containing 1% penicillin-streptomycin solution, 0.5 mM EDTA,
and 25 mM HEPES at a flow rate of 3.5 ml/min for 5 min until the
liver become pale in color with no blood inside. We then switched
to prewarmed digestion buffer [HBSS with calcium-magnesium
containing 1% penicillin-streptomycin solution, 25 mM HEPES,
5 mM CaCl2, and 0.35 mg/ml collagenase X (Fujifilm, Tokyo,
Japan)] for 5 min at a flow rate of 3.5 ml/min. We separated the
liver with 2 sets of forceps and put the dish into the incubator to
digest for another 5 min. We then added 25 ml cold DMEM with
10% FBS and passed the liver through a 100 mm strainer (352360;
Thermo Fisher Scientific, Waltham, MA USA) and isolated a
sample pellet by centrifugation at 50 g for 3 min at 4°C. The liver
was subsequently washed 2 times in 25 ml washing buffer
(DMEM with 1% penicillin-streptomycin solution) and a sample
pellet was isolated by centrifugation at 50 g for 3 min at 4°C. The
viability of isolated hepatocytes was determined by trypan blue
exclusion. Hepatocytes were plated in collagen I-coated 6 well
(5 3 105 cells/well) and 12 well (2 3 105 cells/well) plates in
William E medium (BE02-019; Lonza Group, Basel, Switzerland)
with 10% FBS and 1% penicillin-streptomycin solution and
then were incubated at 37°C in an atmosphere of 5% CO2. At 2 h
after plating, the plating medium was replaced with fresh culture
medium consisting of DMEM supplemented with 1.5% FBS,
1 mM sodium pyruvate, and 1% penicillin-streptomycin solution.
Body temperature in cold response
To test resistance to cold exposure, mice were individually caged
and exposed to 4°C for 6 h with free access to water. Anal tem-
perature was monitored at the beginning and hourly after the
start of cold exposure.
Serum collection and parameters determination
Blood was collected from the orbital sinus, and samples were
incubated at room temperature for 30 min to allow clotting. Se-
rum samples were obtained after centrifuging at 3500 rpm at 4°C
for 20 min. Finally the supernatant (serum) was collected and
stored at 280°C. Serum total Tg, total cholesterol (TC), HDL, and
LDL were measured by using biochemical testing kits (Roche
Holding AG, Basel, Switzerland). Serum glucose levels were
measured using commercial kits (Jiancheng, Nanjing, China).
Serum insulin levels were determined using a rat/mouse insulin
ELISA kit (MilliporeSigma) according to the manufacturer’s
instructions. Homeostasis model assessment of insulin resis-
tance (HOMA-IR) was calculated using the following equation:
YE ET AL.
HOMA-IR = [fasting plasma insulin (mIU/L)] 3 fasting plasma
glucose (mM)/22.5.
Fecal and liver lipids analysis
Fecal lipids were extracted as previously described (31). Feces
were collected from mice housed individually for 5 consecutive
days. Feces were dried for 1 h at 70°C, and 100 mg aliquots of
feces were weighed, incubated with 2 ml of chloroform-methanol
(2:1,v/v) for 30 min at 60°C with constant agitation, and then
centrifuged (5 min at 5000 rpm). Water (1 ml) was added to the
supernatant; following vortex, phase separation was induced
by low-speed centrifugation (2000 rpm for 10 min). The lower
chloroform phase was then removed and transferred to a new
tube, and the sample was dried under nitrogen. Then samples
were resuspended in 500 ml chloroform, evaporated to dryness,
and finally resuspended in 500 ml 100% ethanol. For analysis of
liver lipids, 100 mg aliquots of tissue were homogenized with
2 ml chloroform-methanol and then agitated overnight on an or-
bital shaker at 4°C. The homogenate was then centrifuged (5 min at
5000 rpm), 0.9% NaCl solution was added to the liquid phase, and
the samples were vortexed. Phase separation was induced by
centrifugation (2000 rpm for 10 min), and the bottom phase was
moved to a new tube and then processed as previously described.
The levels of TC, Tg, and FFA in the fecal and liver lipid extracts
were measured using commercial kits following the manufac-
turer’s instructions and normalized to feces or tissue weights.
Glucose and insulin tolerance tests
For glucose tolerance tests (GTTs), mice were tested after an
overnight period having food withheld. Glucose (40% solution,
1.5 g/kg body weight; MilliporeSigma) was intraperitoneally
injected, and tail vein blood glucose levels were measured at 0, 30,
60, 90, and 120 min after injection with a OneTouch Ultra Blood
Glucose Monitoring System (LifeScan, Milpitas, CA, USA). For
insulin tolerance tests (ITTs), mice were briefly starved for 4 h and
then intraperitoneally injected with human insulin (0.75 U/kg
body weight; NovoRapid; Novo Nordisk, Bagsværd, Denmark).
Glucose concentrations in blood were measured after 0, 30, 60, 90,
and 120 min. Area under the curves (AUCs) for GTT and ITT
was calculated by trapezoidal approximation, using Prism 6.0
(GraphPad, La Jolla, CA, USA).
RNA isolation and quantitative RT-PCR analysis
Total RNA was extracted by using an RNAiso reagent kit
(Takara, Otsu, Shiga, Japan). Reverse transcription was per-
formed on each RNA sample (1 mg) by using PrimeScript RT
Reagent Kit with gDNA eraser (Takara) with a final reaction
volume of 20 ml. For quantitative RT-PCR (qRT-PCR) analysis,
the reaction mixture was run in an iQ5 real time PCR machine
(Bio-Rad, Hercules, CA, USA) instrument using SYBR Premix
Ex Taq II (Takara). All expression levels were normalized to
the corresponding glyceraldehyde-3-phosphate dehydrogenase
mRNA levels and analyzed using the 22DDCt method. The qRT-
PCR cycles were as follows: step 1, preparative denaturation (30 s
at 95°C); step 2, 40 cycles of denaturation (5 s at 95°C) and
annealing (30 s at 60°C); and step 3, dissociation following the
manufacturer’s protocol. PCR primers used in the real-time PCR
analysis are listed in Supplemental Table S1.
Semiquantification PCR
The total RNA was extracted by using an RNAiso Reagent Kit.
The cDNA were synthesized using PrimeScript RT Reagent Kit.
GPR109A REGULATES ENERGY HOMEOSTASIS
PCR was performed in 20 ml reaction mixture according to the
instruction of KOD-Plus (Toyobo, Osaka, Japan). Primers used
for GPR109A are indicated in Supplemental Table S1. PCR was
performed using an initial denaturation at 94°C for 5 min, fol-
lowed by 28 cycles at 94°C for 30 s, 53°C for 30 s, and 68°C for 30 s.
After 28 cycles, an additional elongation step was performed at
68°C for 10 min. Amplified PCR products were run on 1.2%
agarose gels containing ethidium bromide.
Histologic analysis
To examine hepatic steatosis, liver samples were embedded in
Tissue-Tek (Sakura Finetek, Torrance, CA, USA) and frozen and
sectioned (10 mm) and stored at 280°C until use. The sections
were stained with hematoxylin and Oil red O (MilliporeSigma)
for lipid deposition. For histology, adipose tissues were fixed in
10% neutral-buffered formalin, followed by dehydration with
graded ethanol (80–100%) and embedding in paraffin. Then the
tissues were sliced into 6-mm pieces using microtome, depar-
affinized in xylene, and passed through 80–100% ethanol. And
hematoxylin and eosin (H&E) staining was performed using
the standard techniques. For transmission electron microscopy
analysis, liver and muscle tissues were cut into 3 mm3 fragments
and fixed by immersion in 2.5% glutaraldehyde in phosphate
buffer (0.1 M, pH 7.4) overnight at 4°C. After that, they were
washed in 0.1 M phosphate buffer 3 times. Then, they were
postfixated with 1% osmium tetroxide (OsO4) in 0.1 M phos-
phate buffer for 2 h at room temperature. Subsequently, they
were rinsed with 0.1 M phosphate buffer for 3 times again. Tis-
sues were dehydrated through graded alcohols (30, 50, 70, 80, 90,
95, and 100%) for 30 min. Last they were embedded in Epon 812.
Ultrathin sections (70–90 nm in thickness) of Epon-embedded
samples were placed on copper grids and stained with uranyl
acetate and lead citrate. Sections were examined with a Hitachi
model H-7650 electron microscope (Hitachi, Chiyoda, Japan).
Measurement of enzymes activities
To determine the activities of enzymes involving in lipogenesis in
liver and white adipose tissue (WAT), the enzyme source fraction
was prepared as follows. Briefly, a 10% (w/v) homogenate was
prepared in phosphate buffered solution (pH 7.2–7.4) and then
centrifuged at 2000–3000 rpm for 20 min and the supernatant was
transferred into a new tube. Samples were stored at 280°C until
use. All enzymes, such as pyruvate dehydrogenase (PDH), acetyl
CoA carboxylase (ACC), acetyl CoA carboxylase 1 (ACC1), and
diacylglycerol acyltransferase 2 (DGAT2), were measure by
ELISA (Jing Ma, Shanghai, China).
Western blot analysis
Proteins were extracted from tissues or cultured cells by ho-
mogenizing or scraping in ice-cold RIPA buffer [50 mM Tris (pH
7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate,
0.1% SDS, 2 mM EDTA] (Beyotime, Shanghai, China) containing
1 mM sodium orthovanadate and protease inhibitors (Complete
Mini; Roche). Tissue samples were transferred to microcentrifuge
tubes and rotated on a rocking platform for 40 min at 4°C after
homogenization on ice. The homogenate was cleared by centri-
fugation at 4°C for 20 min at 12,000 rpm and the supernatant was
transferred into a new tube. Protein concentration in the super-
natant was determined using the BCA Protein Assay Kit (Probe
Gene, Jiangsu, China). Equal amounts of protein samples were
subjected to SDS-PAGE and transferred to PVDF membranes
(MilliporeSigma). Membranes were blocked with 5% bovine se-
rum albumin in tris-buffered saline containing 0.1% Tween-20
(TBST) for 1 h at room temperature and incubated overnight at
4767
4°C with primary antibodies against GPR109A (Santa Cruz
Biotechnology, Dallas, TX, USA) and b-tubulin (Cell Signaling
Technology, Danvers, MA, USA), respectively. The following
day, the membranes were washed with TBST and incubated
with a horseradish peroxidase–conjugated secondary antibody
(1: 2000) for 2 h at room temperature. Proteins were visualized
with an ECL reagent (Beyotime) by the Tanon 5200 Chemilu-
minescent Imaging System (Tanon, Shanghai, China).
Statistical analysis
Statistical analysis was performed using Prism 6.0 (GraphPad).
All of the data were expressed as the mean 6 SEM. All experi-
ments were performed in triplicate. Unpaired, 2-tailed Student’s
t test was used for comparisons between 2 groups for all figures.
Values of P , 0.05 were considered significant.
RESULTS
GPR109A-deficient mice are susceptible to DIO
To explore GPR109A function in lipid metabolism, parallel
experiments in mice with GPR109A (WT) and without
GPR109A (Gpr109a2/2) were performed. We recorded
weight gain weekly when both WT and Gpr109a2/2 male
mice were fed either a NCD or HFD for 12 wk. As the
feeding progressed, starting from ;10–12 wk of age, the
GPR109A-knockout mice exhibited significantly increased
body weight compared with the WT mice, without any
significant difference in food intake between the two
groups (Fig. 1A– F). After 12 wk of NCD feeding, adipose
tissue weights and organ weights were examined. Our
data demonstrated that GPR109A-deficient mice dis-
played a significant increase in adipose tissue weights
[including epididymal WAT (eWAT), perirenal WAT
(pWAT), and inguinal WAT] and liver weight but not in
BAT or other organ weights (kidney, lung, heart, and
spleen) when compared to WT mice (Fig. 1G, H and Sup-
plemental Fig. S1A, B). Moreover, H&E-stained histologic
sections of eWAT and electron microscope observation of
liver and muscle revealed that GPR109A-deficient mice
had a striking visible increase in the amount and size of
lipid droplets in liver, muscle, and eWAT compared with
the WT group (Fig. 1I). Taken together, it is more likely that
GPR109A plays an essential role in lipid metabolism.
GPR109A exerts inhibitory effects on HFD-
induced obesity upon activation by niacin
To further validate the role of GPR109A in the antiobesity
action, WT, and Gpr109a2/2 mice were fed a 6-wk HFD to
induce obesity. A HFD supplemented with niacin (HFD +
niacin, 50 mM) or vehicle (HFD + water) was then in-
troduced during the following 8 wk. In WT mice groups,
treatment with niacin resulted in a significant reduction
in body weight, but weekly food intake remained un-
changed as compared to vehicle-treated mice (Fig. 2A, B).
As anticipated, the mice maintained on the HFD exhibited
a progressive increase in body weight gain (Fig. 2A, C).
While lack of GPR109A prevented antiobesity actions of
niacin (Fig. 2C, D), mice fed a HFD in response to treatment
4768 Vol. 33 April 2019 The FASEB Journal x www.fasebj.org
with niacin exhibited a significant reduction in adipose
tissue weights (including eWAT, pWAT, and inguinal
WAT) and liver weight, but not in BAT or other organ
weights (kidney, lung, heart, and spleen) with respect to
vehicle-treated mice (Fig. 2E, F and Supplemental Fig. S1C,
D), which indicated that niacin protected the mice from
DIO through GPR109A. Furthermore, histologic exami-
nation showed that niacin treatment led to a marked de-
crease in lipid accumulation in liver, muscle, and eWAT
(Fig. 2G).
Niacin has profound and unique beneficial effects on
blood lipid (11). We examined the effects of niacin treat-
ment on metabolic parameters in serum of experimental
mice. As shown in Fig. 3, treatment with niacin led to a
significant reduction in serum TC, LDL, and HDL, with
a characteristic of high ratio of HDL/LDL in mice fed an
HFD, but Tg remained unchanged in respect to vehicle-
treated mice (Fig. 3A). The effect of niacin on TC, LDL,
HDL, and the ratio of HDL/LDL was not observed in
Gpr109a2/2 mice (Fig. 3B). We also performed a GTT and
ITT and documented that niacin-treated mice were less
glucose tolerant but exhibited insulin tolerance compara-
ble to control mice (Fig. 3C). No changes on GTT and ITT
were detected in niacin-treated Gpr109a2/2 mice (Fig. 3D).
Moreover, quantitative analysis showed that niacin treat-
ment results in a significant reduction in blood insulin (Fig.
3E); however, HFD-fed mice treated with niacin exhibited
a marked decrease in HOMA-IR values compared to
vehicle-treated mice (Fig. 3F). As expected, GPR109A-
knockout mice exhibited significantly higher fasting glu-
cose and insulin levels compared with the WT mice, which
inevitably resulted in higher HOMA-IR levels (Supple-
mental Fig. S2A–C), whereas no significant difference in
fasting glucose and insulin levels (including HOMA-IR
values) was observed in niacin- and vehicle-treated
Gpr109a2/2 mice when fed an HFD (Supplemental Fig.
S2D–F). Taken together, these results suggest that niacin is
likely to play a beneficial role in insulin sensitivity via
GPR109A.
GPR109A inhibits de novo lipogenesis in liver
in response to niacin
Our data clearly showed that upon activation by niacin,
GPR109A exerts its inhibitory effects on lipid accumula-
tion in adipose tissue, liver, and muscle when fed an HFD.
Moreover, quantitative analysis revealed that HFD-fed
mice displayed a significant increase in hepatic Tg content,
whereas niacin supplement led to a marked reduction in
Tg content in the liver; in contrast, no difference in TC and
FFA levels was observed (Fig. 4A). In addition, the de-
crease in the hepatic insulin level was observed in mice
with niacin treatment (Fig. 4B), coincident with the result
in serum (Fig. 3E). However, animals remain unaltered in
food intake. We then investigated whether or not niacin
plays a role in the regulation of hepatic de novo lipogenesis
via GPR109A. We first used qRT-PCR to quantitatively
analyze the GPR109A expression in mouse liver.
It has been reported that GPR109A is highly expressed
in adipocytes and activated immune cells (11, 14–16). Our
YE ET AL.
Figure 1. GPR109A-deficient animals display obesity. A) Body weights were recorded in mice fed a NCD (n = 7–8). B) Food intake
was recorded. C ) Representative images of fat mass are shown. D) Body weights were recorded in mice fed an HFD (n = 7–8). E )
Food intake was recorded. F ) Representative images of fat mass are shown. G) Fat pads and organ weights were recorded in mice
fed a NCD (n = 7–8). H ) Fat pads and organ weights were recorded in mice fed an HFD (n = 7–8). I ) Representative SEM images
in the liver (left panels; scale bars, 2 mm) and muscle (middle panels; scale bars, 0.5 mm) and H&E staining of eWAT (right
panels; scale bars, 100 mm) (n = 3 mice/group). iWAT, inguinal WAT; ns, not significant; WT, wild-type. All the data are
presented as means 6 SEM. Significance determined by unpaired, 2-tailed Student’s t test. *P , 0.05, **P , 0.01, ***P , 0.001,
****P , 0.0001.

GPR109A REGULATES ENERGY HOMEOSTASIS 4769

Figure 2. GPR109A exerts inhibitory effects on HFD-induced obesity upon activation by niacin. A–D) Body weight (A) and food
intake (B) were recorded weekly in WT mice (n = 10–11); body weight (C ) and food intake (D) were recorded weekly in
Gpr109a2/2 mice (n = 10). E ) Fat pads and organ weights were recorded after WT mice were anesthetized (n = 10–11). F ) Fat
pads and organ weights were recorded after Gpr109a2/2 mice were anesthetized (n = 10). G) Representative SEM images in the
liver (left panels; scale bars, 5 mm) and muscle (middle panels; scale bars, 0.5 mm) and H&E staining of eWAT (right panels; scale
bars, 100 mm) (n = 3 mice/group). iWAT, inguinal WAT; NA, niacin; ns, not significant; WT, wild type. All the data are presented as
means 6 SEM. Significance was determined by unpaired, 2-tailed Student’s t test. *P , 0.05, **P , 0.01, ****P , 0.0001.

data clearly showed that when fed an HFD, mice exhibited
a significant increase in lipid accumulation in the liver,
whereas niacin supplementation resulted in a significant
reduction of lipid accumulation in the liver. To confirm the
role of GPR109A in the liver, we examined the expression
of GPR109A in different tissues and organs. High-level
expression of GPR109A mRNA was detected in eWAT
and BAT (Fig. 4C). Interestingly, although relatively lower
basal expression of GPR109A was detected, upon in-
troduction of an HFD, GPR109A mRNA was markedly
up-regulated in the liver but not in other organs (Fig. 4C,
D and Supplemental Fig. S3A). Moreover, niacin sup-
plementation showed no significant effect on GPR109A
expression in liver or other tissues, except in BAT (Fig. 4D
and Supplemental Fig. S3A). In contrast, GPR109A was
completely absent in the liver from Gpr109a2/2 mice
(Supplemental Fig. S3B,C). GPR109A expression in the
liver was also confirmed at the protein level by Western
blot (Fig. 4E).
However, our data on GPR109A expression in mouse
liver could not discriminate between the hepatocytes and
Kupffer cells. Therefore, to confirm the expression of
GPR109A in hepatocytes, we further analyzed the ex-
pression of GPR109A in the mice primary hepatocytes. As
anticipated, endogenous expression of GPR109A in both
mRNA and protein levels was detected in the primary

4770 Vol. 33 April 2019 The FASEB Journal x www.fasebj.org YE ET AL.

Figure 3. Niacin improves the dyslipoidemia and insulin sensitivity via GPR109A. A) Serum lipid parameters, including total Tg,
TC, HDL, and LDL were measured in WT mice using biochemical testing kits (n = 7–8). B) Serum lipid parameters were
measured in Gpr109a2/2 mice (n = 7–8). C ) GTT and ITT results and the corresponding AUC in WT mice (n = 5–6). D) GTT
and ITT results and the corresponding AUC in Gpr109a2/2 mice (n = 5–6). E ) Serum insulin level during GTT was measured by
ELISA in WT mice (n = 5–6). F ) HOMA-IR index was calculated in WT mice as stated in the Materials and Methods section (n =
5–6). All the data are presented as mean 6 SEM. NA, niacin; ns, not significant; WT, wild type. Significance was determined by
unpaired, 2-tailed Student’s t test. *P , 0.05, **P , 0.01, ***P , 0.0001.

hepatocytes (Fig. 4F). To confirm the role of GPR109A in
the regulation of de novo lipogenesis in the liver, we used
ELISA to quantitatively analyze the activities of key en-
zymes involved in fatty acid biosynthesis, ACC, ACC1,
and PDH (32). As shown in Fig. 4G, in WT mice fed a HFD,
treatment with niacin resulted in a significant decrease in
the enzyme activities of ACC, ACC1, and PDH in the liver,
whereas no change in eWAT was observed (Fig. 4G).
However, in Gpr109a2/2 mice, no difference in the enzyme
activities of PDH and ACC in the liver following treat-
ment with niacin was detected (Fig. 4H). Our data also
showed that niacin supplementation led to no statistically

GPR109A REGULATES ENERGY HOMEOSTASIS 4771

Figure 4. Activated GPR109A directly inhibits hepatocyte lipogenesis. A) Liver lipid content, including the levels of Tg, TC, and
FFA, were measured (n = 5–6). B) ELISA analysis revealed the insulin level reduced significantly in the mice treated with niacin
(n = 5). C ) GPR109A gene expression was measured by qRT-PCR in muscle, liver, intestine, eWAT, and BAT of the mice fed a
NCD, HFD, or HFD with 50 mM niacin, respectively (n = 8–10). Gene expression in muscle served as the statistical controls. D)
Expression of GPR109A was measured in the liver isolated from the WT mice fed a NCD or HFD with or without niacin by
(continued on next page)

4772 Vol. 33 April 2019 The FASEB Journal x www.fasebj.org YE ET AL.

significant changes in the activity of DGAT2 (Fig. 4G),
which directly catalyzes the formation of Tgs from diac-
ylglycerol and Acyl-CoA in fat, liver, and skin cells (33). In
addition, these data were further confirmed by quan-
titative determination of mRNA transcripts of sterol reg-
ulatory element–binding protein-1c (SREBP-1c), a key
transcriptional activator of lipogenic genes and its down-
stream target genes, ACC1, fatty acid synthase (FAS), and
stearoyl-CoA desaturase-1 (SCD-1) by real time RT-PCR.
As revealed in Fig. 4I, administration of niacin resulted in a
significant decrease in mRNA levels of SREBP-1c and its
target genes, ACC1, FAS, and SCD-1 in the liver compared
to the vehicle-treated mice. Our data suggest the inhibitory
effects of niacin-GPR109A on hepatic de novo lipogenesis;
however, detailed mechanism involved in this inhibition
needs further exploration.
Agonist-activated GPR109A triggers BAT
and/or browning WAT thermogenic activity
improved cristae organization in HFD-fed WT mice (Fig.
5D). Furthermore, we assayed the expression of genes
involved in BAT thermogenesis by real time PCR analysis.
As shown in Fig. 5E, the mRNA expression of uncoupling
protein 1 (UCP1), a critical player in allowing electrons to
be released as heat rather than stored, and PR domain
containing 16 (PRDM16), a well-known BAT tran-
scriptional regulator, were significantly up-regulated in
HFD-fed WT mice in response to treatment with niacin.
However, no difference was detected in niacin-treated
Gpr109a2/2 mice (Fig. 5F). Additionally, we also ob-
served that niacin showed the potential to elevate the
expression of beige-specific genes including Cd137, Tbx1,
and Tmem26 in subcutaneous WAT (sWAT) and the ex-
pression of peroxisome proliferator-activated receptor g
coactivator 1-a (PGC-1a) and Tbx1 in eWAT but not in
pWAT in HFD-fed WT mice (Fig. 5G) or Gpr109a2/2 mice
(Fig. 5H). These data collectively suggest that nia-
cin stimulates energy expenditure through GPR109A in
HFD-fed mice.

Obesity is an abnormal accumulation of lipid in adipose

tissue caused by an imbalance between energy intake and
expenditure. In the HFD-induced obesity mouse model,
niacin supplementation led to a significant reduction in
body weight but no change in daily food intake, indicating
that niacin is more likely to promote energy expenditure.
To validate this hypothesis, we first monitored the core
body temperature by measuring the anal temperature of
mice fed an HFD and treated with vehicle or niacin upon
acute cold exposure. GPR109A-deficient mice showed a
continuous decline in body temperature and reached 32°C
after acute cold exposure at 4°C for 6 h, whereas WT mice
were able to maintain their body temperature at 33.5°C
(Fig. 5A). Moreover, WT mice fed an HFD and treated
with niacin revealed significantly higher body tem-
peratures than that of vehicle-treated mice; however, no
significant difference was observed between the niacin-
and vehicle-treated group in GPR109A-deficient mice
in response to acute cold exposure (Fig. 5A) and in WT
mice without acute cold exposure (Fig. 5B). These data
collectively confirm cold tolerance in niacin-treated WT
mice.
BAT is the major site essential to generate heat (ther-
mogenesis) for the maintenance of body temperature.
Therefore, we examined the ultrastructure of BAT
mitochondria. Transmission electron microscopy dem-
onstrated that BAT from Gpr109a2/2 mice exhibited a
significant reduction in the number and size of mito-
chondria with aberrant cristae compared to WT mice (Fig.
5C). In addition, niacin treatment caused a remark-
able increase in number and size of mitochondria with
Niacin-activated GPR109A leads to a reduction
in dietary fatty acid absorption
Recent studies have demonstrated that GPR109A is
expressed in colonic and intestinal epithelial cells and
mediates the local control of intestinal glucose absorp-
tion (34) and the tumor-suppressive effects of the bac-
terial fermentation product butyrate in the colon (35).
Accordingly, we further examined the role of GPR109A
expression in colonic and intestinal epithelial cells in the
control of intestinal fatty acid absorption. We collected
feces from mice housed individually from 5 consecutive
days and measured fecal lipid contents. As indicated in
Fig. 6, the weights of feces normalized to body weight in
the niacin-treated mice when fed a HFD compared to
the control mice (Fig. 6A). Further analysis showed that
total lipid, Tg, TC, and FFA in feces were significantly
increased when HFD-fed mice treated with niacin were
compared to the control mice (Fig. 6A). However, total
lipid in feces was reduced in HFD-fed Gpr109a2/2 mice
treated with niacin; no difference in Tg, TC, and FFA
was observed between niacin-treated mice and control
mice (Fig. 6B). This was confirmed by RT-PCR quanti-
tative analysis of 3 key proteins [fatty acid transport
protein 4 (FATP4), intestinal-type fatty acid–binding
protein (I-FABP), and NCP1L1] involved in the control
of fatty acid and cholesterol absorption. The mRNA
levels of the 3 genes were obviously decreased in the
WT niacin group but not in the Gpr109a2/2 mice (Fig.
6C). These findings suggest that niacin is likely to

quantitative analyses (n = 8–10). E ) The relative gene expression of GPR109A in the liver was calculated by Western blot (n = 4).
F ) Expression of GPR109A was measured in primary mice hepatocytes by semiquantification PCR (left panel) and Western blot
(right panel) (n = 3). G) Comparing the activities of enzymes involving in lipogenesis (PDH, ACC, ACC1, and DGAT2) of WT
mice fed an HFD with or without niacin in the liver and eWAT (n = 5). H ) The activities of PDH and ACC in the liver were
measured in Gpr109a2/2 mice fed an HFD (n = 7, 9). I ) The mRNA expression analysis of genes involved in de novo lipogenesis in
the liver, and the data of the WT (vehicle) group served as the statistical controls (n = 8). NA, niacin; ns, not significant; WT, wild-
type. All data are expressed as means 6 SEM. ANOVA followed by unpaired, 2-tailed Student’s t test. Significant differences
between groups are indicated with asterisks. *P , 0.05, **P , 0.01, ***P , 0.001.

GPR109A REGULATES ENERGY HOMEOSTASIS 4773

Figure 5. Niacin treatment triggers BAT thermogenesis via GPR109A. A) To test resistance to cold exposure, mice were exposed
to 4°C for 6 h; anal temperature monitored hourly (n = 5, 6). B) Anal temperature monitored weekly under room temperature
(n = 5). C ) Electron micrographs of the BAT in WT and Gpr109a2/2 mice. Scale bars, 1 mm. D) Electron micrographs of the
BAT at the end of niacin treatment. Scale bars, 1 mm. E ) The mRNA expression of UCP1, PGC-1a, and PRDM16 in BAT of WT
mice (n = 10, 11). F ) The mRNA expression of UCP1, PGC-1a, and PRDM16 in Gpr109a2/2 mice (n = 6, 7). G) The mRNA
expression of brown or beige genes (UCP1, PRDM16, PGC-1a, Cd137, Tbx1, and Tmem26) in pWAT, sWAT and eWAT in WT
mice (n = 10, 11). H ) The mRNA expression of brown or beige genes in pWAT, sWAT, and eWAT in Gpr109a2/2 mice (n = 6,
7). NA, niacin; ns, not significant; WT, wild type. All values are presented as mean 6 SEM. ANOVA followed by unpaired, 2-
tailed Student’s t test. Significant differences between groups are indicated with asterisks. *P , 0.05, **P , 0.01, ***P , 0.001.

4774 Vol. 33 April 2019 The FASEB Journal x www.fasebj.org YE ET AL.

Figure 6. Activated GPR109A decreases fatty acid absorption. Feces were collected over a period of 5 d from WT mice (n =
7, 9) as well as Gpr109a 2/2 mice (n = 6) fed an HFD. A) Fecal mass was normalized to body weight (mg/g). Fecal lipids
were extracted, and the fecal lipid concentration was expressed as milligrams of lipids per gram of feces weight. Total Tg,
TC, and FFA were measured in WT mice. B) Feces lipid indicators were measured in Gpr109a 2/2 mice. C ) We performed
qRT-PCR using total mRNA isolated from intestines and examined the mRNA expression of indicated genes involved
in fatty acid absorption (FATP4 and I-FABP) and cholesterol absorption (NPC1L1). The data from the vehicle group
served as the statistical controls. NA, niacin; ns, not significant; WT, wild type. All values are presented as mean 6 SEM.
ANOVA followed by 2-tailed Student’s t test. Significant differences between groups are indicated with asterisks. *P , 0.05,
**P , 0.01.

exhibit the potential to inhibit the dietary fat absorption
through GPR109A, thereby causing the decrease in fat
deposit and body weight.
DISCUSSION
Since the identification of GPR109A, a Gi/o-coupled
receptor, as the molecular target for niacin (20, 22, 36),
most studies have focused on its pronounced hypo-
lipidemic and cardioprotective action. Recent investi-
gations have also shed light on the anti-inflammatory
and anti-oxidative effects of GPR109A (22, 37–40).
However, little attention has been paid to the potential
of this receptor to favorably modulate de novo lipogen-
esis and energy homeostasis. In the present study, we
explored the physiologic roles of niacin-GPR109A in
the regulation of lipid metabolism and energy homeo-
stasis in an HFD-induced murine model of obesity
combined with GPR109A-deficient mice. The most
important observation is that niacin supplementation
prevented the progression of dietary obesity in HFD-
fed WT mice but not in HFD-fed GPR109A-deficient
mice. Mechanistically, we demonstrated that niacin-
mediated reduction in body weight and fat mass was
caused by fine-tuning of de novo lipogenesis in liver,
energy expenditure, and intestinal fatty acid absorption
through GPR109A.
During the breeding process of GPR109A-knockout
mice obtained from Dr. Offermanns, we observed that
the deletion of GPR109A resulted in obvious increases in
body weight and fat mass compared to WT mice. In
contrast, lactate receptor GPR81-deficient mice gain
significantly less body weight than WT mice under HFD-
feeding conditions, although Gi/o-coupled GPR81 ex-
erts the same activity in the inhibition of lipolysis in
WAT (41). This prompted us to initiate this study to
evaluate the exact role of GPR109A in the regulation of
energy homeostasis. Recent studies indicated that niacin
treatment remarkably reduced body weight in the obese
mice (42). Previous investigations have demonstrated
that the ketone body b-hydroxybutyrate (bOHB), as an
important metabolic intermediate and also a gut bacte-
rial fermentation-yielded product, which is the only
known endogenous ligand for GPR109A (18), causes
reductions of body weight and fat content (43, 44).
Moreover, niacin and bOHB were also found to improve
the alcoholic or nonalcoholic fatty liver disease in rats
and mice (45–51). Using a combination of GPR109A-
deficient mice with HFD-induced model of obesity, we
provided further evidence that niacin treatment led to a
significant reduction in body weight and fat mass

GPR109A REGULATES ENERGY HOMEOSTASIS 4775

through GPR109A when fed a NCD or HFD. These
findings suggest that GPR109A has the potential to
control energy homeostasis, preventing weight gain.
Our results clearly revealed that niacin treatment re-
markably reduced liver weight in WT mice fed an HFD but
not in GPR109A-deficient mice. Ultrastructural examina-
tion validated that GPR109A-deficient mice had a striking
visible increase in the amount and size of lipid droplets in
the liver compared with the WT group, and niacin treat-
ment led to a significant reduction in the amount and size
of lipid droplets in the liver of WT mice when fed an HFD.
These findings suggest that GPR109A plays an important
role in the regulation of de novo lipogenesis in liver. The
liver has long been recognized as the major site of de novo
lipogenesis, responsible for the conversion of excess di-
etary carbohydrate into Tgs. De novo lipogenesis turnover
accounts for ;20% of lipid turnover within adipose tissue
(52); however, de novo lipogenesis in nonadipose tissues
leads to ectopic lipid accumulation responsible for meta-
bolic stress and insulin resistance (53). Although expres-
sion of GPR109A in the liver remains controversial, the
abundance of GPR109A mRNA detected was higher in the
liver in lean-type than in fat-type cows (54). GPR109A has
been found to be expressed in the murine liver, but the
basal expression is low (55). In the current study, we pro-
vided evidence to confirm that GPR109A mRNA and
protein were detected in the murine liver at a relatively
lower level; however, mice fed an HFD exhibited signifi-
cantly enhanced expression of GPR109A in the liver. In
addition, the expression of GPR109A in primary mice
hepatocytes was also detected. Further biochemical anal-
ysis of key enzymes involved in lipogenesis demonstrated
that niacin exhibited inhibitory effects on PDH (the gate-
keeping enzyme of pyruvate flux into acetyl-CoA) and
ACC1 (the key regulatory step of fatty acid synthesis) (56,
57). These findings were verified by real-time qRT-PCR for
genes encoding SREBP-1c (a key transcriptional activator
of lipogenic genes) and lipogenic enzymes (ACC1, FAS,
and SCD-1) (58–60). Niacin has been shown to directly
inhibit the activity of DGAT2, the key enzyme that cata-
lyzes the final step in Tg synthesis in the liver (30, 61).
However, our results strongly suggest that niacin ex-
erts its inhibitory effects on hepatic lipogenesis through
GPR109A.
It is noteworthy that mice treated with niacin
exhibited a remarkable reduction in body weight and fat
mass without affecting food intake, consistent with
previous observations that bOHB supplementation eli-
cited body weight loss but without a decrease in food
intake (43, 44). It suggests that energy expenditure is
likely to contribute to niacin-induced body weight loss.
Results derived from cold response experiments showed
that thermogenic function was enhanced in WT mice
treated with niacin but not in GPR109A knockout mice.
Real-time PCR analysis demonstrated that upon treat-
ment with niacin, the expression of thermogenic genes
UCP1 (a thermogenic protein exclusively expressed
in brown or beige adipocytes) (62, 63), PGC-1a (a
master regulator of mitochondrial biogenesis) (64), and
PRDM16 (a well-known BAT transcriptional regulator)
(65) were significantly increased in mice fed an HFD.
4776 Vol. 33 April 2019 The FASEB Journal x www.fasebj.org
Interestingly, we found that mitochondria in BAT of
GPR109A knockout mice were smaller in size with ab-
normal cristae structures compared to WT mice, and
niacin treatment led to significant improvement in mi-
tochondrial size and cristae structure of mice fed an
HFD. Additionally, niacin stimulated a beige fat ther-
mogenic program of the sWAT and eWAT. These data
strongly suggest that niacin treatment triggers BAT
and/or browning WAT thermogenic activity in mice
through GPR109A.
Importantly, GPR109A has been shown to functionally
express in colonic and intestinal epithelial cells (34, 35).
Niacin has been found to locally control glucose uptake at
the level of the small intestine through GPR109A (34). This
prompts us to examine whether or not GPR109A is in-
volved in the control of uptake of sterols and fatty acids in
the level of the small intestine. To our surprise, niacin
supplementation caused a significant increase of total
lipids, Tgs, cholesterols, and FFAs in the feces of WT mice
fed an HFD, but no change was observed in HFD-fed
GPR109A-deficient mice. Further gene expression anal-
ysis showed that genes encoding FATP4, I-FABP, and
Niemann-Pick C1-Like 1 (NPC1L1) exhibited a significant
reduction in expression in the intestinal tissues. It is estab-
lished that polytopic transmembrane protein NPC1L1 is
essential for intestinal absorption of cholesterol (66),
whereas proteins FATP4 and I-FABP play a role in dietary
fat absorption and fatty acid transport, respectively (67,
68). Taken together, these findings suggest that in addi-
tion to the control of glucose uptake, GPR109A expressed
in the small intestine is likely involved in the local regu-
lation of intestinal absorption of Tgs, cholesterols, and
FFAs.
Consistent with previous studies (43, 69–71), we also
observed that the HFD-induced obese mice treated with
niacin exhibited an obvious reduction in serum insu-
lin concentration and glucose tolerance compared to
the vehicle-treated mice. However, our data demon-
strated that treatment with niacin led to a significant im-
provement in insulin resistance in HFD-fed obese mice
(HOMA-IR). GPR109A has been shown to be functionally
expressed in both human and murine islet b-cells (72, 73).
Chen et al. (42) have shown that niacin exerts its inhibitory
actions on b-cell function via GPR109A-mediated signal-
ing but without affecting islet architecture. Nevertheless, a
recent study demonstrated that GPR109A expression was
down-regulated dramatically in islet b-cells of type 2 di-
abetic patients as well as in diabetes/diabetes mice, sug-
gesting that GPR109A signaling plays an important role in
the prevention of diabetes (72). Moreover, an early study
observed that a short-term niacin analog administration
resulted in an improved glucose tolerance in prediabetic or
diabetic patients (74). In addition, our data clearly dem-
onstrated that niacin treatment caused a significant re-
duction in hepatic de novo lipogenesis and ectopic lipid
accumulation in the liver, adipose tissue, and muscle of
mice fed an HFD, leading to a beneficial effect on insulin
resistance. The effects of niacin on glucose homeostasis
remain controversial, and the exact roles of GPR109A in
the regulation of insulin resistance now deserve thorough
evaluation.
YE ET AL.
 In summary, the data presented in this report have
demonstrated the novel role of niacin in the regulation of
energy homeostasis by fine-tuning of hepatic de novo
lipogenesis, energy expenditure, and intestinal fat ab-
sorption via GPR109A-mediated signaling. Our findings
provide a critical clue to the potential new therapeutic
strategies that could treat obesity, diabetes, and car-
diovascular disease. As the debate continues about
the favorable effects of niacin-GPR109A on the devel-
opment and progression of atherosclerosis and its
complications, it will be vital to further perform a more
comprehensive assessment of niacin-GPR109A in the
regulation of energy homeostasis in addition to anti-
inflammation.
ACKNOWLEDGMENTS
The authors thank Dr. Stefan Offermanns (Max Planck
Institute for Heart and Lung Research, Bad Nauheim, Germany)
for providing GPR109A KO (C57BL) breeders and critical
comments. Junying Li (Center of Analysis and Measurement,
Zhejiang University), Qiong Huang (Core Facilities, School of
Medicine, Zhejiang University), and Shenghui Hong (Laboratory
Animal Center, Zhejiang University) are acknowledged for their
technical assistance. This work was supported by grants from the
Ministry of Science and Technology of China (2012CB910402 to
N.Z.), the National Natural Science Foundation of China (81173106,
31200621, and 31771543 to N.Z. and Y.S.), the Zhejiang Provincial
Natural Science Foundation of China (LY15C050001 to N.Z.), and
the Siyuan Foundation (to N.Z. and Y.S.). The authors declare no
conflicts of interest.
AUTHOR CONTRIBUTIONS
Y. Shi and N. Zhou designed and supervised the research;
L. Ye, Z. Cao, X. Lai, and W. Wang performed in vivo
experiments; L. Ye, Z. Cao, X. Lai, W. Wang, Z. Guo,
L. Yan, and Y. Wang performed in vitro experiments; and
L. Ye, Y. Shi, and N. Zhou contributed to data interpretation
and wrote the manuscript.
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Received for publication September 13, 2018.
Accepted for publication December 3, 2018.
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