European Journal of Medicinal Chemistry 143 (2018) 114e122

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European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Design, syntheses and lipid accumulation inhibitory activities of novel

resveratrol mimics
Chanjuan Li 1, Bao Cheng 1, Sai Fang, Huihao Zhou, Qiong Gu**, Jun Xu*
School of Pharmaceutical Sciences, Sun Yat-Sen University, 132 East Circle Road at University City, Guangzhou 510006, China

a r t i c l e i n f o a b s t r a c t

Article history: Hispidine was initially discovered from Ficus Hispida for cardiovascular protection. In this paper, hispi-
Received 1 September 2017 dine derivatives, which contain a novel resveratrol-like scaffold, have been designed, synthesized, and
Received in revised form assayed as agents against lipid accumulations in 3T3-L1 pre-adipocytes. Six hispidine derivatives have
20 October 2017
the activity of reducing TG in 3T3-L1 adipocytes in dosage-dependent manner. The most active com-
Accepted 5 November 2017
Available online 7 November 2017
pound can reduce the lipid accumulation up to 78.4% at 10 mM qPCR and Western blotting results
demonstrate that the two most active compounds inhibit both lipodenesis and adipogenesis in 3T3-L1
cells through (1) increasing the phosphorylations of AMPK and ACC, promoting SIRT1 expression. These
Keywords:
Lipid accumulation
three proteins are key regulators for lipogenesis and energy metabolism. (2) Decreasing the expressions
AMPK pathway of PPARg, sREBP-1c, and FABP4, which are pivotal regulators for adipogenesis. Overall, this work proves
PPARg pathway that hispidine derivatives diminish the lipid accumulation in 3T3-L1 cell line by downregulating lipo-
SIRT1 genic and adipogenic pathways.
Adipocyte © 2017 Elsevier Masson SAS. All rights reserved.

1. Introduction resveratrol and hispidine-like scaffold, were designed and syn-

Hispidine, which is extracted from Ficus Hispida, and resveratrol
are structurally mimics (Fig. 1). Local people make Ficus Hispida
juice as a substitute for milk. The stem and leaf of the plant are also
used as natural therapeutic agents against hypoglycemia, diar-
rhoeal, and cardiovascular risk [1e4]. The mechanisms of actions of
hispidine derivatives are not well understood.
Resveratrol, the naturally occurring polyphenol found in red
wine and grape skin, was initially reported as a potent anti-tumor
agent [5]. From then on, resveratrol was further reported for
many pharmacological activities such as, cardio-protection, anti-
aging, anti-oxidation, anti-cancer, anti-inflammation [6e8], anti-
obesity and, anti-diabetes [9]. The mechanism of actions of
resveratrol are reported to be associated with Silent information
regulator 1 (SIRT1) activation [10], Adenosine 5-monophosphate-
activated protein kinase (AMPK) phosphorylation and, Acetyl CoA
carboxylase (ACC) phosphorylation [11].
Considering the structures and physiological benefits of both
resveratrol and hispidine, derivatives, which contain a novel
* Corresponding author.
** Corresponding author.
E-mail addresses: guqiong@mail.sysu.edu.cn (Q. Gu), junxu@biochemomes.com
(J. Xu).
1
Authors contribute equally to this article.
thesized in pursuing to find lipid accumulation inhibitory agents.
3T3-L1 cell line, the in vitro model recapitulating both pre-
adipocyte differentiation (adipogenesis) and lipid accumulation
(lipogenesis) of adipocyte differentiation, was adopted in this study
for screening [12]. The differentiation of 3T3-L1 pre-adipocytes is
mainly regulated by peroxisome proliferator-activated receptor g
(PPARg) pathway [13]. Increased PPARg expression can stimulate
sterol regulatory element-binding protein-1c (sREBP-1c) and fatty
acid-binding protein 4 (FABP4) expressions, which induce adipo-
cyte differentiation in the early stage [14e16]. The lipogenesis and
energy metabolism are mainly orchestrated by AMPK pathway [17].
Activated AMPK down-regulates ACC activity through phosphory-
lating ACC, resulting in inhibited lipogenesis and increased energy
metabolism in SIRT1 related manner [18].
To understand the mechanisms of actions of these hispidine
derivatives on 3T3-L1 pre-adipocytes differentiation, experiments
were further designed to study how hispidine derivatives diminish
lipid accumulation and, through which pathway.
2. Chemistry
The synthetic route for hispidine derivatives are shown in
Scheme 1 and Scheme 2. Commercially available 4-hydroxy-6-
methyl-2H-pyran-2-one was first reacted with K2CO3 and

https://doi.org/10.1016/j.ejmech.2017.11.017
0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.
 C. Li et al. / European Journal of Medicinal Chemistry 143 (2018) 114e122
Fig. 1. Structures of hispidine and resveratrol.
Scheme 1. Syntheses of 1e14. Reagents and conditions: (a) K2CO3, acetone, (CH3)2SO4, r.t., overnight, yield: 85%. (b) aldehydes Mg(OCH3)2, reflux, N2, overnight, yield: 32%e59%.
Scheme 2. Syntheses of 15e23. Reagents and conditions: (c) excessive liquid NH3,
EtOH, 120
C, in the sealed tube, 36 h, yield: 19%e51%.
(CH3)2SO4 at room temperature in acetone overnight to give com-
pound 2. Compound 2 was then reacted with different aldehydes in
the presence of Mg(OCH3)2 and was refluxed under N2 to give
compounds 3e14. Compounds 3e14 were reacted with excessive
liquid NH3 (28%e30%) in ethanol at 120
C in sealed tube to provide
target compounds 15e23.
3. Experiments
3.1. Chemistry
All chemicals and solvents were purchased from commercial
sources and were used without further purification. Column
chromatography (CC) was carried out on silica gel (200e300 mesh,
Qingdao Ocean Chemical Company, China), and thin-layer chro-
matography (TLC) analyses were carried out on silica gel GF254
glass plates. 1H NMR and 13C NMR were recorded in CDCl3, Acetone-
d6, or Methanol-d4 at room temperature on the Bruker spectrom-
eter. Electrospray ionizationemass spectrometry (ESI-MS) spectra
were recorded on an Agilent G1946D mass spectrometer.
3.1.1. 4-methoxy-6-methyl-2H-pyran-2-one (2)
A mixture of 4-hydroxy-6-methyl-2H-pyran-2-one (2.60 mmol)
and K2CO3 (2.60 mmol) and (CH3)2SO4 (2.60 mmol) in acetone
(15 mL) was stirred at room temperature overnight. The mixture
was filtered. The filtrate was concentrated in vacuum. The residue
was purified with silica gel chromatography using chloroform as
eluent to obtain target compound 2 as white solid. Yield: 86%; 1H
NMR (400 MHz, CDCl3) d 5.71 (s, 1H), 5.34 (s, 1H), 3.73 (s, 3H), 2.14
(s, 3H).
115
3.1.2. General procedure for the preparation of compounds 3-14
Compound 2 (2.40 mmol) and appropriate aldehydes
(2.40 mmol) in the presence of Mg(OCH3)2 (10 mL) was refluxed
under N2 overnight. After the reaction was completed as indicated
by TLC, the mixture was concentrated under reduced pressure. The
residue was purified with silica gel chromatography to obtain
target compounds 3e14.
3.1.2.1. (E)-4-methoxy-6-styryl-2H-pyran-2-one (3). Yield 56%; m.p.
147.2e149.3
C; white solid; 1H NMR (400 MHz, CDCl3) d 7.58 (d,
J ¼ 7.1 Hz, 2H), 7.44 (s, 1H), 7.37 (d, J ¼ 7.6 Hz, 1H), 7.31 (s, 2H), 6.59
(dd, J ¼ 16.0, 4.1 Hz, 1H), 5.95 (s, 1H), 5.50 (s, 1H), 3.82 (s, 3H).
3.1.2.2. (E)-4-methoxy-6-(2-methoxystyryl)-2H-pyran-2-one (4).
Yield: 57%; m.p. 134.0e136.1
C; white solid; 1H NMR (400 MHz,
CDCl3) d 7.79 (d, J ¼ 16.1 Hz, 1H), 7.49 (d, J ¼ 7.0 Hz, 1H), 7.30 (dd,
J ¼ 15.5, 8.3 Hz, 2H), 7.11e6.85 (m, 3H), 6.69 (t, J ¼ 14.5 Hz, 1H), 5.91
(s, 1H), 5.46 (s, 1H), 3.90 (s, 3H), 3.83 (s, 3H). 13C NMR (100 MHz,
CDCl3) d 171.2, 164.2, 159.4, 158.0, 131.4, 130.5, 128.2, 124.3, 120.7,
119.4, 111.1, 100.9, 88.6, 55.8, 55.4. LCMS (ESI) m/z: 258.1 [M þ H]þ.
3.1.2.3. (E)-4-methoxy-6-(3-methoxystyryl)-2H-pyran-2-one (5).
Yield: 54%; white solid; 1H NMR (400 MHz, CDCl3) d 7.46e7.33 (m,
1H), 7.19 (dd, J ¼ 11.1, 8.5 Hz, 1H), 7.03 (d, J ¼ 7.6 Hz, 1H), 6.93 (d,
J ¼ 10.9 Hz, 1H), 6.82 (dd, J ¼ 8.2, 2.2 Hz, 1H), 6.49 (d, J ¼ 15.9 Hz,
1H), 5.87 (d, J ¼ 1.8 Hz, 1H), 5.42 (d, J ¼ 1.8 Hz, 1H), 3.76 (s, 3H), 3.75
(s, 3H). 13C NMR (100 MHz, CDCl3) d 171.0, 163.9, 159.9, 158.5, 136.6,
135.7, 129.9, 120.1, 118.9, 115.2, 112.5, 101.4, 88.9, 55.9, 55.3. LCMS
(ESI) m/z: 258.1 [M þ H]þ.
3.1.2.4. (E)-4-methoxy-6-(4-methoxystyryl)-2H-pyran-2-one (6).
Yield 59%; m.p. 152.5e155.3
C; white solid; 1H NMR (400 MHz,
CDCl3) d 7.37 (d, J ¼ 5.9 Hz, 1H), 7.32 (s, 2H), 6.82 (d, J ¼ 8.2 Hz, 2H),
6.37 (d, J ¼ 15.9 Hz, 1H), 5.81 (s, 1H), 5.39 (s, 1H), 3.75 (s, 3H), 3.74 (s,
3H). 13C NMR (100 MHz, CDCl3) d 171.2, 164.1, 160.7, 159.1, 135.4,
135.4, 128.9, 128.0, 116.4, 114.4, 114.4, 100.4, 88.3, 55.8, 55.3. LCMS
(ESI) m/z: 258.1 [M þ H]þ.
3.1.2.5. (E)-6-(2-chlorostyryl)-4-methoxy-2H-pyran-2-one (7).
Yield 43%; m.p. 115.6e116.8
C; light yellow solid; 1H NMR
(400 MHz, CDCl3) d 7.76 (d, J ¼ 16.0 Hz, 1H), 7.51 (dd, J ¼ 6.1, 3.4 Hz,
1H), 7.33 (dd, J ¼ 5.8, 3.5 Hz, 1H), 7.20 (d, J ¼ 2.7 Hz, 1H), 7.18 (d,
J ¼ 2.9 Hz, 1H), 6.51 (d, J ¼ 16.0 Hz, 1H), 5.92 (d, J ¼ 2.0 Hz,1H), 5.44
(d, J ¼ 2.0 Hz, 1H) 3.78e3.54 (m, 3H). 13C NMR (100 MHz, CDCl3)
d 169.8, 162.7, 157.2, 133.5, 132.5, 130.6, 129.1, 126.0, 120.4, 101.0,
88.3, 54.9. LCMS (ESI) m/z: 263.1 [M þ H]þ.
3.1.2.6. (E)-6-(4-chlorostyryl)-4-methoxy-2H-pyran-2-one (8).
Yield 47%; m.p. 139.0e141.2
C; light yellow solid; 1H NMR
(400 MHz, CDCl3) d 7.37 (d, J ¼ 8.9 Hz, 1H), 7.34 (s, 2H), 7.27 (d,
J ¼ 8.4 Hz, 2H), 6.47 (d, J ¼ 16.0 Hz, 1H), 5.87 (d, J ¼ 1.8 Hz, 1H), 5.42
(d, J ¼ 1.8 Hz, 1H), 3.82e3.63 (m, 3H). 13C NMR (100 MHz, CDCl3)
116 C. Li et al. / European Journal of Medicinal Chemistry 143 (2018) 114e122
d 170.9, 163.7, 158.2, 135.2, 134.3, 133.7, 129.1, 129.1, 128.5, 128.5,
119.1, 101.6, 89.0, 55.9. LCMS (ESI) m/z: 263.1 [M þ H]þ.
3.1.2.7. (E)-6-(4-(dimethylamino)styryl)-4-methoxy-2H-pyran-2-one
(9). Yield 32%; m.p. 145.0e146.7
C; yellow solid; 1H NMR (400 MHz,
CDCl3) d 7.37 (d, J ¼ 2.9 Hz, 1H), 7.31 (s, 2H), 6.62 (d, J ¼ 4.4 Hz, 2H),
6.31 (dd, J ¼ 15.8, 4.2 Hz, 1H), 5.77 (s, 1H), 5.36 (s, 1H), 3.75 (d,
J ¼ 4.4 Hz, 3H), 3.15e2.79 (m, 6H). 13C NMR (100 MHz, CDCl3) d 171.4,
164.4, 159.9, 151.2, 136.3, 128.9, 128.9, 123.2, 113.6, 112.0, 112.0, 99.2,
87.6, 55.7, 40.1, 40.1. LCMS (ESI) m/z: 272.1 [M þ H]þ.
3.1.2.8. (E)-6-(2-cyclohexylvinyl)-4-methoxy-2H-pyran-2-one (10).
Yield 57%; white solid; 1H NMR (400 MHz, CDCl3) d 6.57 (dd,
J ¼ 15.7, 7.0 Hz, 1H), 5.82 (d, J ¼ 15.7 Hz, 1H), 5.70 (d, J ¼ 2.0 Hz, 1H),
5.35 (d, J ¼ 2.0 Hz, 1H), 3.74 (s, 3H), 2.04 (dd, J ¼ 10.9, 7.0 Hz, 1H),
1.67 (d, J ¼ 10.8 Hz, 4H), 1.59 (d, J ¼ 12.7 Hz, 1H), 1.22 (m, 3H), 1.07
(m, 3H). 13C NMR (100 MHz, CDCl3) d 171.2, 164.0, 158.9, 145.0, 118.9,
99.5, 88.0, 55.7, 40.7, 32.0, 25.7. LCMS (ESI) m/z: 235.1 [M þ H]þ.
3.1.2.9. 4-methoxy-6-((1E,3E)-4-phenylbuta-1,3-dien-1-yl)-2H-py-
ran-2-one (11). Yield: 43%; light yellow solid; 1H NMR (400 MHz,
CDCl3) d 7.38 (d, J ¼ 7.2 Hz, 2H), 7.27 (d, J ¼ 7.7 Hz, 3H), 7.25e7.15 (m,
3H), 6.86e6.71 (m, 2H), 6.08 (d, J ¼ 15.1 Hz, 1H), 5.80 (s, 1H), 5.40 (s,
1H), 3.74 (d, J ¼ 2.7 Hz, 3H). 13C NMR (100 MHz, CDCl3) d 170.0,
163.0, 157.6, 137.2, 135.4, 135.2, 127.7, 127.7, 127.6, 126.0, 125.9, 125.9,
121.0, 99.9, 87.7, 54.8. LCMS (ESI) m/z: 255.1 [M þ H]þ.
3.1.2.10. (E)-4-methoxy-6-(2-(5-methylfuran-2-yl)vinyl)-2H-pyran-
2-one (12). Yield 43%; m.p. 148.5e150.4
C; light yellow solid; 1H
NMR (400 MHz, CDCl3) d 7.11 (d, J ¼ 15.6 Hz, 1H), 6.33 (dd, J ¼ 9.2,
6.2 Hz, 2H), 5.99 (d, J ¼ 2.7 Hz, 1H), 5.80 (s, 1H), 5.38 (d, J ¼ 1.9 Hz,
1H), 3.73 (d, J ¼ 10.1 Hz, 3H), 2.27 (s, 3H). 13C NMR (100 MHz, CDCl3)
d 171.1, 164.0, 158.9, 154.6, 150.2, 122.7, 114.9, 108.8, 100.5, 88.2, 55.8,
13.8. LCMS (ESI) m/z: 233.2 [M þ H]þ.
3.1.2.11. (E)-6-(2-(furan-2-yl)vinyl)-4-methoxy-2H-pyran-2-one
(13). Yield 41%; m.p. 143.0e144.7
C; light yellow solid; 1 HNMR
(400 MHz, CDCl3) d 7.39 (s, 1H), 7.21 (t, J ¼ 4.7 Hz, 1H), 7.17 (d,
J ¼ 4.8 Hz, 1H), 6.50 (d, J ¼ 15.7 Hz, 2H), 6.40 (d, J ¼ 3.1 Hz, 1H), 5.86
(s, 1H), 5.41 (d, J ¼ 2.9 Hz, 1H), 3.76 (s, 3H). 13C NMR (100 MHz,
CDCl3) d 170.0, 162.9, 157.4, 150.5, 142.9, 121.5, 115.5, 112.3, 111.3,
100.2, 87.6, 54.9. LCMS (ESI) m/z: 219.1 [M þ H]þ.
3.1.2.12. (E)-4-methoxy-6-(2-(thiophen-2-yl)vinyl)-2H-pyran-2-one
(14). Yield 37%; m.p. 166.1e168.2
C; light yellow solid; 1H NMR
(400 MHz, CDCl3) d 7.54 (d, J ¼ 15.6 Hz, 1H), 7.24 (d, J ¼ 4.9 Hz, 1H),
7.11 (d, J ¼ 3.0 Hz, 1H), 6.96 (t, J ¼ 3.8 Hz, 1H), 6.30 (d, J ¼ 15.6 Hz,
1H), 5.83 (s, 1H), 5.40 (s, 1H), 3.74 (s, 3H). 13C NMR (100 MHz, CDCl3)
d 171.0, 163.8, 158.3, 140.6, 129.5, 128.6, 128.1, 127.1, 117.7, 100.9, 88.6,
55.9. LCMS (ESI) m/z: 235.2 [M þ H]þ.
3.1.3. General procedure for the preparation of compounds 15-23
Compounds 3e14 (1.2 mmol) were reacted with excessive liquid
NH3 (28%), stirred in the ethanol at 120
C, in sealed tubes, for 36 h.
Each mixture was concentrated in vacuum. The residue was puri-
fied with silica gel chromatography using chloroform/methanol (V:
V ¼ 20:1) as eluent to obtain target compounds 15e23.
3.1.3.1. (E)-4-hydroxy-6-styrylpyridin-2(1H)-one (15). Yield: 28%;
light yellow solid; 1H NMR (400 MHz, Methanol-d4) d 7.57 (d,
J ¼ 7.1 Hz, 2H), 7.42 (s, 1H), 7.38 (dd, J ¼ 9.6, 4.8 Hz, 3H), 7.33 (dd,
J ¼ 5.2, 1.8 Hz, 1H), 6.79 (d, J ¼ 16.0 Hz, 1H), 6.09 (d, J ¼ 1.9 Hz, 1H),
5.14 (d, J ¼ 1.9 Hz, 1H). 13C NMR (100 MHz, Methanol-d4) d 167.8,
162.6, 160.0, 136.9, 136.0, 130.3, 129.9, 129.9, 128.5, 128.5, 120.5,
101.9, 83.5. LCMS (ESI) m/z: 214.1 [M þ H]þ.
3.1.3.2. (E)-4-hydroxy-6-(2-methoxystyryl)pyridin-2(1H)-one (16).
Yield: 27%; m.p. 248.0e249.7
C; light yellow solid; 1H NMR
(400 MHz, Methanol-d4) d 7.57 (d, J ¼ 7.3 Hz, 2H), 7.31 (t, J ¼ 7.4 Hz,
1H), 7.01 (d, J ¼ 8.3 Hz, 1H), 6.96 (m, 1H), 6.86 (d, J ¼ 16.6 Hz, 1H),
6.10 (s, 1H), 5.48 (s, 1H), 3.90 (s, 3H). 13C NMR (100 MHz, Methanol-
d4) d 165.4, 164.2, 159.2, 157.2, 143.9, 129.6, 127.9, 126.6, 124.0, 120.3,
120.0, 110.5, 97.7, 54.2. LCMS (ESI) m/z: 244.2 [M þ H]þ.
3.1.3.3. (E)-4-hydroxy-6-(3-methoxystyryl)pyridin-2(1H)-one (17).
Yield: 23%; m.p. 168.4e171.1
C; light yellow solid; 1H NMR
(400 MHz, Acetone-d6) d 7.33 (s, 1H), 7.30 (d, J ¼ 7.3 Hz, 1H), 7.21 (d,
J ¼ 6.7 Hz, 2H), 6.91 (d, J ¼ 7.4 Hz, 1H), 6.86 (d, J ¼ 16.0 Hz, 1H), 6.28
(s, 1H), 6.08 (s, 1H), 5.08 (s, 1H), 3.84 (s, 3H). 13C NMR (100 MHz,
Acetone-d6) d 163.4, 161.1, 160.0, 159.3, 138.1, 134.4, 130.6, 121.4,
120.7, 115.7, 113.1, 101.6, 84.4, 55.5. LCMS (ESI) m/z: 244.1 [M þ
H]þ.
3.1.3.4. (E)-4-hydroxy-6-(4-methoxystyryl)pyridin-2(1H)-one (18).
Yield: 31%; m.p. 144.6e146.5
C; light yellow solid; 1H NMR
(400 MHz, Acetone-d6) d 7.58 (d, J ¼ 7.9 Hz, 2H), 7.29 (d, J ¼ 15.9 Hz,
1H), 6.96 (d, J ¼ 7.7 Hz, 2H), 6.69 (d, J ¼ 15.9 Hz, 1H), 6.24 (s, 2H),
6.02 (s, 1H), 5.05 (s, 1H), 3.83 (s, 3H). 13C NMR (100 MHz, Acetone-
d6) d 163.5, 161.5, 160.2, 159.7, 134.1, 129.7, 129.7, 129.3, 118.8, 115.1,
115.1, 100.6, 84.0, 55.6. LCMS (ESI) m/z: 244.1 [M þ H]þ.
3.1.3.5. (E)-6-(2-chlorostyryl)-4-hydroxypyridin-2(1H)-one (19).
Yield: 19%; m.p. 213.1e215.4
C; light yellow solid; 1H NMR
(400 MHz, Acetone-d6) d 7.84 (s, 1H), 7.70 (d, J ¼ 5.9 Hz, 1H), 7.48 (d,
J ¼ 2.9 Hz, 1H), 7.37 (d, J ¼ 2.6 Hz, 2H), 6.91 (d, J ¼ 7.7 Hz, 1H), 6.33 (s,
2H), 6.16 (s, 1H), 5.11 (d, J ¼ 5.4 Hz, 1H). 13C NMR (100 MHz,
Acetone-d6) d 163.2, 159.9, 158.7, 134.5, 131.0, 130.8, 129.4, 128.3,
128.0, 124.0, 102.6, 84.7. LCMS (ESI) m/z: 248.1 [M þ H]þ.
3.1.3.6. (E)-6-(2-cyclohexylvinyl)-4-hydroxypyridin-2(1H)-one (20).
Yield: 51%; m.p. 173.2e175.3
C; light yellow solid; 1H NMR
(400 MHz, CDCl3) d 6.60 (dd, J ¼ 15.7, 7.0 Hz, 1H), 5.83 (d, J ¼ 15.7 Hz,
1H), 5.61 (s, 1H), 5.11 (s, 1H), 4.94 (s, 2H), 2.08 (d, J ¼ 7.3 Hz, 1H), 1.71
(d, J ¼ 10.2 Hz, 4H), 1.23 (t, J ¼ 16.8 Hz, 3H), 1.11 (t, 3H). 13C NMR
(100 MHz, CDCl3) d 164.7, 159.4, 158.7, 144.8, 119.3, 98.4, 84.9, 40.8,
32.1, 32.1, 25.9, 25.7, 25.7. LCMS (ESI) m/z: 220.1 [M þ H]þ.
3.1.3.7. (E)-4-hydroxy-6-(2-(5-methylfuran-2-yl)vinyl)pyridin-
2(1H)-one (21). Yield: 34%; m.p. 181.7e183.5
C; yellow solid; 1H
NMR (400 MHz, Acetone-d6) d 6.78 (d, J ¼ 15.7 Hz, 1H), 6.30 (d,
J ¼ 2.7 Hz, 1H), 6.18 (d, J ¼ 15.7 Hz, 1H), 5.99 (s, 2H), 5.88 (s, 1H), 5.76
(s, 1H), 4.77 (s, 1H), 2.61 (s, 3H). 13C NMR (100 MHz, Acetone-d6)
d 163.4, 160.2, 159.3, 155.0, 151.4, 121.8, 117.0, 115.0, 109.5, 100.8,
84.0, 13.7. LCMS (ESI) m/z: 218.1 [M þ H]þ.
3.1.3.8. (E)-6-(2-(furan-2-yl)vinyl)-4-hydroxypyridin-2(1H)-one
(22). Yield: 36%; m.p. 174.5e176.9
C; yellow solid; 1H NMR
(400 MHz, Acetone-d6) d 7.62 (s, 1H), 7.12 (d, J ¼ 15.8 Hz, 1H), 6.72 (t,
J ¼ 11.6 Hz, 1H), 6.56 (d, J ¼ 15.7 Hz, 2H), 6.28 (s, 2H), 6.07 (d,
J ¼ 1.2 Hz, 1H), 5.06 (d, J ¼ 1.7 Hz, 1H). 13C NMR (100 MHz, Acetone-
d6) d 163.4, 160.1, 158.9, 152.8, 144.9, 121.7, 118.7, 113.4, 113.1, 101.4,
84.3. LCMS (ESI) m/z: 204.1 [M þ H]þ.
3.1.3.9. (E)-4-hydroxy-6-(2-(thiophen-2-yl)vinyl)pyridin-2(1H)-one
(23). Yield: 32%; m.p. 201.7e203.8
C; yellow solid; 1H NMR
(400 MHz, Methanol-d4) d 7.52 (d, J ¼ 15.7 Hz, 1H), 7.44 (d,
J ¼ 5.0 Hz, 1H), 7.27 (d, J ¼ 3.2 Hz, 1H), 7.06 (dd, J ¼ 5.0, 3.7 Hz, 1H),
6.53 (d, J ¼ 15.7 Hz, 1H), 6.04 (d, J ¼ 1.7 Hz, 1H), 5.11 (d, J ¼ 1.9 Hz,
1H). 13C NMR (100 MHz, Methanol-d4) d 167.8, 162.5, 159.7, 142.0,
130.5, 129.1, 128.9, 128.3, 119.4, 101.6, 83.4. LCMS (ESI) m/z: 220.1
[M þ H]þ.
 C. Li et al. / European Journal of Medicinal Chemistry 143 (2018) 114e122
3.2. Cell culture and adipocyte differentiation
Cells (HEK293T, 3T3-L1) were cultured in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% fetal bovine serum
(FBS), 100 U/mL penicillin and 100 mg/mL streptomycin at 37
C in 5%
CO2. Adipocyte differentiation was carried out on 3T3-L1 in accor-
dance with Ji-Ming Ye [19]. Briefly, 2 days after full confluence (day
0), the medium was changed to differentiation medium (DMEM
supplemented with 10% FBS, 100 U/mL penicillin, 100 mg/mL strep-
tomycin, 2 mg/mL insulin, 100 ng/mL dexamethasone, 0.5 mM 3-
isobutyl-1-methylxanthine (IBMX) and 10 ng/mL biotin) for 3 days.
Then (day 3), differentiation medium was switched to post-
differentiation medium (DMEM supplemented with 10% FBS, 100
U/mL penicillin, 100 mg/mL streptomycin, 2 mg/mL insulin) for
additional 3 days. At the end (day 6), cells were used for further
analysis. Tested compounds were dissolved in dimethyl sulfoxide
(DMSO) and supplemented at indicated concentrations throughout
the course of differentiation. Berberine (BBR) was used as positive
control. 0.1% DMSO was used as vehicle control.
3.3. Triglycerides (TGs) assay
3T3-L1 cells were collected at the end of differentiation and
washed twice with ice-cold PBS buffer (0.2 M NaCl, 10 mM
Na2HPO4, 3 mM KCl, 2 mM KH2PO4, pH 7.4). Then the cells were
lysed, the levels of intracellular triglycerides were determined us-
ing commercial TG assay kit (Jiancheng Bioengineering Institution,
Nanjing, China) according to the manufacture's protocol. Results
Fig. 2. (A) TG assay results for 11 active hispidine derivatives. 3T3-L1 pre-adipocytes were treated with 10 mM of 21 compounds on days 0 and 3, for 3 days during the differ-
entiation. The levels of intracellular TG were determined using commercial TG assay kit. Data are represented as mean ± SD of three independent experiments (*p < 0.05 vs vehicle,
**p < 0.01 vs vehicle). (B) The structures of compounds with best TG-lowing effect.
117
were presented as the relative TG content compared to the positive
control cells.
3.4. Oil red O (ORO) staining
Cells were fixed with 10% (v/v) formaldehyde in PBS for 1 h at
room temperate and stained with freshly prepared ORO working
solution for 30 min followed by three washes with water. Plates
were pictured using a microscope (Nikon, Tokyo, Japan) equipped
with a CCD digital camera (Nikon, Tokyo, Japan). Constant
condenser and light intensity settings were used throughout the
imaging process.
3.5. Cytotoxicity assay
HEK293T cells were seeded at the density of 5000 per well into
96-well plates to grow overnight. Then the cells were treated with
various concentrations of compounds for 24 h. 0.5% DMSO as vehicle
control. MTT (0.05 mg) was added to each well and incubated for 4 h,
after which 100 mL DMSO was added to each well. The absorbance of
wells was recorded at 492 nm after 20 min of shaking. The inhibition
percentage was calculated as (Aexperiment eAblank)/(Acontrol e Ablank).
3.6. Real-time quantitative PCR
Total RNA was isolated using RNAiso plus (TaKaRa, Dalian, China)
according to the manufacturer's protocol. Total RNA (1 mg) was
converted to cDNA using ReverTra Ace qPCR RT Master Mix (Toyobo,
118 C. Li et al. / European Journal of Medicinal Chemistry 143 (2018) 114e122

Osaka, Japan) according to the manufacturer's protocol. cDNA and
SYBR Green was used to amplify specific target genes by Real-time
PCR Master Mix (Toyobo, Osaka, Japan). The following PCR primer
sequences were used: b-actin, sense 50 -TGGAATCCTGTGGCATC-
CATGAAA-30 and antisense 50 -TAAAACGCAGCTCAGTAACAGTCC-3’;
AMPK, sense 50 -AAGCCGACCCAATGACATCA-30 and antisense 50 -
CTTCCTTCGTACACGCAAAT-3’; ACC, sense 50 -AGGATTTGCTGTTTCT-
CAGAGCTT-30 and antisense 50 -CAGGATCTACCCAGGCCACAT-3’; SIR
T1, sense 50 -TTGTGAAGCTGTTCGTGGAG-30 and antisense 50 -GCGT
GGAGGTTTTTCAGTA-3’; PPARg, sense 50 -TGCTGTTATGGGTGAAACT
CTG-30 antisense 50 -GAAATCAACTGTGGTAAAGGGC-3’; sREBP-1c,
sense 50 -CAGCTCAGAGCCGTGGTGA-3'and antisense 50 -TGTGTGCAC
TTCGTAGGGTC-3’; FABP4, sense 50 -GTCACCATCCGGTCAGAGAGTAC-
3'and antisense50 -TCGTCTGCGGTGATTTCATC-3’.
3.7. Western blotting
Cell lysates were prepared in lysis buffer containing 1% (v/v)
NP-40, 0.25% (w/v) deoxycholate, 0.15% (w/v) SDS, protease in-
hibitor (Beyotime, Shanghai, China), and phosphatase inhibitor
cocktail (Beyotime, Shanghai, China). Protein concentrations were
quantified using Pierce Rapid Gold BCA Protein Assay Kit (Ther-
moFisher scientific, Waltham, USA). Protein samples were dena-
ture in SDS sample buffer and then subjected to SDS-PAGE and
blotted onto polyvinylidene difluoride (Millipore, Darmstadt,
Germany) membranes. The membranes were then blocked with
5% (w/v) skim milk in TBST (Tris-buffered saline containing 0.1% (v/
v) Tween 20) for 2 h and then incubated with primary antibodies
overnight at 4
C. After three times washing in TBST, the mem-
branes were then incubated with anti-mouse or anti-rabbit IgG
secondary antibodies (conjugated to Horseradish Peroxidase) at
room temperature for 2 h. Immunoreactive signals were detected
using enhanced chemiluminescence reagents with Chem-
iluminescence imager (Tanon 5200, Shanghai, China). Densitom-
etry analysis was performed using Quantity One Software (Bio-Rad
Laboratories, USA) and quantified to the loading control a-Tublin.
Antibodies used including phospho (Thr172)- and total-AMPKa
(Cell Signaling, Danvers, USA), phospho (Ser79)- and total- ACC
(Cell Signaling, Danvers, USA), SIRT1 (Cell Signaling, Danvers,
USA), PPARg (Sigma, St. Louis, USA), sREBP-1c (Santa Cruz,
Dallas, USA), FABP4 (Abcam, Cambridge, USA), a-Tublin (Sigma,
St. Louis, USA).

Fig. 3. (A) Hispidine derivatives dose-dependently reduce lipid accumulation in 3T3-L1 cells. (B) Compounds 20 and 21 are superior to BBR and resveratrol in terms of reducing TG
accumulation. Pictures were taken on day-6 with 10 magnification. UN: undifferentiated cells. Vehicle: differentiated cells without compounds. 293T cells received treatment of
compounds for 24 h to test their cytotoxicity. Data are represented as mean ± SD of three independent experiments (*p < 0.05 vs vehicle, **p < 0.01 vs vehicle).
 C. Li et al. / European Journal of Medicinal Chemistry 143 (2018) 114e122 119

4. Results
4.1. Identifying hispidine derivatives as lipid-lowering agents
through 3T3-L1 cell-based assays
The 3T3-L1 cells were treated with differentiation cocktail
together with 21 compounds at the concentration of 10 mM. 11
compounds exhibited the ability of the down-regulating TG accu-
mulations in 3T3-L1 cells (Fig. 2A). Among the 11 active com-
pounds, 6 compounds significantly exhibited greater activities than
BBR (a well-known positive control agent for 3T3-L1 assay). Com-
pound 20 was the best TG accumulation reducer, which reduced
78.4% TG accumulation. The chemical structures of the 6 most
active compounds are depicted in Fig. 2B.
4.2. Measuring the inhibitory efficacies of lipid accumulation
in 3T3-L1 cells
To further evaluate the inhibitory efficacies of these compounds
on lipid accumulation, the 3T3-L1 pre-adipocytes were treated
with differentiation cocktail together with 6 active compounds
using different concentrations (10, 3.3, 1.1, and 0.37 mM) for 6 days.
All 6 active compounds significantly reduced the intracellular lipid
accumulation in a dosage-dependent manner (Fig. 3A). Compounds
20 and 21 are superior to others in terms of reducing TG accumu-
lation. ORO staining was applied for visual inspecting the quantity
of lipid, which was stained in red. The agents tested in 3T3-L1 as-
says were BBR, resveratrol, and compounds 20 and 21. In Fig. 3B,
there is almost no lipid (red spots) can be observed in the cells
treated with BBR, resveratrol, compound 20 or 21 at 10 mM. There is
significantly reduced lipid in the cells treated with those agents at
1 mM. Previous cytotoxicity tests demonstrated that 1 or 10 mM are
far away below the CC50 of these agents. Specifically, compound 21
is not toxic to 3T3-L1 cells at up to 50 mM (Fig. 3B).
4.3. Hispidine derivatives suppressed adipogenic transcript factors
expressions
PPARg and sREBP-1c are the main transcription factors
involved in adipocyte differentiation. FABP4 is a marker of
adipocyte differentiation. To elucidate the mechanism of actions
of compounds 20 and 21 in the differentiation of 3T3-L1 pre-
adipocyte, we investigated compounds 20 and 21 effects on the
mRNA and protein expressions of adipogenic genes PPARg,
sREBP-1c, and downstream transcriptional factor FABP4 through
qPCR and Western blotting analyses. The experiments demon-
strate that when treated with differentiation cocktail, PPARg,
sREBP-1c and FABP4 mRNA were significantly up-regulated. After
treated with compound 20 or 21 for 3 days, the mRNA expres-
sions of PPARg, sREBP-1c and FABP4 were significantly sup-
pressed comparing with that of vehicle (Fig. 4CeE). Consistently,
PPARg, sREBP-1c and FABP4 protein expression levels were
increased when treated with differentiation cocktail. After treated
with compound 21, PPARg, sREBP-1c and FABP4 protein expres-
sion levels were significantly attenuated (Fig. 4A and B). These
results indicate that compounds 20 and 21 can suppress the

Fig. 4. Hispidine derivatives blocked PPARg-FABP4 pathway. 3T3-L1 pre-adipocytes were treated with differentiation cocktail with or without compound 20 or 21 at day 0 for 3
days. Then the cells were harvested for qPCR and Western blotting analyses. Un: undifferentiated cells. Dif. cocktail: differentiation cocktail. (A) PPARg, sREBP-1c, and FABP4 protein
levels. (B) Quantitative PPARg, sREBP-1c, and FABP4 protein levels. (C)e(E) mRNA levels for PPARg (C), sREBP-1c (D), and FABP4 (E). Data are represented as mean ± SD of 3 in-
dependent experiments (*p < 0.05 vs vehicle, **p < 0.01 vs vehicle).
120 C. Li et al. / European Journal of Medicinal Chemistry 143 (2018) 114e122

Fig. 5. Hispidine derivatives suppress lipogenesis by up-regulating AMPK pathway. 3T3-L1 pre-adipocytes were treated with differentiation cocktail with or without compounds 20
or 21 at day 0 for 3 days. Then the cells were harvested for qPCR and Western blotting analysis. Un: undifferentiated cells. Dif. cocktail: differentiation cocktail. (A) Protein expression
levels of the phosphorylated AMPKa, total AMPKa, phosphorylated ACC, total ACC, and SIRT1. (B) Quantitative AMPKa, total AMPKa, phosphorylated ACC, total ACC, and SIRT1
protein levels. (C) The mRNA levels for AMPK, ACC, and SIRT1. Data are represented as mean ± SD of 3 independent experiments (*p < 0.05 vs vehicle, **p < 0.01 vs vehicle).

differentiation of 3T3-L1 pre-adipocytes by inhibiting the mRNA
and protein expressions of adipogenic genes.
4.4. Hispidine derivatives suppress lipogenesis by up-regulating
AMPK pathway
AMPK activation can inhibit lipogenesis and induce energy
metabolism. To prove compounds 20 and 21 up-regulating AMPK
pathway, qPCR and Western blotting analyses were performed on
3T3-L1 cells treated with compound 20 or 21. In Fig. 5, the mRNA
and protein levels of t-AMPK were up-regulated when treated with
differentiation cocktail. Treatment with compound 20 or 21 caused
increased AMPKa phosphorylation. Consequently, ACC (the
substrate of AMPK) phosphorylation was increased. Specifically,
compounds 20 and 21 increased the AMPKa phosphorylation by
3.45 and 3.10 fold, and that of ACC by 2.98 and 4.12 fold. In short, the
mRNA expression was not increased, the phosphorylation was
increased instead.
AMPKa activation has many therapeutic benefits because of its
enhancing oxidative metabolism and mitochondrial biogenesis.
This process relies on SIRT1 [10], which is required for AMPKa
activation during mitochondrial biogenesis as well as energy
metabolism [17]. Here, the mRNA and SIRT1 protein expressions
were also studied. Results indicate that SIRT1 mRNA and protein
expression levels in differentiated 3T3-L1 cells are significantly
higher than pre-adipocytes. After the 3T3-L1 cells were treated

Table 1
Correlations between molecular properties and activity.

Descriptor code Description R2 Significance

ASA Water accessible surface area of hydrophobic atoms 0.444 0.001

ASA_H Total hydrophobic surface area 0.444 0.001
vdw_area Van der Waals (VDW) surface area 0.380 0.003
a_acc Number of H-bond acceptor atoms 0.379 0.003
vsa_acc VDW acceptor surface area 0.379 0.003
vdw_vol VDW volume 0.361 0.004
vsa_hyd VDW hydrophobic surface area 0.341 0.005
a_hyd Number of hydrophobic atoms 0.219 0.033
E Potential energy 0.204 0.400
logP Log octanol/water partition coefficient 0.128
logS Log solubility in water 0.120
 C. Li et al. / European Journal of Medicinal Chemistry 143 (2018) 114e122
Fig. 6. Pathway of compounds 20 and 21 blocking adipogenesis and lipogenesis.
with compounds 20 or 21, SIRT1 mRNA and protein expression
levels are even higher. This means that SIRT1 involves in AMPK
activation during pre-adipocytes differentiation (Fig. 5A, B).
5. Discussion
In this study, hispidine derivatives, which contain a novel
resveratrol-like scaffold, have been designed, synthesized, and
assayed as agents against lipid accumulations in 3T3-L1 pre-
adipocytes. The structure-activity relationship can be drawn
through 3D and 2D molecular property descriptors calculation using
Molecular Operating Environment (MOE). According to R2 and sta-
tistical significance, 8 molecular property descriptors (descriptors
concerning surface area, conformation dependent charge, pharma-
cophore feature, and physical properties) are found to be signifi-
cantly related to their TG inhibitory activities. Potential energy, logP,
and logS are also considered related (Table 1). Increased hydropho-
bic surface, H-bond acceptor, potential energy, logP, and decreased
logS reduces TG inhibitory activity (Figs. S44AeS44K, right), indi-
cating that these hispidine derivatives should keep a balance in
hydrophilicity and lipophilicity (logP ~2). Compared to hydroxyl
group, hispidine derivatives with a methoxyl group at C-4 position
can significantly increase hydrophobic surface, H-bond acceptor,
VDW acceptor surface, and logP, and decrease logS (Fig. S44, left).
Consequently, this reduced TG inhibitory activity.
The differentiation of pre-adipocytes into mature adipocytes
consists of adipogenesis and lipogenesis [12,20], and compounds
20 and 21 can block both processes. Based on the results, we can
construct the pathway of compounds 20 and 21 blocking adi-
pogenesis and lipogenesis. As shown in Fig. 6, we conclude (1)
the hispidine derivatives (compounds 20 and 21) increase AMPK
phosphorylation and SIRT1 expression, down-regulate ACC ac-
tivity through phosphorylating ACC. Consequently, the com-
pounds inhibit lipogenesis and increase energy metabolism in
3T3-L1 pre-adipocytes differentiation process. (2) The hispidine
derivatives inhibit PPARg, sREBP-1c, and FABP4 expressions. The
adipogenesis is mainly regulated by PPARg pathway [13]. sREBP-
1c is a downstream factor of PPARg, and regulates the expression
121
of lipogenic proteins like ACC. FABP4 is another downstream
factor of PPARg and a biomarker of adipocyte differentiation [21].
Decreased PPARg, sREBP-1c, and FABP4 expressions result in
inhibited adipogenesis in 3T3-L1 pre-adipocyte differentiation
process.
In conclusion, the hispidine derivatives can block both adipo-
genesis and lipogenesis through increasing the phosphorylation of
AMPK and ACC, up-regulating SIRT1, down-regulating PPARg,
sREBP-1c, and FABP4. Hispidine derivatives can be promising leads
for the therapeutic solutions against obesity and type 2 diabetes.
Author's contributions
J.X. and Q.G. designed research. CJ.L., B.C., and S.F. performed
research, all authors analyzed data, J.X. CJ.L., and B.C. wrote the
paper, all authors approved the final version.
Competing interests
The authors declare no competing financial interest.
Acknowledgements
This work was funded by the Science and Technology Planning
Project of Guangdong Province (No. 2016A020217002), the Medical
Scientific Research Foundation of Guangdong Province (A2016133),
the National Natural Science Foundation of China (No. 81603030),
the National Natural Science Foundation of China (No. 81473138),
the National Natural Science Foundation of China (No. 81603026),
and the 111 Project (No. B16047).
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.ejmech.2017.11.017.
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