Small molecule IVQ, as a prodrug of gluconeogenesis inhibitor QVO, efficiently ameliorates glucose homeostasis in type 2 diabetic mice

Article abstract of DOI:10.1038/s41401-018-0208-2

Gluconeogenesis is a major source of hyperglycemia in patients with type 2 diabetes mellitus (T2DM), thus targeting gluconeogenesis to suppress glucose production is a promising strategy for anti-T2DM drug discovery. In our preliminary in vitro studies, we found that a small-molecule (E)-3-(2-(quinoline-4-yl)vinyl)-1H-indol-6-ol (QVO) inhibited the hepatic glucose production (HGP) in primary hepatocytes. We further revealed that QVO suppressed hepatic gluconeogenesis involving calmodulin-dependent protein kinase kinase β- and liver kinase B1-adenosine monophosphate-activated protein kinase (AMPK) pathways as well as AMPK-independent mitochondrial function-related signaling pathway. To evaluate QVO’s anti-T2DM activity in vivo, which was impeded by the complicated synthesis route of QVO with a low yield, we designed and synthesized 4-[2-(1H-indol-3-yl)vinyl]quinoline (IVQ) as a prodrug with easier synthesis route and higher yield. IVQ did not inhibit the HGP in primary hepatocytes in vitro. Pharmacokinetic studies demonstrated that IVQ was quickly converted to QVO in mice and rats following administration. In both db/db and ob/ob mice, oral administration of IVQ hydrochloride (IVQ-HCl) (23 and 46 mg/kg every day, for 5 weeks) ameliorated hyperglycemia, and suppressed hepatic gluconeogenesis and activated AMPK signaling pathway in the liver tissues. Furthermore, IVQ caused neither cardiovascular system dysfunction nor genotoxicity. The good druggability of IVQ has highlighted its potential in the treatment of T2DM and the prodrug design for anti-T2DM drug development.

Full text of DOI:10.1038/s41401-018-0208-2

www.nature.com/aps
ARTICLE
Small molecule IVQ, as a prodrug of gluconeogenesis inhibitor
QVO, efficiently ameliorates glucose homeostasis in type 2
diabetic mice
Ting-ting Zhou1, Tong Zhao2, Fei Ma3, Yi-nan Zhang2, Jing Jiang2, Yuan Ruan2, Qiu-ying Yan2, Gai-hong Wang3, Jin Ren3,
Xiao-wei Guan2, Jun Guo2, Yong-hua Zhao4, Ji-ming Ye5, Li-hong Hu2, Jing Chen3 and Xu Shen2
Gluconeogenesis is a major source of hyperglycemia in patients with type 2 diabetes mellitus (T2DM), thus targeting
gluconeogenesis to suppress glucose production is a promising strategy for anti-T2DM drug discovery. In our preliminary in vitro
studies, we found that a small-molecule (E)-3-(2-(quinoline-4-yl)vinyl)-1H-indol-6-ol (QVO) inhibited the hepatic glucose production
(HGP) in primary hepatocytes. We further revealed that QVO suppressed hepatic gluconeogenesis involving calmodulin-dependent
protein kinase kinase β- and liver kinase B1-adenosine monophosphate-activated protein kinase (AMPK) pathways as well as AMPK-
independent mitochondrial function-related signaling pathway. To evaluate QVO’s anti-T2DM activity in vivo, which was impeded
by the complicated synthesis route of QVO with a low yield, we designed and synthesized 4-[2-(1H-indol-3-yl)vinyl]quinoline (IVQ)
as a prodrug with easier synthesis route and higher yield. IVQ did not inhibit the HGP in primary hepatocytes in vitro.
Pharmacokinetic studies demonstrated that IVQ was quickly converted to QVO in mice and rats following administration. In both
db/db and ob/ob mice, oral administration of IVQ hydrochloride (IVQ-HCl) (23 and 46 mg/kg every day, for 5 weeks) ameliorated
hyperglycemia, and suppressed hepatic gluconeogenesis and activated AMPK signaling pathway in the liver tissues. Furthermore,
IVQ caused neither cardiovascular system dysfunction nor genotoxicity. The good druggability of IVQ has highlighted its potential
in the treatment of T2DM and the prodrug design for anti-T2DM drug development.
Keywords: type 2 diabetes mellitus; hepatic gluconeogenesis; AMPK signaling pathway; (E)-3-(2-(quinoline-4-yl)vinyl)-1H-indol-6-ol
(QVO); 4-[2-(1H-indol-3-yl)vinyl]quinoline (IVQ); prodrug
Acta Pharmacologica Sinica (2019) 0:1–12; https://doi.org/10.1038/s41401-018-0208-2
INTRODUCTION
homeostasis of the whole body, including skeletal muscles,
adipose tissues, pancreatic islets, and the liver [4, 5]. Structurally,
AMPK is a heterotrimeric complex with one catalytic α subunit and
two regulatory units β and γ, containing two isoforms of α (α1
and α2) and β (β1 and β2), and three isoforms of γ subunits (γ1, γ2,
and γ3) [6, 7]. It is activated by elevated AMP upon energy
shortage, causing inhibition of the ATP-consuming anabolic
process and stimulation of the ATP-producing process [4]. AMPK
activity can be directly regulated by AMP binding to the
regulatory γ subunit or indirectly by phosphorylating the catalytic
α subunit on Thr172 through other kinases, including calmodulin-
dependent protein kinase kinase β (CaMKKβ) and liver kinase B1
(LKB1) [5]. AMPK inactivation is associated with several metabolic
disorders, including T2DM, obesity and insulin resistance [5].
Because of its crucial role in maintaining energy homeostasis,
AMPK is always accepted as a potent target for drug development
against metabolic disease [7].
Diabetes mellitus (DM) is a chronic disease with high morbidity
and mortality [1]. Type 2 diabetes mellitus (T2DM), accounting for
over 90% of DM cases, is a chronic metabolic disease with a
complex pathogenesis that is characterized by insulin resistance,
pancreatic β-cell dysfunction, and elevated glucose production [2].
Physiologically, the liver has a pivotal role in maintaining glucose
homeostasis, and hepatic gluconeogenesis is stimulated to
maintain the blood glucose level in the normal range during
prolonged starvation [3], while uncontrolled regulation of
gluconeogenesis is believed to be one of the major factors
leading to metabolic disorders [2]. Accumulated evidence has
demonstrated that gluconeogenesis is a main source of contin-
uous hepatic glucose production (HGP) in the diabetic condition,
contributing to 50%–60% of the elevated glucose level in T2DM
patients [1]. Therefore, targeting gluconeogenesis inhibition is a
promising strategy for anti-diabetic drug discovery.
Adenosine monophosphate-activated protein kinase (AMPK), as
a major energy sensor, functions potently in maintaining glucose
In the current study, we found that the small-molecule (E)-3-(2-
(quinoline-4-yl)vinyl)-1H-indol-6-ol (QVO; Fig. 1a) inhibited
1Wuxi School of Medicine, Jiangnan University, Wuxi 214122, China; 2State Key Laboratory Cultivation Base for TCM Quality and Efficacy, School of Medicine and Life Sciences,
Nanjing University of Chinese Medicine, Nanjing 210023, China; 3Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; 4Institute of
Chinese Medical Sciences, University of Macau, Macau, China and 5School of Health and Biomedical Sciences, RMIT University, PO Box 71, Melbourne, VIC 3083, Australia
Correspondence: Li-hong Hu (lhhu@njucm.edu.cn) or Jing Chen (jingchen@simm.ac.cn) or Xu Shen (xshen@njucm.edu.cn)
These authors contributed equally: Ting-ting Zhou and Tong Zhao
Received: 23 May 2018 Accepted: 23 December 2018
© CPS and SIMM 2019

IVQ ameliorated glucose homeostasis and activated AMPK
TT Zhou et al.
2
hepatic gluconeogenesis in primary hepatocytes according to
the screening platform against the laboratory in-house com-
pound library. In addition, we identified that 4-[2-(1H-indol-3-yl)
vinyl]quinoline (IVQ), as a prodrug of QVO, could be converted to
QVO in vivo, and IVQ hydrochloride (IVQ-HCl) effectively
suppressed hepatic gluconeogenesis and ameliorated glucose
homeostasis in T2DM model mice. IVQ-HCl decreased fasting
blood glucose and glycated hemoglobin (HbA1c) levels and
improved glucose and pyruvate tolerance in both db/db and ob/
ob mice. Moreover, IVQ exerted no obvious cardiovascular
system dysfunction or apparent genotoxicity, suggesting the
potential druggability of IVQ. Mechanistic studies have demon-
strated that QVO probably suppressed gluconeogenesis invol-
ving the CaMKKβ- and LKB1-AMPK pathways and AMPK-
independent mitochondrial function-related signaling pathway.
Our current work has highlighted the promising strategy of
targeting gluconeogenesis inhibition and the potential of IVQ in
the treatment of T2DM.
ice. The kinase reaction was initiated upon adding ATP (50 μM)
and substrate SAMS (100 μM; Sangong, Shanghai, China) at room
temperature for 30 min, avoiding light. The generation of ADP
reflected AMPK enzyme activity, and ADP was detected by the
ADP Hunter Plus assay kit (Discover X, CA, USA). The fluorescent
signal was detected using an M5 multi-detection reader (Mole-
cular Devices, CA, USA) at an excitation wavelength of 530 nm and
an emission wavelength of 590 nm.
Western blot assay
Western blot assays were performed as previously described [10].
Briefly, cell lysates were separated by SDS-PAGE and were
transferred to nitrocellulose membranes (GE Health, CHI, USA).
The membranes were incubated with the corresponding primary
antibodies at 4 °C overnight and were subsequently incubated
with secondary antibodies for 2 h at room temperature. Visualiza-
tion of the signals on the membranes was obtained using the
ImageQuant LAS 4000 mini system (GE Health) and West-Dura
detection system (Thermo Scientific).
MATERIALS AND METHODS
Materials and reagents
Glucagon, STO609, and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-
H-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich
(Shanghai, China). QVO was obtained from
compound library (SPECS, Zoetermeer, Netherlands). IVQ-HCl
was prepared as indicated in the Supplementary Materials section.
Antibodies against phospho-AMPK (Thr172), AMPK and LKB1 were
from Cell Signaling Technology (MA, USA), and glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) was from Kangcheng Bio-
tech (Shanghai, China). The antibody against phospho-TORC2
(Ser171) was obtained from Bioss Antibodies, Inc. (MA, USA). LKB1-
siRNA and AMPK-siRNA were purchased from Thermo Scientific
(MA, USA). Lipofectamine RNAiMAX and all media for cell culture,
fetal bovine serum and antibiotic supplements were from
Invitrogen (CA, USA).
ATP and MMP assays
ATP and MMP assays were performed as previously described [11].
For the ATP assay, hepatocytes were incubated with QVO (1, 10,
20 μM) for 4 h. The concentration of ATP was detected using the
ATP assay kit (Beyotime Company, S0027, Shanghai, China)
according to the manufacturer’s protocol.
The mitochondrial membrane potential (MMP) level was
detected using the MMP assay kit with JC-1 (Beyotime Company,
C2006) according to the manufacturer’s instructions. The hepato-
cytes were incubated with the corresponding compounds for 4 h
and then were incubated with JC-1 (10 µg/mL) for 20 min at 37 °C.
After washing twice with JC-1 buffer, the fluorescent signal was
detected using an M5 multi-detection reader at 490/530 (ex/em)
and 525/590 (ex/em). The data were shown as the relative ratio
between the red fluorescent signal and green fluorescent signal.
a commercial
MTT assay
Cell culture
The cells were incubated with different concentrations of QVO
(1, 10, 20 μM) for 24 h, followed by the addition of 200 μL of
DMSO to dissolve the formazan crystals after incubation with
MTT (0.5 mg/mL) at 37 °C for 4 h. The absorbance at 490 or 570
nm was measured using an M5 spectrophotometer (Molecular
Devices).
Primary hepatocytes were isolated from nine-week-old male
C57BL/6 mice using a two-step collagenase perfusion method as
previously described [8]. Freshly isolated mouse hepatocytes were
cultured in Williams’ E medium supplemented with 10% FBS, 100
U/mL of penicillin, and 100 mg/mL of streptomycin.
HGP and q-PCR assays
Seahorse assay
HGP and q-PCR assays were conducted as described previously [8].
For the HGP assay, hepatocytes were treated with serum-free MEM
containing the corresponding compounds and glucagon (10 nM)
for 16 h. The cells were then incubated with compounds and
glucagon (10 nM) in glucose production detection buffer (glucose-
free DMEM without phenol red supplemented with sodium lactate
(20 mM) and sodium pyruvate (2 mM)) for 6 h. Detection buffer
was collected to detect the glucose level using a colorimetric
glucose assay kit (Nanjing Jiancheng, Nanjing, China). The primers
for q-PCR were obtained from Sangon Biotech (Shanghai, China)
and were as follows:
G6Pase (+): 5'-TAATTGGCTCTGCCAATGGCGATC-3';
G6Pase (−): 5'-ATCAGTCTGTGCCTTGCCCCTGT-3';
PEPCK (+): 5'-CTGCATAACGGTCTGGACTTC-3';
PEPCK (−): 5'-CAGCAACTGCCCGTACTCC-3';
GAPDH (+): 5'-ACAGCAACAGGGTGGTGGAC-3';
GAPDH (−): 5'-TTTGAGGGTGCAGCGAACTT-3'.
Hepatocytes were seeded at 5000 cells/well in MEM with 10% FBS
and 1% penicillin-streptomycin in XF96 cell culture microplates
(Seahorse Biosciences, CA, USA) overnight. The cells were starved
for 4 h with MEM containing QVO (1, 10, 20 μM) or metformin
(2 mM, as a positive control). Next, the cells were cultured in
−
175 μL of XF assay base medium (HCO3 free-modified DMEM
(Seahorse Bioscience) supplemented with 1 mM L-glutamine and
1 mM pyruvate) at 37 °C with no CO2 supplementation for 1 h to
allow temperature and pH equilibration, followed by measure-
ment of the baseline cellular respiration using the Seahorse XF96
instrument (Seahorse Bioscience). This device detected the
oxygen consumption rate (OCR) in 96-well plates after sequentially
adding to each well 25 μL of the corresponding compounds, an
ATP coupler (oligomycin, 1 μM), an uncoupling agent [carbonyl
cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), 0.5 μM],
and a mixture of antimycin A (1 μM) and rotenone (1 μM)
according to the instructions of the Seahorse XF Cell Mito Stress
kit (Seahorse Biosciences).
AMPK enzymatic activity assay
Recombinant AMPK activity was assessed using a modified non-
radioactive approach as published previously [9]. Briefly,
recombinant-AMPK α2β1γ1 (400 ng/mL, Invitrogen) was pre-
incubated with QVO (20 μM) or A-769662 (1 μM) for 30 min on
Mitochondrial respiratory complex activity assay
Mitochondria were obtained as described previously [12]. Briefly,
hepatocytes were harvested, and cell pellets were homogenized in
buffer A (83 mM sucrose, 10 mM Mops, pH = 7.2). The same volume
Acta Pharmacologica Sinica (2019) 0:1 – 12

IVQ ameliorated glucose homeostasis and activated AMPK
TT Zhou et al.
3
of buffer B (250 mM sucrose, 30 mM Mops) was added to the
sample, and the homogenate was centrifuged at 1000 × g/min for
5 min. The supernatant was then re-centrifuged at 12 000 × g/min
for 2 min to obtain the mitochondrial fraction. The mitochondria
were suspended in buffer D (1 M 6-aminohexanoic acid, 50 mM Bis-
Tris·HCl, pH = 7.0) after washing in buffer C (320 mM sucrose, 1 mM
EDTA, 10 mM Tris·HCl, pH = 7.4). The highly enriched mitochondrial
fractions were used to detect the activity of mitochondrial
respiratory complexes using the mitochondrial respiratory chain
complex I (NADH dehydrogenase), complex II (succinate dehydro-
sodium pyruvate (2 mM) were used as gluconeogenic substrates.
As indicated in Fig. 1b, QVO (1, 10, 20 μM) efficiently suppressed
glucagon-stimulated HGP in primary hepatocytes.
QVO inhibits gluconeogenic rate-limiting enzymes G6Pase and
PEPCK: Next, we tested the activity of QVO in suppressing
gluconeogenesis by q-PCR to investigate the mRNA levels of the
gluconeogenic rate-limiting enzymes glucose-6-phosphatase
(G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) in
hepatocytes. Incubation of QVO (1, 10, 20 μM) with glucagon
efficiently reduced the mRNA levels of G6Pase and PEPCK
compared with the results with glucagon treatment (Fig. 1c, d).
Therefore, these results demonstrated that QVO inhibits hepatic
gluconeogenesis.
genase), complex III (cytochrome
c reductase), complex IV
(cytochrome c oxidase), and complex V (mitochondrial F0F1-ATP
synthase) activity assay kits (GENMED, Shanghai, China) with the
M5 spectrophotometer. Complex I was measured as rotenone-
sensitive NADH-CoQ reductase by monitoring the oxidation of
NADH at 340 nm in the presence of coenzyme Q. Complex II was
measured at 600 nm by succinate-mediated phenazine methosul-
fate reduction of dichloroindophenol by succinate dehydrogenase.
Complex III was measured at 550 nm by succinate-mediated
cytochrome c reduction. Complex IV was measured by the oxidation
of reduced cytochrome c at 550 nm. Complex V was measured as
oligomycin-sensitive ATP synthase by monitoring the oxidation of
NADH at 340 nm in the presence of pyruvate.
QVO inhibits hepatic gluconeogenesis involving AMPK
QVO activates AMPK phosphorylation: Next, we investigated
the underlying mechanism of QVO in inhibiting gluconeogenesis.
As shown in Fig. 2a, Western blotting indicated that treatment
with QVO (1, 10, 20 μM) for 4 h dose dependently activated
AMPK phosphorylation but had no effects on total AMPK
in primary hepatocytes. In addition, the AMPK agonist A-769662
(1 μM, as a positive control) effectively increased the recombinant-
AMPK enzyme activity, but QVO (20 μM) had no effects on
AMPK enzyme activity (Fig. 2b), suggesting that QVO is an
indirect activator of AMPK and activates AMPK phosphorylation.
QVO inhibits hepatic gluconeogenesis partially via AMPK
phosphorylation: Because we have determined that QVO
inhibited hepatic gluconeogenesis and stimulated AMPK phos-
phorylation, we next examined whether the inhibition of QVO
against hepatic gluconeogenesis was required by its AMPK
activation. As shown in Fig. 2c, co-incubation with the AMPK
inhibitor compound C (20 μM) for 4 h impeded QVO-induced
AMPK phosphorylation in hepatocytes. However, in the HGP
assay, we noticed that compound C mildly antagonized the
effect of QVO on HGP reduction and the decrease rates of HGP
caused by QVO in the absence (18.33%) or presence (15.38%) of
compound C were close (Fig. 2d). Next, we conducted q-PCR
using AMPK-siRNA. As shown in Fig. 2e and f, AMPK-siRNA
obviously antagonized the inhibition of QVO against G6Pase,
although QVO still reduced the PEPCK mRNA level.
Animal experiments
All animals received humane care, and all animal-related protocols
were approved by the Institutional Animal Care and Use Committees
at Shanghai Institute of Materia Medica, Chinese Academy of
Sciences. db/db and ob/ob male mice were obtained from the
Jackson Laboratory (CA, USA) and were housed in a room
maintained at 20–25 °C, 50% relative humidity and a 12-h light/12-
h dark cycle with food and water ad libitum. Eight-week-old male
mice were divided into three groups by fasting blood glucose and
body weight. Vehicle or IVQ-HCl (23, 46 mg/kg) was orally
administered by gavage needle daily for 5 weeks (n = 8). Fasting
blood glucose levels from 6-h fasted mice were measured weekly.
For the oral glucose tolerance test (OGTT)/pyruvate tolerance test
(PTT), the mice were fasted for 16 h at the fourth/fifth week, and
glucose (1.5 g/kg)/pyruvate (1.5 g/kg) was administered orally/
intraperitoneally. The glucose levels were measured from tail vein
blood samples at 0, 15, 30, 60, 90, and 120 min by ACCU-CHEK
(Roche, Basel, Switzerland). At the termination of the assay, the mice
were sacrificed, and liver tissues were stored at −80 °C for analysis.
For pharmacokinetic assay in vivo, the mice were administered
IVQ-HCl (30 mg/kg, po.; 5 mg/kg, iv.), and rats were administered
IVQ-HCl (20 mg/kg, po.; 10 mg/kg, iv.). The plasma samples were
then collected to detect the concentrations of IVQ at 0.25, 0.5, 1, 2,
4, 8, and 24 h, finally fitting out the related pharmacokinetic
parameters. The metabolite of IVQ was detected and confirmed by
standard samples of QVO in mouse plasma after oral administra-
tion. The QVO concentration was determined in the liver tissues of
the mice and rats at the peak time.
Because the main downstream target of AMPK related to
hepatic gluconeogenesis is the cAMP-responsive element-binding
protein-regulated transcription co-activator 2 (TORC2) [13], we
detected the effect of QVO on TORC2 phosphorylation at Ser171.
As shown in Fig. 2g, QVO (20 µM) and metformin (2 mM, as a
positive control) increased the TORC2 phosphorylation level.
These results suggested that QVO might inhibit hepatic
gluconeogenesis involving the AMPK pathway.
QVO activates AMPK phosphorylation involving CaMKKβ and LKB1
pathways
QVO activates AMPK phosphorylation involving the CaMKKβ and
LKB1 pathways: We next investigated the potential pathways
involved in the QVO-induced AMPK activation. Considering that
AMPK activity is closely related to the phosphorylation on Thr172
of α subunit by upstream kinases CaMKKβ and LKB1 [14], we
wondered whether CaMKKβ or LKB1 signaling participated in the
QVO-induced AMPK phosphorylation. As shown in Fig. 3a, QVO-
induced AMPK phosphorylation was partially attenuated by the
CaMKKβ inhibitor STO609 (2 μg/mL) [15] in hepatocytes. Addi-
tionally, STO609 (2 μg/mL) weakened QVO-induced HGP reduction
from 12.25% to 8.66% (Fig. 3b) and STO609 antagonized the
inhibition of QVO against the G6Pase mRNA level (Fig. 3c). Thus,
these results demonstrated that the CaMKKβ pathway is at
least involved in the regulation of QVO against AMPK
phosphorylation.
Statistical analysis
All values are presented as the mean SEM. Significant differences
of two groups were compared by two-tailed unpaired Student’s t-
test, and multiple treatment groups were compared within
individual experiments by ANOVA. A P-value < 0.05 was considered
to be statistically significant. Significant differences were shown as
*P < 0.05, **P < 0.01, and ***P < 0.001. Unless otherwise indicated, all
experiments were repeated at least three times.
RESULTS
QVO inhibits hepatic gluconeogenesis
QVO inhibits hepatic glucose production: Initially, we found that
QVO (Fig. 1a) could inhibit HGP based on the previously reported
screening platform [8]. In the assay, sodium lactate (20 mM) and
To investigate the potential participation of LKB1 signaling in
QVO-induced AMPK activation, we carried out the RNA
Acta Pharmacologica Sinica (2019) 0:1 – 12

IVQ ameliorated glucose homeostasis and activated AMPK
TT Zhou et al.
4
Fig. 1 QVO inhibits hepatic gluconeogenesis. a Chemical structure of QVO. b QVO (1, 10, 20 μM) inhibited the glucagon-induced HGP in a
dose-dependent manner. c, d Primary hepatocytes were cultured with QVO (1, 10, 20 μM) for 24 h, and then co-incubated with glucagon
(10 nM) together for another 2 h, finally harvested for q-PCR assay to determine the mRNA levels of G6Pase (c) and PEPCK (d). All data were
obtained from three independent experiments and presented as mean SEM (*P < 0.05, **P < 0.01, ***P < 0.001)
interference assay to knockdown LKB1 in primary hepatocytes as
described previously [9]. As demonstrated in Fig. 3e, LKB1
knockdown efficiently reduced QVO-induced AMPK phosphoryla-
tion, suggesting that LKB1 signaling was also involved in the
regulation of QVO against AMPK phosphorylation. Moreover, we
conducted q-PCR in hepatocytes transfected with LKB1-siRNA. As
shown in Fig. 3f and g, LKB1-siRNA at least partially blocked the
inhibition effect of QVO on gluconeogenesis genes. STO609 and
LKB1-siRNA together completely revised the stimulation of QVO
on AMPK phosphorylation (Fig. 3h).
reduce ΔΨm with 4-h treatment, and QVO (20 μM) decreased
ΔΨm in a relatively mild manner compared with CCCP (10 μM, a
positive control) [19] in primary hepatocytes. In addition, the
MTT assay (Fig. 4c) revealed that QVO (1, 10, 20 μM) rendered no
effects on cell viability in primary hepatocytes. These results
indicated that QVO inhibits mitochondrial function without
inducing cell toxicity.
QVO inhibits the activities of mitochondrial respiratory complexes I
and III: To determine whether ATP reduction was implicated in
cellular mitochondrial respiration, we examined oxygen consump-
tion in primary hepatocytes and found that QVO (1, 10, 20 μM)
dose dependently inhibited respiration (Fig. 4d). Reports have
indicated that the activation of AMPK leads to enhanced
mitochondrial biogenesis [20] and mitochondrial function
inhibition-induced energy reduction, in turn, is an upstream
signal to activate AMPK [21]. However, a recent report showed
that the mitochondrial respiratory chain complex I inhibitor
rotenone induced glycolysis and reduced hepatic glucose output
via a non-AMPK pathway, although rotenone also activated AMPK
[22]. Thus, we assessed the potential effects of QVO on
mitochondrial respiratory complexes. In the assay, the primary
hepatocytes were incubated with QVO (20 μM) or the correspond-
ing positive compounds (metformin vs. complex I, phenformin vs.
complex II [23], antimycin A vs. complex III [24], phenformin vs.
complex IV [23], oligomycin vs. complex V [24]) for 4 h, and
mitochondria were isolated to determine the activity of respiratory
complexes. As shown in Fig. 4e, QVO (20 μM) inhibited the
activities of mitochondrial respiratory complexes I and III instead
of those of complex II, IV or V.
Taken together, these results indicated that the two upstream
signals CaMKKβ and LKB1 are involved in the regulation of QVO
against AMPK and gluconeogenesis.
QVO inhibits the mitochondrial function-related signaling
pathway
QVO inhibits mitochondrial function: To date, the suppression of
AMPK activation against hepatic gluconeogenesis is under
much debate. An increasing number of reports have indicated
that AMPK is not probably requisite for hepatic gluconeogenesis
regulation in related genetic loss-of-function studies [16, 17].
For example, metformin inhibits hepatic gluconeogenesis in an
LKB1- and AMPK-independent manner via a decrease in the
hepatic energy state involving decreasing ATP level [18].
Therefore, we next detected the influence of QVO on the ATP
level. The results in Fig. 4a showed that QVO (1, 10, 20 μM)
decreased the ATP content in a dose-dependent manner. Given
that ATP is primarily generated in mitochondria and a decrease
in the mitochondrial membrane potential (ΔΨm) is accompanied
by reduced ATP production [19], we evaluated the influence of
QVO on ΔΨm. As indicated in Fig. 4b, QVO (1, 10, 20 μM) could
These results thus implied that QVO inhibits the mitochondrial
function-related signaling pathway.
Acta Pharmacologica Sinica (2019) 0:1 – 12

IVQ ameliorated glucose homeostasis and activated AMPK
TT Zhou et al.
5
Fig. 2 QVO inhibits hepatic gluconeogenesis involving AMPK activation. a Primary hepatocytes were cultured with QVO (1, 10, 20 μM) for 4 h,
and then collected for Western blot assay with antibodies against p-AMPK and AMPK. b A-769662 (1 μM), as a positive control, increased the
activity of recombinant AMPK enzyme. QVO (20 μM) exhibited no influence on the recombinant AMPK activity. c Primary hepatocytes were
cultured with QVO (20 μM) with or without compound C (20 μM) for 4 h, and then collected for Western blot assay with antibodies against p-
AMPK and AMPK. d Compound C (20 μM) involved HGP assay was conducted. e, f AMPK-siRNA (100 pM)-transfected primary hepatocytes were
cultured with QVO (20 μM) for 24 h, and then co-incubated with glucagon (10 nM) together for another 2 h, finally harvested for q-PCR assay
to determine the mRNA levels of G6Pase (e) and PEPCK (f). g Primary hepatocytes were cultured with QVO (20 μM) and metformin (2 mM) for
4 h, and then collected for Western blot assay with antibodies against p-TORC2. All data were obtained from three independent experiments
and presented as mean SEM (*P < 0.05, **P < 0.01, ***P < 0.001; ns, no significance)
IVQ, as a prodrug of QVO, efficiently ameliorates hyperglycemia in
db/db and ob/ob mice
IVQ functions as a prodrug of QVO: To evaluate the activity of QVO
in anti-T2DM in vivo, we synthesized an increased amount of QVO
for animal experiments. Given the complicated synthesis route
and low yield of QVO (Fig. 5a) and that the hydroxylation pathway
is a principal metabolic pathway associated with cytochrome P450
enzymes (CYPs) activation [25, 26], we next carried out the
relevant prodrug design and speculated that IVQ, as a potential
prodrug with easier synthesis route and higher yield (Fig. 5b),
could be converted into QVO after metabolic reaction (Fig. 5c).
Accordingly, we performed the related pharmacokinetic assay
in vivo. To improve the solubility of IVQ, IVQ-HCl was administered
instead of IVQ in vivo [27]. The results demonstrated that IVQ was
Acta Pharmacologica Sinica (2019) 0:1 – 12

IVQ ameliorated glucose homeostasis and activated AMPK
TT Zhou et al.
6
Fig. 3 QVO activates AMPK phosphorylation involving CaMKKβ and LKB1 pathways. a Primary hepatocytes were cultured by QVO (20 μM)
with or without CaMKKβ inhibitor STO609 (2 μg/mL) for 4 h, and then collected for Western blot assay with antibodies against p-AMPK and
AMPK. b STO609 (2 μg/mL) involved HGP assay was conducted. c, d STO609 (2 μg/mL) involved q-PCR assay was conducted. e Primary
hepatocytes were transfected with LKB1-siRNA (100 pM) for 24 h, and then treated with QVO (20 μM) for 4 h, finally collected for Western blot
assay with antibodies against LKB1, p-AMPK, and AMPK. f, g AMPK-siRNA (100 pM)-transfected primary hepatocytes were cultured with QVO
(20 μM) for 24 h, and then co-incubated with glucagon (10 nM) together for another 2 h, finally harvested for q-PCR assay to determine the
mRNA levels of G6Pase (f) and PEPCK (g). h LKB1-siRNA (100 pM)-transfected primary hepatocytes were treated with QVO (20 μM) and STO609
(2 μg/mL) for 4 h, finally collected for Western blot assay with antibodies against p-AMPK and AMPK. All data were obtained from three
independent experiments and presented as mean SEM (*P < 0.05, **P < 0.01, ***P < 0.001, ns, no significance)
Acta Pharmacologica Sinica (2019) 0:1 – 12

IVQ ameliorated glucose homeostasis and activated AMPK
TT Zhou et al.
7
Fig. 4 QVO inhibits mitochondrial function-related signaling pathway. a Primary hepatocytes were treated with QVO (1, 10, 20 μM) for 4 h, and
then ATP content was detected. b Primary hepatocytes were treated with QVO (1, 10, 20 μM) or CCCP (10 μM, a positive control) for 4 h, and
then ΔΨm values were detected. c Primary hepatocytes were treated with QVO (1, 10, 20 μM) for 24 h, and then used for MTT assay to detect
cell viability. d Primary hepatocytes were treated with QVO (1, 10, 20 μM) or metformin (as a positive control) for 4 h, and then oxygen
consumption of respiration was detected according to Seahorse XF cell mito stress test kit on Seahorse XFe96. e Primary hepatocytes were
treated with QVO (20 μM) or corresponding positive controls (metformin vs. complex I, phenformin vs. complex II, antimycin A vs. complex III,
phenformin vs. complex IV, oligomycin vs. complex V) for 4 h, and collected for mitochondria isolation to detect the activity of mitochondrial
respiratory complexes. All data were obtained from three independent experiments and presented as mean SEM (**P < 0.01, ***P < 0.001, ns,
no significance)
quickly metabolized with a short half-life in mice and rats (Table 1).
We also found that IVQ itself could not reduce the HGP stimulated
by glucagon (Fig. 5d). In addition, QVO was the main metabolite of
IVQ in mice orally administered IVQ-HCl (Fig. 5e). We also
determined that IVQ could be converted to QVO in mice and
rats (Supplemental Table 1), and the half-life of QVO was longer
than that of IVQ (Supplemental Table 2). In addition, the QVO
concentration reached 129.84 15.42 and 281.30 54.87 ng/g
(mean SEM, n = 3) in the liver tissues of mice and rats at the
peak time of QVO. These results indicated that IVQ functions as a
prodrug of QVO.
IVQ ameliorates hyperglycemia in db/db and ob/ob mice:
Because QVO has been found to be capable of inhibiting
hepatic gluconeogenesis in hepatocytes, we next examined the
capability of its prodrug IVQ in ameliorating hyperglycemia
against T2DM model mice. It was found that IVQ-HCl adminis-
tration obviously decreased the fasting blood glucose and
HbA1c levels (Fig. 6a, b) and improved the glucose (Fig. 6c, d)
and pyruvate tolerance (Fig. 6e, f) in both model mice.
Therefore, all results demonstrated that IVQ ameliorates
hyperglycemia in db/db and ob/ob mice.
IVQ suppresses hepatic gluconeogenesis and activates the AMPK
signaling pathway in db/db and ob/ob mice
IVQ suppresses hepatic gluconeogenesis in db/db and ob/ob mice:
Given that IVQ improved pyruvate tolerance in vivo, we further
assessed its capability in suppressing gluconeogenesis in the liver
tissues of db/db and ob/ob mice. In addition, as shown in Fig. 7a–d,
IVQ-HCl treatment reduced the mRNA levels of G6Pase and PEPCK
in either of the model mice, indicating that IVQ suppresses hepatic
gluconeogenesis in db/db and ob/ob mice.
IVQ activates the AMPK pathway in db/db and ob/ob mice: Next,
we also evaluated the potential of IVQ in regulating the AMPK
signaling pathway in the liver tissues of db/db and ob/ob mice. As
indicated in Fig. 7e and f, IVQ-HCl treatment increased the
phosphorylation level of AMPK in the liver tissues of either model
mice, thereby suggesting that IVQ activates the AMPK pathway in
db/db and ob/ob mice.
Thus, all above results suggested that IVQ suppresses hepatic
gluconeogenesis and activates AMPK phosphorylation in db/db
and ob/ob mice.
IVQ exhibits no apparent serum-related toxicity in db/db and ob/
ob mice
Given the highly anti-diabetic activity of IVQ, we also detected
serum-related toxicity in db/db and ob/ob mice. The results
Taken together, these above results suggested that IVQ, as a
prodrug of QVO, efficiently ameliorates hyperglycemia in db/db
and ob/ob mice.
Acta Pharmacologica Sinica (2019) 0:1 – 12

IVQ ameliorated glucose homeostasis and activated AMPK
TT Zhou et al.
8
Fig. 5 The prodrug design of QVO based on CYPs system. a Synthetic route of QVO. b Synthetic route of IVQ. c Prodrug design of QVO based
on CYPs system. d IVQ involved HGP assay was conducted. The data were obtained from three independent experiments and presented as
mean SEM (**P < 0.01, ns, no significance). e QVO was the main metabolite of IVQ in mice orally administered with IVQ (30 mg/kg, n = 3)
after 15 min
indicated that IVQ-HCl treatment failed to influence the serum
alanine aminotransferase (ALT, Supplemental Fig. 1a), aspartate
aminotransferase (AST, Supplemental Fig. 1b), albumin (ALB,
Supplemental Fig. 1c), total bilirubin (TB, Supplemental Fig. 1d),
or total protein (TP, Supplemental Fig. 1e) levels in both model
mice, demonstrating that the administration of IVQ (23, 46 mg/kg
every day) for 5 weeks exhibits no apparent serum-related toxicity
in T2DM model mice.
on cardiovascular system function in beagle dogs and geno-
toxicity on Salmonella typhimurium strains using the electro-
cardiogram and ames assays. The electrocardiogram assay
indicated that the single-dose oral treatment of IVQ-HCl (10,
30, 100 mg/kg) exerted no changes in arrhythmia or drug-
correlated changes in electrocardiogram validation marks,
including the heart rate, RR interval, PR interval, QRS duration,
and QT interval within 24 h (Supplemental Table 3), and the
ames assay showed that there were no significant differences in
the revertant colony numbers between IVQ (1–1000 μg/plate)
and the negative control in Salmonella typhimurium strains TA98
and TA100 (Supplemental Table 4). These results demonstrated
that IVQ exhibits no apparent cardiovascular system dysfunction
or genotoxicity.
IVQ exhibits no apparent cardiovascular system dysfunction or
genotoxicity
Because adverse effects on the cardiovascular system and
genotoxicity are the main causes of failure in the development
of new drugs [28, 29], we preliminarily assessed the effect of IVQ
Acta Pharmacologica Sinica (2019) 0:1 – 12

IVQ ameliorated glucose homeostasis and activated AMPK
TT Zhou et al.
9
DISCUSSION
Table 1. Pharmacokinetic assay of IVQ-HCl in mice and rats by
The liver, as one of the major organs responsible for glucose
metabolism regulation, is closely related to T2DM, metabolic
syndrome and fatty liver [30]. Suppressing excessive hepatic
gluconeogenesis is believed to be an ideal strategy to reduce
blood glucose [31]. In the current study, we screened laboratory
in-house compounds and discovered that the small-molecule
compound QVO, as a gluconeogenesis inhibitor, effectively
suppressed HGP. Mechanistic studies demonstrated that QVO
activated the CaMKKβ- and LKB1-AMPK pathways and inhibited
the mitochondrial function pathway. IVQ, as a prodrug of QVO,
patently ameliorated glucose homeostasis in T2DM model mice.
The successful findings of QVO and its prodrug IVQ in suppressing
hepatic gluconeogenesis and ameliorating glucose homeostasis
have confirmed that targeting gluconeogenesis inhibition should
be a promising strategy for T2DM treatment. Our work has also
highlighted the potential druggability of IVQ in the treatment of
T2DM.
intravenous injection (iv.) or oral administration (po.)
Mice (mean SEM)
Rats (mean SEM)
po.(20 mg/kg)
Parameters
iv.(5 mg/kg) po.(30 mg/kg) iv.(10 mg/kg)
AUC(0–t) (ng/mL*h) 1735.3 32.2
AUC(0–∞) (ng/mL*h) 1740.6 30.4
5420 1893.9 8358.4 943.4 3942.6 201.4
6535.5 962.2 8394.3 937.4 3957.6 211.4
MRT(0–t) (h)
VZ (L/kg)
CLZ (L/h/kg)
T1/2Z (h)
0.58 0.2
10.63 9.1
2.87 0.1
2.54 2.2
—
9.71 3.5
138 123.6
4.78 0.6
2.47 1.7
0.5 0.2
1.19 0.1
1.61 0.1
1.22 0.1
0.94 0.1
—
3.58 0.6
18.87 2.2
5.08 0.3
2.62 0.4
Tmax (h)
0.25 0
Cmax (ng/mL)
F%
4100.2 471.5 1078.9 298.6 8151.7 325.2 1812.3 116.3
52.1 18.2 23.6 1.2
—
—
The dosages for mice were 5 mg/kg (iv.) and 30 mg/kg (po.), and for rats
were 10 mg/kg (iv.) and 20 mg/kg (po.). Data were shown as mean SEM,
n = 3
AMPK is widely expressed in mammalian tissues, plays an
important role in controlling energy homeostasis and glucose
Fig. 6 IVQ efficiently ameliorates hyperglycemia in db/db and ob/ob mice. a, b Fasting blood glucose and HbA1c levels were detected in db/db
mice (a) and ob/ob mice (b) with treatment of IVQ-HCl (23, 46 mg/kg every day) (n = 8). c, d OGTT assay was performed in db/db mice (c) and
ob/ob mice (d) after treatment with IVQ-HCl (23, 46 mg/kg every day) for 4 weeks (n = 8). e, f PTT assay was performed in db/db mice (e) and
ob/ob mice (f) after treatment with IVQ-HCl (23, 46 mg/kg every day) for 5 weeks (n = 8). Veh: vehicle; H: 46 mg/kg; L: 23 mg/kg. All data were
presented as mean SEM (**P < 0.01, ***P < 0.001)
Acta Pharmacologica Sinica (2019) 0:1 – 12

IVQ ameliorated glucose homeostasis and activated AMPK
TT Zhou et al.
10
Fig. 7 IVQ inhibits hepatic gluconeogenesis and activates AMPK in db/db and ob/ob mice. a–d Liver tissues of db/db and ob/ob with treatment
of IVQ-HCl (23, 46 mg/kg every day) for 5 weeks were used for q-PCR assay to detect the mRNA levels of G6Pase and PEPCK (n = 8). e, f Liver
tissues of db/db mice (e) or ob/ob (f) mice with treatment of IVQ-HCl (23, 46 mg/kg every day) for 5 weeks were used for Western blot assay
with antibodies against p-AMPK and AMPK (n = 4). Veh: vehicle; H: 46 mg/kg; L: 23 mg/kg. All data were presented as mean SEM (*P < 0.05,
**P < 0.01, ***P < 0.001)
metabolism, and has become an attractive potential therapeutic
target for many diseases, including diabetes, cancer, heart disease,
viral infection, and inflammatory disorders [32], although there are
no AMPK direct activators in clinical use due to the poor
pharmacokinetic profiles or off-target effects of the agents [33,
34]. Thus, further study on AMPK regulation might help to
expound new activation mechanisms or develop compounds with
better pharmacokinetic profiles [33].
AMPK activity can be directly regulated by binding AMP to the
regulatory γ subunit or indirectly by phosphorylation on Thr172 of
the α subunit by upstream kinases [5]. It is reported that binding
AMP to the γ subunit greatly stimulates LKB1-dependent AMPKα
phosphorylation on Thr172, and AMP binding inhibits
dephosphorylation through protein phosphatases, such as PP2A
and PP2C [35]. The downstream targets of AMPK include acetyl-CoA
carboxylase related to fatty acids oxidation [33]; tuberous sclerosis
complex 2 related to mammalian target of rapamycin complex 1
and protein synthesis [36]; 3-hydroxy-3-methylglutaryl-coenzyme A
reductase related to cholesterol synthesis [37]; peroxisome
proliferator-activated receptor-gamma co-activator 1α related to
mitochondrial biogenesis [38] and TORC2 related to hepatic
gluconeogenesis [13]. However, the AMPK-independent mechanism
of hepatic gluconeogenesis suppression by metformin suggests the
dispensability of AMPK in hepatic gluconeogenesis [39]. It is
highlighted that metformin inhibits hepatic gluconeogenesis in an
LKB1- and AMPK-independent manner via a decrease in the hepatic
Acta Pharmacologica Sinica (2019) 0:1 – 12

IVQ ameliorated glucose homeostasis and activated AMPK
TT Zhou et al.
11
energy state involving decreasing ATP levels through the inhibition
of complex I [18]. Moreover, moderate inhibition against mitochon-
drial function is believed to be beneficial to anti-diabetes [19], and
inhibition of mitochondrial complex I stimulates glucose consump-
tion and decreases HGP independently of AMPK activation [22]. To
date, both AMPK-dependent and AMPK-independent mechanisms
of metformin for hepatic gluconeogenesis inhibition have been
determined, a key factor is the dosage of metformin used [39]. A
potential explanation for this discrepancy may be that AMP might
have an additional AMPK-independent effect, lowering cAMP and
reducing the expression of gluconeogenic enzymes [40]. Herein, the
small molecule QVO, as an indirect AMPK activator, activated AMPK
phosphorylation and reduced the ATP level, indicating that QVO
might suppress hepatic gluconeogenesis via AMPK-dependent and
-independent mechanisms. The precise underlying mechanism of
QVO in AMPK and mitochondrial respiratory chain remains to be
investigated. Additionally, QVO, as an AMPK activator, might have
more potential in drug discovery against metabolic diseases.
CYPs are important biological enzymes in the body with many
molecules as substrates for enzymatic reactions [41]. They are major
enzymes involved in drug metabolism, accounting for approxi-
mately 75% of the total metabolism [42] and the hydroxylation
pathway is a primary metabolic pathway related to CYPs activation
[26]. The current study suggested that QVO, as a main metabolite of
IVQ, functioned in hepatic gluconeogenesis inhibition, although
more studies related to QVO are needed. Our results have implied
the potential of CYPs in drug design—for example, by using
different CYPs to achieve targeted drug delivery, design prodrugs,
promote efficacy or decrease the adverse effects of drugs [43].
In conclusion, the small molecule QVO probably suppressed
hepatic gluconeogenesis involving CaMKKβ- and LKB1-AMPK
pathways and mitochondrial function-related signaling pathway.
IVQ, designed as a prodrug of QVO, efficiently ameliorated glucose
homeostasis in db/db and ob/ob mice without obvious cardiovas-
cular system dysfunction or genotoxicity. Our current work has
addressed the advantage of CYPs in drug design and potential of
IVQ in the treatment of T2DM.
2. An H, He L. Current understanding of metformin effect on the control of
hyperglycemia in diabetes. J Endocrinol. 2016;228:R97–106.
3. Rui L. Energy metabolism in the liver. Compr Physiol. 2014;4:177–97.
4. Weikel KA, Ruderman NB, Cacicedo JM. Unraveling the actions of AMP-activated
protein kinase in metabolic diseases: systemic to molecular insights. Metabolism.
2016;65:634–45.
5. Ramesh M, Vepuri SB, Oosthuizen F, Soliman ME. Adenosine monophosphate-
activated protein kinase (AMPK) as a diverse therapeutic target: a computational
perspective. Appl Biochem Biotechnol. 2016;178:810–30.
6. Yao F, Zhang M, Chen L. Adipose tissue-specialized immunologic features might
be the potential therapeutic target of prospective medicines for obesity. J Dia-
betes Res. 2017;2017:4504612.
7. Ewart MA, Kennedy S. Diabetic cardiovascular disease—AMP-activated protein
kinase (AMPK) as a therapeutic target. Cardiovasc Hematol Agents Med Chem.
2012;10:190–211.
8. Zhou TT, Ma F, Shi XF, Xu X, Du T, Guo XD, et al. DMT efficiently inhibits hepatic
gluconeogenesis involving Galphaq signaling pathway.
J Mol Endocrinol.
2017;59:151–69.
9. Tang X, Zhuang J, Chen J, Yu L, Hu L, Jiang H, et al. Arctigenin efficiently
enhanced sedentary mice treadmill endurance. PLoS One. 2011;6:e24224.
10. Zhou TT, Quan LL, Chen LP, Du T, Sun KX, Zhang JC, et al. SP6616 as a new
Kv2.1 channel inhibitor efficiently promotes beta-cell survival involving
both PKC/Erk1/2 and CaM/PI3K/Akt signaling pathways. Cell Death Dis. 2016;
7:e2216.
11. Guo XD, Sun GL, Zhou TT, Wang YY, Xu X, Shi XF, et al. LX2343 alleviates cognitive
impairments in AD model rats by inhibiting oxidative stress-induced neuronal
apoptosis and tauopathy. Acta Pharmacol Sin. 2017;38:1104–19.
12. Lopez-Fabuel I, Le Douce J, Logan A, James AM, Bonvento G, Murphy MP, et al.
Complex I assembly into supercomplexes determines differential mitochondrial
ROS production in neurons and astrocytes. Proc Natl Acad Sci
U S A.
2016;113:13063–8.
13. Jiang SJ, Dong H, Li JB, Xu LJ, Zou X, Wang KF, et al. Berberine inhibits hepatic
gluconeogenesis via the LKB1-AMPK-TORC2 signaling pathway in streptozotocin-
induced diabetic rats. World J Gastroenterol. 2015;21:7777–85.
14. Wang G, Xu X, Yao X, Zhu Z, Yu L, Chen L, et al. Latanoprost effectively ame-
liorates glucose and lipid disorders in db/db and ob/ob mice. Diabetologia.
2013;56:2702–12.
15. Pyla R, Osman I, Pichavaram P, Hansen P, Segar L. Metformin exaggerates
phenylephrine-induced AMPK phosphorylation independent of CaMKKbeta and
attenuates contractile response in endothelium-denuded rat aorta. Biochem
Pharmacol. 2014;92:266–79.
16. Rena G, Pearson ER, Sakamoto K. Molecular mechanism of action of metformin:
old or new insights? Diabetologia. 2013;56:1898–906.
17. Cameron AR, Logie L, Patel K, Bacon S, Forteath C, Harthill J, et al. Investigation of
salicylate hepatic responses in comparison with chemical analogues of the drug.
Biochim Biophys Acta. 2016;1862:1412–22.
18. Foretz M, Hebrard S, Leclerc J, Zarrinpashneh E, Soty M, Mithieux G, et al. Met-
formin inhibits hepatic gluconeogenesis in mice independently of the LKB1/
ACKNOWLEDGEMENTS
This work was supported by the National Natural Science Foundation of China (Grant
numbers 81473141, 81703806, 81800430), NSFC-TRF collaboration projects (Grant
numbers NSFC 81561148011), the Priority Academic Program Development of
Jiangsu Higher Education Institutions (Integration of Chinese and Western Medicine),
the Fundamental Research Funds for the Central Universities (Grant number
JUSRP11863), and Project funded by China Postdoctoral Science Foundation (Grant
number 2018M642172).
AMPK pathway via
a decrease in hepatic energy state. J Clin Invest.
2010;120:2355–69.
19. Qiu BY, Turner N, Li YY, Gu M, Huang MW, Wu F, et al. High-throughput assay for
modulators of mitochondrial membrane potential identifies a novel compound
with beneficial effects on db/db mice. Diabetes. 2010;59:256–65.
20. O’Neill HM, Maarbjerg SJ, Crane JD, Jeppesen J, Jorgensen SB, Schertzer JD, et al.
AMP-activated protein kinase (AMPK) beta1beta2 muscle null mice reveal an
essential role for AMPK in maintaining mitochondrial content and glucose uptake
during exercise. Proc Natl Acad Sci U S A. 2011;108:16092–7.
21. Lin SC, Hardie DG. AMPK: sensing glucose as well as cellular energy status. Cell
Metab. 2018;27:299–313.
22. Hou WL, Yin J, Alimujiang M, Yu XY, Ai LG, Bao YQ, et al. Inhibition of mito-
chondrial complex I improves glucose metabolism independently of AMPK
activation. J Cell Mol Med. 2018;22:1316–28.
AUTHOR CONTRIBUTIONS
XS, JC, and TTZ were responsible for the conception and design of the study. TTZ, TZ,
YR, QYY, and GHW designed and performed experiments. FM, YNZ, JJ, and LHH
contributed to the acquisition of IVQ-HCl. TTZ and XS were responsible for drafting
the manuscript. JR, XWG, JG, YHZ, and JMY gave advice on the manuscript
preparation. XS is the guarantor of this work and, as such, has full access to all the
data in the study and takes responsibility for the integrity of the data and accuracy of
the data analysis. All authors approved the manuscript.
23. Drahota Z, Palenickova E, Endlicher R, Milerova M, Brejchova J, Vosahlikova M,
et al. Biguanides inhibit complex I, II and IV of rat liver mitochondria and modify
their functional properties. Physiol Res. 2014;63:1–11.
24. Choi EM, Lee YS. Mitochondrial defects and cytotoxicity by antimycin A on cul-
tured osteoblastic MC3T3-E1 cells. Food Chem Toxicol. 2011;49:2459–63.
25. Bojic M, Sedgeman CA, Nagy LD, Guengerich FP. Aromatic hydroxylation of sal-
ADDITIONAL INFORMATION
The online version of this article (https://doi.org/10.1038/s41401-018-0208-2)
contains supplementary material, which is available to authorized users.
Competing interests: The authors declare no competing interests.
icylic acid and aspirin by human cytochromes P450. Eur J Pharm Sci.
2015;73:49–56.
26. Tekes K, Kalasz H, Hasan MY, Adeghate E, Darvas F, Ram N, et al. Aliphatic and
aromatic oxidations, epoxidation and S-oxidation of prodrugs that yield active
drug metabolites. Curr Med Chem. 2011;18:4885–900.
27. Thakuria R, Delori A, Jones W, Lipert MP, Roy L, Rodriguez-Hornedo N. Pharma-
ceutical cocrystals and poorly soluble drugs. Int J Pharm. 2013;453:101–25.
REFERENCES
1. Lemes Dos Santos PF, Dos Santos PR, Ferrari GS, Fonseca GA, Ferrari CK.
Knowledge of diabetes mellitus: does gender make a difference? Osong Public
Health Res Perspect. 2014;5:199–203.
Acta Pharmacologica Sinica (2019) 0:1 – 12

IVQ ameliorated glucose homeostasis and activated AMPK
TT Zhou et al.
12
28. Xing G, Lu J, Hu M, Wang S, Zhao L, Zheng W, et al. Effects of group housing on
ECG assessment in conscious cynomolgus monkeys. J Pharmacol Toxicol Meth-
ods. 2015;75:44–51.
37. Clarke PR, Hardie DG. Regulation of HMG-CoA reductase: identification of the site
phosphorylated by the AMP-activated protein kinase in vitro and in intact rat
liver. EMBO J. 1990;9:2439–46.
29. Luo Q, Li Y, Zhang Z.
A
systematic assessment of genotoxicity on
38. Jager S, Handschin C, St-Pierre J, Spiegelman BM. AMP-activated protein kinase
(AMPK) action in skeletal muscle via direct phosphorylation of PGC-1alpha. Proc
Natl Acad Sci U S A. 2007;104:12017–22.
pivaloylacylation-7ADCA-a wide existing antibiotic impurity. Int J Clin Exp Med.
2014;7:4260–71.
30. Kuang JR, Zhang ZH, Leng WL, Lei XT, Liang ZW. Dapper1 attenuates hepatic
gluconeogenesis and lipogenesis by activating PI3K/Akt signaling. Mol Cell
Endocrinol. 2017;447:106–15.
39. Cao J, Meng S, Chang E, Beckwith-Fickas K, Xiong L, Cole RN, et al. Low
concentrations of metformin suppress glucose production in hepatocytes
through AMP-activated protein kinase (AMPK).
J
Biol Chem. 2014;
31. Zheng J, Woo SL, Hu X, Botchlett R, Chen L, Huo Y, et al. Metformin and metabolic
diseases: a focus on hepatic aspects. Front Med. 2015;9:173–86.
32. Grahame Hardie D. AMP-activated protein kinase: a key regulator of energy
balance with many roles in human disease. J Int Med. 2014;276:543–59.
289:20435–46.
40. Miller RA, Chu Q, Xie J, Foretz M, Viollet B, Birnbaum MJ. Biguanides suppress
hepatic glucagon signalling by decreasing production of cyclic AMP. Nature.
2013;494:256–60.
33. Coughlan KA, Valentine RJ, Ruderman NB, Saha AK. AMPK activation:
a
41. Ji L, Faponle AS, Quesne MG, Sainna MA, Zhang J, Franke A, et al. Drug
metabolism by cytochrome p450 enzymes: what distinguishes the pathways
leading to substrate hydroxylation over desaturation? Chemistry.
2015;21:9083–92.
42. Guengerich FP. Cytochrome p450 and chemical toxicology. Chem Res Toxicol.
2008;21:70–83.
therapeutic target for type 2 diabetes? Diabetes Metab Syndr Obes. 2014;
7:241–53.
34. Olivier S, Foretz M, Viollet B. Promise and challenges for direct small molecule
AMPK activators. Biochem Pharmacol. 2018;153:147–58.
35. Kim J, Yang G, Kim Y, Kim J, Ha J. AMPK activators: mechanisms of action and
physiological activities. Exp Mol Med. 2016;48:e224.
36. Inoki K, Zhu T, Guan KL. TSC2 mediates cellular energy response to control cell
growth and survival. Cell. 2003;115:577–90.
43. Huttunen KM, Mahonen N, Raunio H, Rautio J. Cytochrome P450-activated pro-
drugs: targeted drug delivery. Curr Med Chem. 2008;15:2346–65.
Acta Pharmacologica Sinica (2019) 0:1 – 12